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Journal of Controlled Release
journal homepage: www.elsevier.com/locate/jconrel
Review article
Human intratumoral therapy: Linking drug properties and tumor transportof drugs in clinical trialsAric Huanga,1, Melissa M. Pressnalla,1, Ruolin Lua, Sebastian G. Huayamaresc, J. Daniel Griffina,c,Chad Groerd, Brandon J. DeKoskya,b, M. Laird Forresta, Cory J. Berklanda,b,c,⁎
a Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, KS, USAbDepartment of Chemical and Petroleum Engineering, University of Kansas, Lawrence, KS, USAc Bioengineering Graduate Program, University of Kansas, Lawrence, KS, USAdHylaPharm, LLC, Lawrence, KS, USA
A B S T R A C T
Cancer therapies aim to kill tumor cells directly or engage the immune system to fight malignancy. Checkpoint inhibitors, oncolytic viruses, cell-based im-munotherapies, cytokines, and adjuvants have been applied to prompt the immune system to recognize and attack cancer cells. However, systemic exposure of cancertherapies can induce unwanted adverse events. Intratumoral administration of potent therapies utilizes small amounts of drugs, in an effort to minimize systemicexposure and off-target toxicities. Here, we discuss the properties of the tumor microenvironment and transport considerations for intratumoral drug delivery.Specifically, we consider various tumor tissue factors and physicochemical factors that can affect tumor retention after intratumoral injection. We also reviewapproved and clinical-stage intratumoral therapies and consider how the molecular and biophysical properties (e.g. size and charge) of these therapies influencesintratumoral transport (e.g. tumor retention and cellular uptake). Finally, we offer a critical review and highlight several emerging approaches to promote tumorretention and limit systemic exposure of potent intratumoral therapies.
1. Introduction
Recent clinical successes of human intratumoral (IT) therapies havestimulated a wave of new trials investigating IT therapies alone and intandem with other immuno-oncology agents. IT delivery refers to thedirect injection of a drug/formulation into a tumor. IT therapy offersunique anti-cancer benefits since direct injection bypasses systemictrafficking and tumor penetration [1], and delivering small IT dosesreduces severe adverse events (AEs) associated with systemic deliveryof cancer therapies [2–6]. IT administration of immunostimulants, forexample, can work synergistically with checkpoint inhibitors makingnonresponsive ‘cold’ tumors ‘hot’ by recruiting and activating tumorinfiltrating lymphocytes [4,7,8]. Intuitively, the design of IT therapiesis significantly different than that of systemic cancer medications, asthese localized interventions aim for retention at the administration siteor draining lymph nodes with limited systemic exposure. In this review,we highlight transport mechanisms involved in IT delivery, review re-cent IT clinical trials, and deduce relationships between biophysicalproperties of IT therapeutics, potential effects on IT transport, andclinical results. Finally, we highlight emerging strategies for promotingtumor retention of IT therapies.
2. Overview of current cancer therapies
To begin, the palette of cancer therapies is briefly reviewed prior todelving into the current landscape of IT therapies. Physicians have ra-diation therapy, chemotherapy, and immunotherapy interventions attheir disposal. Below, we only briefly touch on radiation and che-motherapy. Then, we offer a deeper background on cancer im-munotherapies due to the potential synergies with IT therapies, whichis the key topic of this review.
2.1. Radiation therapy
Radiation therapy employs highly focused energy to kill or damagetumor cells [9–11]. Radiation can be used to treat tumors alone, or incombination with other cancer treatments such as chemotherapy, im-munotherapy, and surgery [10–12]. For instance, radiation can be usedto shrink the tumor before surgery or to eliminate residual tumor cellspost-surgery. Despite efforts to minimize radiation damage to non-cancerous normal tissues, damage to normal tissues is common, leadingto side effects such as fatigue, hair loss, and skin irritation.
https://doi.org/10.1016/j.jconrel.2020.06.029Received 12 May 2020; Received in revised form 23 June 2020; Accepted 25 June 2020
⁎ Corresponding author.E-mail address: [email protected] (C.J. Berkland).
1 These authors contributed equally to this work.
Journal of Controlled Release 326 (2020) 203–221
Available online 14 July 20200168-3659/ © 2020 Elsevier B.V. All rights reserved.
T
2.2. Chemotherapy
Chemotherapies are cytotoxic, anti-cancer agents that non-selec-tively target quickly proliferating cells [13]. The most common route ofadministration is IV, because most chemotherapeutic drugs exhibit poorand variable oral bioavailability [14,15]. Systemically administeredchemotherapies commonly elicit some degree of unintended side-effectsfrom non-selective cytotoxic action [16,17].
2.3. Immunotherapy
Cancer immunotherapy stems from Coley’s seminal work and har-nesses the body’s own immune mechanisms to fight cancer. Major im-munotherapy classes include checkpoint inhibitors, oncolytic viruses(OVs), cell-based immunotherapies, cytokines and adjuvants [18,19].Immune checkpoint inhibitors block checkpoint receptors to preventsuppressive immune responses, resulting in enhanced anti-tumor re-sponses. Ipilimumab (Yervoy®), an antibody against cytotoxic T-lym-phocyte antigen-4 (CTLA-4), was the first approved checkpoint inhibitorand increased the survival of metastatic melanoma patients [20,21].Other major checkpoint inhibitor targets include the programmed celldeath protein-1 (PD-1) (e.g., Keytruda) or its ligand (PD-L1) (e.g. Imfiazior Opdivo) [21]. However, checkpoint inhibitors are often associated withirAEs and toxicities from over-activation of T lymphocytes [22]. OVs canbe engineered to selectively infect cancer cells and stimulate anti-tumorimmune responses. The oncolytic herpes virus, talimogene laherparepvec(T-Vec), was the first approved OV for the treatment of advanced mela-noma, but toxic side effects caused by genetic manipulation still remain asafety concern [23]. Cytokines are often combined with adjuvants and areimmunomodulators that enhance the host anti-tumor immune responses.Interferon-α and interleukin-2 are two cytokines that have been approvedfor the treatment of several types of leukemia and melanoma [24]. Ad-juvants can stimulate immune responses and are often added to vaccinesto improve immunogenicity [25,26]. For instance, the adjuvant AS04 is aTLR4 agonist used in Cervarix, an approved preventive vaccine for humanpapillomavirus (HPV) [27].
Cellular immunotherapies have provided a range of new and targetedsolutions for tumor cell killing [28]. One rapidly emerging cellular im-mune therapy uses chimeric antigen receptor (CAR) T-cells. A patient’sown T-cells are extracted and transfected with CAR, that binds to tumorantigens, thus addressing T-cell killing directly to antibody recognition.Typically, CAR-T therapy requires a pre-conditioning lymphodepletiontreatment prior to modified T-cell infusion to increase expansion of themodified T cells. The first CAR-T cell therapy, Kymriah® (or tisagenle-cleucel), was approved in 2017 for treating B-cell precursor acute lym-phoblastic leukemia that is refractory or in the second or later relapse[29]. Tisagenlecleucel is composed of an anti-CD19 scFv, a CD8-α hingeregion, 4-1BB (CD137) co-stimulation domains, and a CD3ζ signalingdomain [30]. It is prepared from a patient’s peripheral blood mono-nuclear cells by enriching for T-cells, modifying the T-cells using lenti-viral gene transfer and subsequently activating them with anti-CD3/CD28 [29]. The second and only other approved CAR-T cell therapy,Yescarta® (axicabtagene ciloleucel), was approved by the FDA for use inadults with relapsed or refractory diffuse large B-cell lymphoma a fewmonths after tisagenleceucel [31]. Axicabtagene ciloleucel is also a CD19directed CAR-T cell but differs from tisagenleceucel by using CD28 hingeand CD3ζ co-stimulation domains. CAR-T cells are highly effective forhematologic cancers; but have limited efficacy for solid tumors due tochallenges for in T-cell recruitment, expansion, and survival within theTME [32–34]. The first FDA-approved cancer vaccine Provenge® (sipu-leucel-T) is comprised of autologous T-cells selective for prostate acidphosphatase that is expressed in 95% of prostate cancers [35,36]. Themost common adverse reactions to sipuleucel-T include fever and fatigue[35]. For all cell-based therapies, insufficient cell trafficking, tumor mi-croenvironment, inhibitory cytokines, and regulatory cells are still ob-stacles to more wide-spread efficacy [37].
The surge of immunotherapeutic breakthroughs illustrates the im-mense promise of using the immune system to fight cancer, but eachexample carries systemic exposure risks. Adjuvants and TLR agonists cantrigger intense immune anaphylaxis resembling that of sepsis. Immunetherapies such as checkpoint inhibitors, CAR-T, adoptive T cell therapies,and cancer vaccines can leave patients susceptible to off-target immune-related adverse events. These potential dangers highlight the importanceof new strategies for safer and more specific cancer treatments. IT ad-ministration is a compelling approach to enhance safety.
3. Intratumoral therapy
3.1. A brief history
The first successful IT cancer therapies were administered over 100years ago by Dr. William Coley on patients with inoperable solid tumors.Coley noticed a patient with an inoperable egg-sized sarcoma on the facewas completely cured after suffering a severe infection from a failed skingraft. He proposed that by introducing a bacterial infection at the site ofthe patient’s tumor, an immune response against the malignancy mightbe generated. This intervention proved an unprecedented success, andColey went on to treat many more patients with bacteria-derived, heat-killed toxins. Coley’s Toxins became one of the first examples of cancerimmunotherapy [38,39]. However, with the introduction of modern ra-diation and chemotherapy protocols, Coley’s toxins largely faded into thebackground and are no longer in use [40].
Even though IT therapy most literally means injection directly intotumors, it can more broadly refer to any therapy that is delivered in veryclose anatomical proximity to a tumor with the intention of direct uptakeby tumors or tumor cells. Even under this broad definition, only three ITtherapies are approved today. First used nearly 40 years ago, BacillusCalmette-Guerin (BCG) was approved by U.S. Food and DrugAdministration (FDA) in 1990 and is instilled locally into patients withbladder cancer [41]. This approach applies the same concepts laid out byColey [42]. Imiquimod, a TLR7 agonist, was FDA-approved in 1997.Imiquimod is topically applied for genital warts and basal cell carcinomas,where it can diffuse through the skin into the superficial tumors and tis-sues [43]. Talimogene laherparepvec (T-Vec/Imlygic®) is used to treatmelanoma and was FDA-approved in 2015. T-Vec is an oncolytic herpesvirus designed to kill cancer cells and stimulate an immune response byexpressing granulocyte-macrophage colony-stimulating factor (GM-CSF)[44]. Today, there are many more agents being investigated for IT deliverythat exploit the immune system, including pathogen associated molecularpatterns (PAMPs), monoclonal antibodies (mAbs), cytokines, small mole-cules, viral and gene therapies, and autologous cells [45].
Merck’s $300M acquisition of Immune Design reignited activity andinterest around IT therapy. Leading up to its acquisition, ImmuneDesign disclosed the IT immunotherapy G100 [46,47]. G100 is a stableoil-in-water emulsion containing glucopyranosyl lipid A (GLA), a potenttoll-like receptor 4 (TLR4) agonist that induces activation of localdendritic cells (DCs) to elicit broad, patient-specific anti-tumor immuneresponses [47]. Notably, G100 exhibited abscopal effects, the shrinkageof distal (non-injected) tumors [48]. This highly promising therapyreceived orphan drug designation by the FDA and European MedicinesAgency (EMA) for follicular non-Hodgkin’s lymphoma, further high-lighting IT interventions as a compelling therapeutic approach [49].The efficacy of G100 was even more pronounced when applied incombination with Merck’s anti-PD1 checkpoint inhibitor, Keytruda®,alongside radiation therapy [47].
3.2. Mechanism of intratumoral therapy
Tumor tissue is typified by the aberrant, unchecked proliferation ofcells. The immune system usually recognizes and eliminates nascenttumors, but immunosuppressive mechanisms of tumors can allow ma-lignant cells to proliferate undetected by the immune system. T-
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regulatory cells (Tregs) are attracted to the tumor by chemokines andaid in suppressing antigen presenting cells (APCs) that may otherwisestimulate a response against tumor antigens [50]. Additionally, tumorcells can secrete anti-inflammatory and regulatory cytokines (ie TGFβ,IL-10) that facilitate cancer growth and directly prevent DC activation.Tumor cells can also limit the expression of co-stimulatory molecules(MHC II, CD80, CD86), potentially inducing anergy or senescence ininfiltrating T cells [50,51]. At the other extreme, overstimulation cancause T-cell exhaustion from chronic exposure to tumor antigen [52].Finally, tumor cells can downregulate the expression of tumor antigensover time, evading recognition by cytotoxic T-lymphocytes (CTLs).
Different mechanisms can be used to destroy the primary tumor andsome can even promote clearance of distal tumors (Figure 1). Systemicor local delivery of cytotoxic agents or targeted radiotherapy can beused to damage or destroy tumor cells. Destruction of cancer cells canresult in the release of tumor-derived antigens. Alternatively, im-munostimulants can be used to overcome the suppressive environmentwithin the tumor, which work by recruiting immune cells or by acti-vating the immune system to recognize and attack cancer cells. Immunecells can be activated by immunostimulants in the presence of tumorantigen, traffic to lymph nodes, and then activate tumor antigen spe-cific T-cells via cross-presentation. Antigen-specific T-cells may thencirculate back to the tumor or to distal tumors and instigate tumor-cellkilling. The activation of the innate immune response creates a pro-inflammatory microenvironment and can result in recruitment of ad-ditional immune cells to the tumor. Categories of IT cancer therapiesthat are reviewed in this article is listed in Table 1.
With scientific advances in oncology, cancer mortality rates havedecreased by 29% between 1991 and 2017 [61]. However, over 1.7million cancer cases and over 600,000 cancer deaths occurred in theUnited States in 2019. Cancer therapies face the challenge of accessingtargets deep within tumor tissue, and often suffer from adverse effects.Systemically delivered therapeutics encounter countless obstaclesleading to a very small fraction of the drug reaching the tumor andunwanted distribution to healthy tissues [62]. After reaching the tumor,the drug penetrates the tumor and encounters the tumor micro-environment (TME), which can be heterogenous and differs drasticallyfrom healthy tissue. While IT administration of therapeutic agents canovercome concerns associated with systemic delivery, understandingthe TME for IT therapy becomes paramount for success.
4. Tumor properties to consider for intratumoral therapies
4.1. Tumor microenvironment
The TME is heterogeneous between patients, tumor types, and ofteneven within individual tumors. Overall, tumor tissue is typically distinctfrom normal tissue in that it has poorly organized vasculature withinconsistent vessel diameters and more prevalent branching [63].Tumor cell distance from blood vessels can result in restriction ofoxygen supply causing hypoxia in portions of the tumor [64]. Thepoorly organized vasculature and hypoxic setting creates a micro-environment with increased fluid leakage and elevated interstitial fluidpressure (IFP). Compared to the extravascular space in healthy tissue,
Fig. 1. Intratumoral (IT) immunotherapy mechanisms and invoking an abscopal effect. The administration of cytotoxic drugs (chemotherapy) or radiotherapy caninduce tumor cell killing and release of tumor antigens. Injection of immunotherapy can activate the innate and/or adaptive arms of an immune response, leading toeffects on distal tumors arising from circulating adaptive immune cells.
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tumors tend to have higher extracellular matrix density lacking func-tional lymphatic vessels, which limits interstitial diffusion and thedrainage of fluid from the tissue [65,66]. Collectively, the poorly or-ganized vasculature and increased IFP can decrease uptake of circu-lating therapeutic molecules, which may cause poor prognosis [65].
Intracellular pH is similar between tumors and normal tissue, althoughextracellular pH can be more acidic in tumors [67]. Increased extracellularacidity and anerobic glycolysis alters the pH gradients found in the TMEversus healthy tissue [67,68]. Higher acidity may increase tumor cell in-vasion and metastatic potential while also aiding in evasion of immunesurveillance [69,70]. For drugs that rely on passive diffusion to enter cells,the decreased extracellular pH may cause weakly basic drugs to becomeionized, preventing diffusion across membranes [67,71].
4.2. Intratumoral transport
In addition to the mechanism of action for the active component,the design of IT therapy formulation requires an understanding ofmolecular transport within the TME after the drug/formulation is de-livered. After delivery (injection) into the tumor, the drug/formulationwill undergo several transport and kinetic processes (Figure 2) that willultimately determine retention or elimination of the anti-cancertherapy (Table 2). Major molecular transport and kinetic processeswithin the TME include extracellular binding, cellular uptake and in-tracellular binding, and exfiltration from the TME.
Molecular transport through normal extracellular matrix is based onboth diffusion along a concentration gradient as well as advective con-vection (or bulk transport of mass) along a pressure gradient [72]. How-ever, since elevated IFP makes the bulk transport (i.e. convection) of the ITtherapy negligible in the TME, transport of anti-cancer agents after IT ad-ministration are typically governed by diffusion [65,73]. Though the bloodvessels represent an escape route for the therapeutic agent, the abnormaland poorly organized vasculature of the TME increases retention at thetumor cells that are distant from the vessels [74]. The absence of functionallymphatics in the TME reduces the elimination of the agent, improving ITretention [75,76]. Despite the relatively ineffective lymphatic drainage inthe TME, peritumoral lymphatics are a major route for metastasis and thelocal loss of IT therapeutics [77]. Angiogenesis, which seeks to normalizetumor vasculature leading to increased blood flow and reduced IFP, canresult in decreased retention time [78–80]. Vascular permeability can alsodecrease tumor retention time for small molecules, but this characteristic ispurported to be insignificant for macromolecules [81,82].
Densely packed collagen fibers are characteristic of the TME andpose transport resistance, which likely results in an overall increase inretention of IT therapy, although dense collagen may also restrict accessto sites within the tumor [72,83]. Fibrillar collagen and high IFP
contribute to a high mechanical stress in the tumor [84], which pro-motes diffusion over convection for the IT therapy. Cellular packingdensity can also affect drug diffusion; loosely packed tumor cells canimprove retention at tumor by enabling fast and thorough penetrationof the therapeutic agent [85]. Conversely, regions of tightly packedcells may impair the penetration of the therapeutic agent throughoutthe tumor. Finally, cellular uptake or binding of the therapy can occurby passive diffusion, active uptake, or other mechanisms depending onmolecular properties.
Drug features such as molecular size, charge, and other propertiesinfluence intratumoral residence time (Table 2). Water soluble smallmolecules diffuse more easily in the TME resulting in a lower retentionin the tumor [86], but increases in molecular hydrodynamic radius canreverse this effect. It is critical to balance molecular size such that atherapeutic or its carrier is small enough to diffuse through the TMEwhile avoiding clearance through lymphatic drainage or cellular uptake[87]. Large molecules such as monoclonal antibodies, for instance, canhave limited tumor retention due to endocytic clearance after IT ad-ministration [88]. Drug diffusion and retention deep inside the tumormass is affected by binding kinetics and affinity [89]. Molecular chargemay also be exploited such that the acidic extracellular pH in the tumorhas a positive impact on retention. The lymphatics are a primary me-chanism for clearance of SC injected mAbs, and the clearance rate ismainly dependent on the isoelectric point (pI) [90]. Hence, carefulantibody design utilizing physiologically based pharmacokineticmodels in the development stage could lead to new generations of an-tibody-based therapies with enhanced local tissue and IT retention. Themany factors that influence TME transport offer unique opportunitiesfor the retention of drugs injected IT such that the exploitation of theseabnormalities can be harnessed to maximize therapeutic effects.
5. Intratumoral therapies in clinical trials
Many types of IT therapies are in clinical trials, but this review fo-cuses on trials with posted or published data with only brief considera-tion of clinical trials yet to produce results. While radiation and cells canbe administered locally/intratumorally [91–94], these categories are notcovered in this review. Moreover, many radiation therapies are given incombination with other IT therapeutics. Clinical trials of IT therapies arehighlighted in Table 3 and in the following sections, with a more com-plete summary available in Supplementary Table 1.
5.1. Pathogen-associated molecular patterns
Pathogen-Associated Molecular Patterns (PAMPs) are non-self mo-lecules that activate innate immune responses. PAMPs are recognized
Table 1Categories of IT therapies to treat cancer covered in this article.
Category Mechanism Cells Types Involved Ref
Pathogen-Associated Molecular Patterns(PAMPs)
• Binding toll-like receptors (TLRs), RIG-I-like receptors (RLRs), NOD-like receptors (NLRs),and cell membrane components
• Downstream signaling leading to innate immune response
Immune cells,cancer cells
[53,54]
Cytokines • Binding to specific cell-surface glycoproteins
• Downstream signaling leading to innate immune response
• Direct anti-proliferative activity
Immune cells,cancer cells
[55]
Viruses and Plasmids • Interaction of viral surface proteins with cell surface proteins
• Target cancer cells by exploiting pathways, receptors, and mechanisms that promote tumorgrowth
• Viruses can be used to infect cancer cells or as vehicles for gene delivery
• Cell death and downstream signaling leading to innate immune response
Immune cells,cancer cells
[56]
Monoclonal antibodies (mAbs) • Binding to specific protein on surface of tumor or immune cell
• Checkpoint blockades inhibit immune suppression
• Other mAbs can mark cells for death or aid in immune activation
Immune cells,cancer cells
[57]
Small Molecules • Extra and Intracellular targets, must diffuse or transport through cell membrane
• Cytotoxins → Cause damage to various cell functions
• Targeted drugs → disruption of specific pathways critical for tumor cell progression
Cancer cells,rapidly dividing cells
[58–60]
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by pattern recognition receptors (PRRs) including toll-like receptors(TLRs), nucleotide-binding oligomerization domain (NOD)-like re-ceptors, RIG-I-like receptors (RLR), stimulator of interferon genes(STING) receptors, and C-type lectin receptors (CLR) [112]. ManyPAMP candidates pose a high probability of significant adverse events(AEs) when they enter systemic circulation. While IT administration canreduce the side-effects associated with systemic administration, im-munostimulatory molecules can still leak out of the tumor and into thesystemic circulation to cause AEs.
Unmethylated CpG oligonucleotides are PAMPs that mimic bacterialDNA and trigger an innate immune response upon binding to TLR9[167,168]. PF-3512676 is a class B, linear CpG formulated as a sodiumsalt with a molar mass of 8204 g/mol [169]. The formulation of PF-3512676 is proprietary, however, literature suggests it is un-modified,water-soluble, negatively-charged, and does not form higher orderstructures [97]. Clinical trial results are promising with IT administrationin B-cell lymphoma and mycosis fungoides but interestingly a higherpercentage of AEs were experienced in mycosis fungoides patients re-ceiving the same dose [170]. The differences between the rate of AEscould result from the extreme heterogeneity in vasculature of tumorsacross different types and locations. In a phase II study with lymphomapatients, an increased dose resulted in similar efficacy but more thandoubled the percentage of AEs, likely a result of increased systemic ex-posure [171]. Another presumably unmodified and soluble CpG therapy,SD-101, is a class C CpG. While the structural and formulation
information is proprietary, CpG class C is known to form dimers [172].Several IT trials investigating SD-101 in combination with other anti-cancer modalities exhibited promising abscopal effects; however, therewere grade 1-2 AEs in 100% of patients that includes detriments seen inauthentic pathogen infections such as sepsis, with a high incidence ofgrade 3 or 4 AEs and some serious AEs (SAEs) [173–176].
Many approaches have utilized structurally modified CpG ODNs toincrease immunogenicity and stability [7,98,177]. IMO-2125 exploits aninteresting design. Two strands of class C CpG are linked at the 3’ endconsisting of an 11-mer of CpG on each flanking end to allow formationof intermolecular structure that deters intramolecular interaction[177,178]. Favorable potency may be retained by the exposed 5’ endswhich are pertinent for CpG’s binding mechanism [168,178]. This var-iant is formulated as a sodium salt with a molecular weight (MW) of7712 g/mol and likely forms dimers [95,168]. IMO-2125 has beengranted fast track designation and orphan drug designation by the FDAand has shown promising results in early trials with fewer AEs than mostother IT TLR agonists. Additionally, this modified CpG therapy showsincreased TLR9 activation over unmodified CpG likely due to increasedmetabolic stability from the chemical linkage of the 3’ ends [178].
Another consideration for CpG-based therapies is the type of back-bone. Naturally-occurring CpG has a phosphorodiester (PO) backbone;however, synthetic CpG is often made with a phosphorothioate (PT)backbone to increase stability in vivo, which in turn enhances potency[179]. The creators of MGN1703 purport that the PT backbone of CpG
Fig. 2. Transport and kinetic processes in in-tratumoral injection therapies. The therapeuticagent can diffuse through the TME, enter thecell, be bound by extracellular or intracellularproteins, unbind, or leave the tumor into bloodvessels, lymphatics, peripheral blood, or ad-jacent tissue by diffusion and advective convec-tion. Diffusional transport of the agent back intothe tumor is expected to be minimal.
Table 2Factors affecting transport of therapy out of the tumor after intratumoral injection and probable transport phenomena.
Tumor tissue factors Phenomena
Microvascular permeability Decreases retention at tumor for small moleculesAbnormal vascular architecture May increase or decrease retention at the tumorAbsence of lymphatics Increases retention at the tumorInterstitial fluid pressure (IFP) Increased IFP increases retention time in the tumor but decreases retention close to vesselsSolid stress elevation Increases retention within the tumor, but decreases retention close to vesselsAngiogenesis Decreases retention at the tumorPhysicochemical factors PhenomenaConcentration gradient Increases diffusion out of tumor, decreasing retentionWater solubility Water soluble agents diffuse easily in the TME, decreasing retention in tumorExtracellular pH Effect on retention at tumor depends on the carcinogenic agent's molecular properties (pI, pKa)Fibrillar collagen Dense matrix increases retention at tumorCellular packing density Packing density may increase or decrease retention at tumor
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Table3
Ther
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char
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Cate
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Alte
rnat
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Form
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ion
info
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gage
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Refs
PAM
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soto
limod
/IM
O-2
125
CpG
clas
sC
deri
vativ
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onis
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wo
stra
nds
ofCp
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ked
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ends
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form
sdi
mer
s
•MW
7712
Da
•For
mul
ated
asa
sodi
umsa
lt
•Intr
acel
lula
rTL
Rbi
ndin
g
•Exp
osed
5’en
dsin
crea
sepo
tenc
yN
CT03
0522
05N
CT02
6449
67N
CT03
4455
33N
CT04
2708
64N
CT03
8650
82N
CT04
1962
83
[95]
SD-1
01Cp
Gcl
ass
C,TL
R9ag
onis
t•P
ropr
ieta
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clos
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Nan
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mul
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CpG
mot
ifs
•CpG
alon
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nega
tivel
ych
arge
d
•Intr
acel
lula
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Rbi
ndin
gN
CT02
2547
72N
CT03
0077
32N
CT03
8312
95N
CT02
5218
70N
CT02
7317
42N
CT03
4109
01N
CT02
9279
64N
CT02
2661
47N
CT03
3223
84N
CT01
7453
54N
CT04
0500
85
[96]
PF-3
5126
76/
Aga
tolim
od/C
pG79
09Cp
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B,TL
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onis
t•M
W76
98.2
12D
a
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ssB
CpG
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ually
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alon
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nega
tivel
ych
arge
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•AFM
mea
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men
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sB:
1.2
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•Intr
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Rbi
ndin
gN
CT00
1859
65N
CT00
8805
81N
CT00
2269
93
[97]
CMP-
001
CpG
clas
sA
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ew
ithna
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DN
Aba
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•AFM
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sure
men
ts:1
.1x
10-1
7x
25-9
0nm
•Intr
acel
lula
rTL
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ndin
gN
CT03
5076
99N
CT03
0846
40N
CT03
9836
68N
CT02
6801
84N
CT03
6186
41
[96,
97]
MG
N17
03/L
efito
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CpG
deri
vativ
e,na
tive
DN
Aba
ckbo
ne(P
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•Dum
bbel
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ped
•28
base
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doub
le-s
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mid
dle
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ion
flank
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two
sing
le-s
tran
ded
loop
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ntai
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30nu
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•App
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CT02
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70[9
8–10
0]
Hilt
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/pol
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-LC
TLR3
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)
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PLL
MW
28kD
abu
tra
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13-3
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n
•Net
posi
tivel
ych
arge
d
•Intr
acel
lula
rTL
Rbi
ndin
gN
CT02
4238
63N
CT01
9765
85N
CT03
2621
03N
CT01
9848
92N
CT00
8808
67
[101
]
BO-1
12/
poly
I:C+
poly
alky
lene
imin
eTL
R3ag
onis
t•C
ompl
exed
with
PEI
•PEI
MW
betw
een
17.5
-22.
6kD
a
•Zet
apo
tent
ial3
8m
Vat
pH3.
1
•45-
85nm
part
icle
s
•pol
yI:C
/PEI
ratio
betw
een
2.5-
4.5
•Aqu
eous
form
ulat
ion
with
gluc
ose
orm
anni
tol
•Intr
acel
lula
rTL
Rbi
ndin
gN
CT02
8280
98[1
02,1
03]
G10
0/G
LA-S
EG
LAde
riva
tive,
TLR4
agon
ist
•Sin
gle
phos
phat
egr
oups
and
six
C 14
acyl
chai
ns
•For
mul
ated
ina
squa
lene
inw
ater
emul
sion
•Con
tain
seg
gph
osph
atid
ylch
olin
e(P
C),D
L-α-
toco
pher
ol,a
ndPo
loxa
mer
188
•Par
ticle
size
82.7
-111
nm
•Zet
apo
tent
ial-
17m
V
•Ext
race
llula
rTL
Rbi
ndin
gN
CT02
0356
57N
CT02
1806
98N
CT03
7428
04N
CT02
5014
73N
CT03
9156
78N
CT02
4067
81N
CT03
9821
21N
CT02
3871
25
[104
–109
]
CV81
02TL
R7/8
and
RLR
agon
ist
•ssRN
A-5
47nu
cleo
tides
•Com
plex
edw
ithca
tioni
cpe
ptid
e(C
ys-A
rg12
-Cy
s)th
atis
disu
lfide
-cro
sslin
ked
•Intr
acel
lula
rTL
Ran
dRL
Rbi
ndin
gN
CT03
2910
02[1
10,1
11]
(continuedonnextpage
)
A. Huang, et al. Journal of Controlled Release 326 (2020) 203–221
208
Table3
(continued)
Cate
gory
Ther
apy/
Alte
rnat
ive
Nam
esD
escr
iptio
nof
Act
ive
Char
acte
rist
ics
and
Form
ulat
ion
info
rmat
ion
Mec
hani
smEn
gage
dN
CTs
Refs
MK4
621/
RGT1
00(u
pcom
ing
tria
lsfo
rmul
ate
with
JetP
EI)
RIG
-Iag
onis
t•C
yclic
dinu
cleo
tide
•No
stru
ctur
alin
form
atio
npr
ovid
ed
•New
erfo
rmul
atio
nar
eco
mpl
exin
gw
ithJe
tPEI
whi
chis
alin
earP
EIw
ith1-
3po
sitiv
ech
arge
son
the
nitr
ogen
spec
ies.
•Intr
acel
lula
rTL
Rbi
ndin
gN
CT03
7391
38N
CT03
0650
23[1
12]
Mot
olim
od/V
TX-2
337
TLR8
and
NO
Dag
onis
t•M
W45
8.6
g/m
ol
•No
char
ge•In
trac
ellu
lar
TLR
bind
ing
NCT
0390
6526
MIW
815/
AD
U-S
100
STIN
Gag
onis
t•S
ynth
etic
cycl
icdi
nucl
eotid
e
•For
mul
atio
nun
know
n•In
trac
ellu
lar
bind
ing
NCT
0317
2936
NCT
0267
5439
NCT
0393
7141
MK-
1454
STIN
Gag
onis
t•S
ynth
etic
cycl
icdi
nucl
eotid
e
•For
mul
atio
nun
know
n•In
trac
ellu
lar
bind
ing
NCT
0301
0176
BCG
Der
ivat
ive
ofBC
Gba
cter
ia•L
ive,
atte
nuat
edBC
G
•Gra
mpo
sitiv
e,ro
dsh
aped
•Ave
rage
leng
th2.
36μm
,wid
th0.
474
μm,
volu
me
0.38
9μm
3or
0.90
6μm
diam
eter
•Vac
cine
cont
ains
loos
ely
aggr
egat
edce
llsof
ten
but
nota
lway
s
•TIC
Esu
bstr
ain
isoe
lect
ric
poin
tis
4.4
•Moc
kba
cter
iali
nfec
tion
NCT
0392
8275
NCT
0183
8200
[113
,114
]
Clos
trid
ium
novy
i-NT
Der
ivat
ive
ofcl
ostr
idiu
mba
cter
ia•G
ram
posi
tive,
cont
ain
flage
lla,s
pore
form
ing
•Len
gth
ofov
alsh
ape-
1μm
•Moc
kba
cter
iali
nfec
tion
NCT
0192
4689
[115
,116
]
Cyto
kine
Gra
nulo
cyte
-Mac
roph
age
Colo
nySt
imul
atin
gFa
ctor
(GM
-CSF
)w
hite
bloo
dce
llgr
owth
fact
or•1
4-35
kDa
glyc
opro
tein
•127
amin
oac
ids
•2nm
x3
nmx
4nm
•Imm
unos
timul
ator
ycy
toki
neN
CT00
6000
02[1
17,1
18]
IL-2
Imm
une
cell
sign
alin
gm
olec
ule
•15.
5kD
aan
dis
com
pris
edof
133
amin
oac
ids
•18
MIU
reco
mbi
nant
hum
anIL
-2(P
role
ukin®,
Chir
on,R
atin
gen,
Ger
man
y)w
asdi
ssol
ved
in6
mlg
luco
se(5
%)p
repa
red
with
albu
min
(0.2
%)
solu
tion
•Imm
unos
timul
ator
ycy
toki
neN
CT03
2338
28N
CT00
2045
81N
CT01
4803
23N
CT00
6000
02N
CT01
6724
50
[119
,120
]
PEG
-IL-2
Mod
ified
imm
une
cell
sign
alin
gm
olec
ule
•Cov
alen
tadd
ition
of6–
7kD
apo
ly-e
thyl
ene
glyc
ol(P
EG)
•Imm
unos
timul
ator
ycy
toki
ne[1
21,1
22]
Cyto
kine
/tox
infu
sion
IL-4
(38-
37)-
PE38
KDEL
Imm
une
cell
sign
alin
gm
olec
ule
conj
ugat
edto
ato
xin
•am
ino
acid
s38
–129
ofIL
-4,f
used
via
ape
ptid
elin
ker
toam
ino
acid
s1–
37,w
hich
intu
rnis
fuse
dto
the
PE38
KDEL
toxi
n
•PE3
8KD
ELis
com
pose
dof
amin
oac
ids
253–
364
and
381–
608
ofPE
,with
KDEL
(an
endo
plas
mic
reta
inin
gse
quen
ce),
atpo
sitio
ns60
9–61
2.
•Bin
dto
IL-4
rece
ptor
son
tum
ors
•Pse
udom
onas
exot
oxin
(PE)
isa
cyto
toxi
cag
ent
NCT
0079
7940
NCT
0001
4677
[123
]
IL13
-PE3
8QQ
R(I
L13P
E)Im
mun
ece
llsi
gnal
ing
mol
ecul
eco
njug
ated
toa
toxi
n•IL
-13
conj
ugat
edto
trun
cate
dPE
•Bin
dto
IL-1
3re
cept
ors
ontu
mor
sN
CT00
0647
79[1
24]
Ant
ibod
y/Cy
toki
nefu
sion
darl
euki
n(L
19-IL
2)an
dfib
rom
un(L
19-T
NFα
)L1
9(a
hum
anm
onoc
lona
lant
ibod
yfr
agm
ent)
fuse
dto
anim
mun
ocyt
okin
e
Com
bina
tion
ofim
mun
ece
llsi
gnal
ing
mol
ecul
es•D
arle
ukin
:int
erle
ukin
-2(I
L-2)
isfu
sed
toa
hum
ansi
ngle
-cha
inva
riab
lefr
agm
ent
(scF
v)th
atre
cogn
izes
L19
•Fib
rom
un:t
umor
necr
osis
fact
or-α
(TN
Fα)
fuse
dto
scFv
that
reco
gniz
esL1
9
•Bin
dto
L19
ontu
mor
s
•imm
unos
timul
ator
ycy
toki
neD
arle
ukin
:N
CT01
2530
96D
arom
un(D
arle
ukin
and
Fibr
omun
):N
CT02
0766
33N
CT02
9382
99N
CT03
5678
89
[125
]
Onc
olyt
icvi
rus
ON
YX-0
15A
deno
viru
s•E
1B55
-kD
age
nede
lete
d
•90
-100
nmdi
amet
er•D
estr
oytu
mor
cells
[126
]
DN
X-24
01A
deno
viru
s•E
1Age
nede
letio
n
•RG
D-m
otif
engi
neer
edin
toth
efib
erH
-loop
•RD
G-m
otif
allo
win
tera
ctio
nw
ithα v
β 3an
dα v
β 5in
tegr
ins
enri
ched
ontu
mor
cells
•Des
troy
tum
orce
lls
NCT
0080
5376
NCT
0279
8406
NCT
0219
7169
NCT
0195
6734
[127
]
(continuedonnextpage
)
A. Huang, et al. Journal of Controlled Release 326 (2020) 203–221
209
Table3
(continued)
Cate
gory
Ther
apy/
Alte
rnat
ive
Nam
esD
escr
iptio
nof
Act
ive
Char
acte
rist
ics
and
Form
ulat
ion
info
rmat
ion
Mec
hani
smEn
gage
dN
CTs
Refs
Coxs
acki
evir
usA
21(C
VA21
)co
xsac
kiev
irus
•~31
nmin
diam
eter
•Bin
dto
intr
acel
lula
rad
hesi
onm
olec
ule
1(I
CAM
-1)
and
deca
yac
cele
ratio
nfa
ctor
(DA
F)pr
otei
nson
tum
orce
lls
NCT
0122
7551
NCT
0043
8009
NCT
0023
5482
NCT
0083
2559
NCT
0230
7149
[128
]
HF1
0H
erpe
ssi
mpl
exvi
rus-
1(H
SV-1
)•L
osso
fexp
ress
ion
ofU
L43,
UL4
9.5,
UL5
5,U
L56,
and
LAT
•Ove
rexp
ress
ion
ofU
L53
and
UL5
4
•155
–24
0nm
indi
amet
er
•Des
troy
tum
orce
llsN
CT02
4280
36N
CT01
0171
85N
CT03
1530
85N
CT03
2528
08N
CT02
2728
55N
CT03
2594
25
[129
,130
]
HSV
-171
6H
erpe
ssi
mpl
exvi
rus
•RL1
gene
dele
tion
•155
–24
0nm
indi
amet
er•D
estr
oytu
mor
cells
NCT
0093
1931
NCT
0203
1965
H-1
parv
ovir
us(H
-1PV
,Par
vOry
x)pa
rvov
irus
•180
–250
Åin
diam
eter
•Des
troy
tum
orce
llsN
CT02
6533
13N
CT01
3014
30[1
30–1
32]
mea
sles
viru
sEd
mon
ston
-Zag
reb
vacc
ine
stra
inm
easl
esvi
rus
•120
–25
0nm
indi
amet
er•B
ind
toCD
46th
atar
eex
pres
sed
byso
me
canc
erce
lllin
es
•Des
troy
tum
orce
lls
[133
,134
]
Pela
reor
ep(R
EOLY
SIN®)
reov
irus
•Unm
odifi
edon
coly
ticre
ovir
us
•Typ
e3
Dea
ring
stra
in•D
estr
oytu
mor
cells
•Mec
hani
smun
clea
r,m
aybe
rela
ted
toRa
ssi
gnal
ing
NCT
0052
8684
NCT
0272
3838
[135
,136
]
vvD
D-C
DSR
Vacc
ina
viru
s•V
acci
nia
grow
thfa
ctor
(VG
F)an
dth
ymid
ine
kina
se(T
K)de
lete
d•D
estr
oytu
mor
cells
NCT
0057
4977
[137
]
Onc
olyt
icvi
rus
+ve
ctor
Talim
ogen
ela
herp
arep
vec
(T-V
ec);
Imly
gic™
Type
Iher
pes
sim
plex
viru
s•IC
P34.
5-de
ficie
nt
•ICP4
7-de
ficie
nt
•155
-240
nmin
diam
eter
•Des
troy
tum
orce
lls
•Exp
ress
esG
M-C
SFfo
rim
mun
ostim
ulat
ion
NCT
0028
9016
NCT
0257
4260
NCT
0201
4441
NCT
0076
9704
NCT
0136
8276
NCT
0236
6195
NCT
0275
6845
NCT
0345
8117
NCT
0406
5152
NCT
0308
6642
NCT
0306
4763
NCT
0221
1131
NCT
0245
3191
NCT
0308
8176
NCT
0174
0297
NCT
0325
6344
NCT
0380
2604
NCT
0226
3508
NCT
0406
8181
NCT
0384
2943
NCT
0262
6000
NCT
0250
9507
NCT
0374
7744
NCT
0281
9843
NCT
0116
1498
[130
]
ON
COS-
102
(pre
viou
sly
calle
dCG
TG-
102
and
Ad5
/3-D
24-G
MCS
F)A
deno
viru
s•S
erot
ype
5ad
enov
irus
•Pla
cing
the
Ad3
fiber
knob
into
the
Ad5
back
bone
resu
ltsin
anA
d5/3
chim
era
•24
bpde
letio
nin
Rbbi
ndin
gsi
teof
E1A
for
canc
erce
llre
stri
cted
repl
icat
ion
•Arm
edw
ithgr
anul
ocyt
e-m
acro
phag
eco
lony
-st
imul
atin
gfa
ctor
(GM
-CSF
)
•Ser
otyp
e3
fiber
knob
allo
wen
hanc
edge
nede
liver
yto
canc
erce
lls
•Des
troy
tum
orce
lls
•Exp
ress
esG
M-C
SFfo
rim
mun
ostim
ulat
ion
NCT
0159
8129
NCT
0351
4836
NCT
0300
3676
[138
,139
]
(continuedonnextpage
)
A. Huang, et al. Journal of Controlled Release 326 (2020) 203–221
210
Table3
(continued)
Cate
gory
Ther
apy/
Alte
rnat
ive
Nam
esD
escr
iptio
nof
Act
ive
Char
acte
rist
ics
and
Form
ulat
ion
info
rmat
ion
Mec
hani
smEn
gage
dN
CTs
Refs
Pexa
-Vec
(JX-
594)
Vacc
ina
viru
s•W
yeth
stra
inva
ccin
iam
odifi
edby
inse
rtio
nof
the
hum
anG
M-C
SFan
dLa
c-Z
gene
sin
toth
eva
ccin
iaTK
gene
regi
onun
der
cont
rolo
fthe
synt
hetic
earl
y-la
tepr
omot
eran
dp7
·5pr
omot
er,
resp
ectiv
ely.
•Vir
ion
mor
phol
ogy
and
size
:Env
elop
ed,
bico
ncav
ecor
ew
ithtw
ola
tera
lbod
ies,
bric
k-sh
aped
topl
eo-m
orph
icvi
rion
s,~
360x
270x
250
nmin
size
•Dilu
ted
inbi
carb
onat
e-bu
ffere
dsa
line
•Rep
licat
ion
and
hGM
-CSF
tran
sgen
e
•Des
troy
tum
orce
llsN
CT01
3298
09N
CT01
3875
55N
CT01
1695
84N
CT00
5543
72N
CT02
5627
55N
CT01
1716
51N
CT02
9771
56N
CT03
2940
83N
CT03
0710
94N
CT00
4293
12N
CT00
6254
56
[140
–142
]
Non
-onc
olyt
icvi
rus
+ve
ctor
TG10
42(A
deno
viru
s-in
terf
eron
-γ)
Ade
novi
rus
•Non
repl
icat
ing
(E1
and
E3re
gion
sde
lete
d)
•Ade
novi
rus
type
5(g
roup
C)ve
ctor
•Con
tain
ing
ahu
man
IFN
-γcD
NA
inse
rtun
der
cyto
meg
alov
irus
prom
oter
cont
rol
•Exp
ress
esIF
N-γ
NCT
0039
4693
[143
]
TNFe
rade
Biol
ogic
(AdG
VEG
R.TN
F.11
D)
Ade
novi
rus
•Rep
licat
ion-
defic
ient
aden
ovir
alve
ctor
that
expr
esse
stu
mor
necr
osis
fact
or-α
(TN
Fα)
unde
rth
eco
ntro
lofa
radi
atio
n-in
duci
ble
Egr-
1pr
omot
er
•Exp
ress
esTN
FαN
CT00
0514
67N
CT00
0514
80[1
44,1
45]
Vira
lvec
tor
INXN
-200
1(A
d-RT
S-hI
L-12
)w
ithor
alac
tivat
orIN
XN-1
001
(Vel
edim
ex)
Ade
novi
rus
•Exp
ress
eshu
man
IL-1
2•E
xpre
sses
hum
anIL
-12
NCT
0139
7708
NCT
0242
3902
NCT
0367
9754
NCT
0202
6271
NCT
0333
0197
NCT
0363
6477
NCT
0400
6119
Non
-onc
olyt
icvi
rus
+ve
ctor
aden
ovir
alve
ctor
expr
essi
ngE.coli
PNP
(Ad/
PNP)
and
IVflu
dara
bine
ther
apy
Ade
novi
rus
•Loa
ded
with
aba
cter
ialg
ene
calle
dE.
coli
puri
nenu
cleo
side
phos
phor
ylas
e(P
NP)
•PN
Pco
nver
tsflu
dara
bine
toan
ti-ca
ncer
agen
tflu
oroa
deni
neN
CT01
3101
79[1
46]
aden
ovir
alve
ctor
(Adv
.RSV
-tk)
expr
essi
ngth
ehe
rpes
thym
idin
eki
nase
gene
with
IVG
anci
clov
ir(G
CV)
•Ade
novi
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low
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ipho
spha
te
NCT
0084
4623
[147
]
Plas
mid
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asm
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3453
30
[149
]
EGFR
antis
ense
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ated
~96
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bits
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olife
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n/gr
owth
NCT
0000
9841
[150
]
BC-8
19(a
lso
calle
dD
TA-H
19)
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0bp
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NCT
0071
1997
[151
–153
]
(continuedonnextpage
)
A. Huang, et al. Journal of Controlled Release 326 (2020) 203–221
211
Table3
(continued)
Cate
gory
Ther
apy/
Alte
rnat
ive
Nam
esD
escr
iptio
nof
Act
ive
Char
acte
rist
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and
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ulat
ion
info
rmat
ion
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hani
smEn
gage
dN
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02•C
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exof
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mid
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dlin
ear
poly
mer
sof
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ethy
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imin
e(J
etPE
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hich
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ates
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cyto
toxi
cpr
o-dr
ugge
mci
tabi
ne
NCT
0127
4455
NCT
0280
6687
[154
,155
]
pbi-s
hRN
AST
MN
1LP
prop
riet
ary
bi-fu
nctio
nals
hRN
A(b
i-shR
NA
)pl
atfo
rmto
exec
ute
RNA
inte
rfer
ence
(RN
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-m
edia
ted
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ncin
g
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som
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rrie
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ex
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ende
din
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ent
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gof
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xtro
sein
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er)
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400
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821
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rage
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hmin
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ecul
ean
dch
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bitin
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istin
gof
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ide
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ns
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a
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ear
colo
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7.0
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real
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ody
mol
ecul
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abou
t10
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pres
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hibi
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gnal
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lym
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yte-
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ciat
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4(C
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xic
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ocyt
es(C
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limum
abbi
nds
toCT
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ock
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rysi
gnal
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ase
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cre
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nof
CTLs
toat
tach
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erce
lls.
NCT
0167
2450
[157
,158
]
AD
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13A
nti-C
D40
mA
bs•A
hum
anm
onos
peci
ficIg
G1
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ody
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ulat
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ceeff
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rT-c
ells
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ckth
etu
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.
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0237
9741
[159
]
TTI-6
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0289
0368
[160
]
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9861
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ti-O
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anIg
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cepr
olife
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nof
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mph
ocyt
esth
atat
tack
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oras
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ated
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ens.
NCT
0383
1295
[161
]
Smal
lMol
ecul
eIN
T230
-6Ci
spla
tin,c
hem
othe
rape
utic
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rmul
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odr
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mor
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les
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ffusi
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.
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tes
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cted
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ugin
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ellm
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cks
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nly
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inje
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or,b
utno
n-in
ject
edtu
mor
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dun
seen
mic
ro-m
etas
tase
s.
NCT
0305
8289
[162
]
Cisp
latin
/Epi
neph
rine
inje
ctab
lege
lCi
spla
tin,c
hem
othe
rape
utic
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tain
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mL
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otei
nca
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rm
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x
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atum
oral
inje
ctio
nof
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latin
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inep
hrin
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ject
able
gela
chie
ves
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entr
atio
nsof
cisp
latin
inth
etu
mor
with
very
low
conc
entr
atio
nsin
plas
ma
and
othe
rtis
sues
.
NCT
0000
2659
NCT
0002
2217
[163
]
Para
-tolu
enes
ulfo
nam
ide
(PTS
)•C
7H9N
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nific
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orne
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alin
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ility
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chon
dria
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mag
e,an
din
hibi
tsA
TPbi
osyn
thes
is
NCT
0344
8146
[164
]
Gem
cita
bine
chem
othe
rape
utic
•Anu
cleo
side
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rug,
anan
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ting
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rand
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inhi
bitin
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hesi
s
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0272
3838
NCT
0183
4170
[165
]
PV-1
0Ro
seBe
ngal
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diso
dium
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xant
hene
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onof
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arks
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ogen
icce
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ath
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lon
canc
erce
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es
NCT
0269
3067
NCT
0255
7321
[166
]
A. Huang, et al. Journal of Controlled Release 326 (2020) 203–221
212
causes toxic side effects [99]. They developed a covalently-closed loopof CpG with its native PO backbone to avoid PT-associated toxicity andenhance the stability of native PO. Similarly, CMP-001 is a CpG class Awith the native PO backbone that is modified to assemble into quad-ruplexes [7,168]. Clinical results for both of these compounds arepending, which may provide a new precedent for future trials em-ploying modified and native backbones of CpG.
Formulation with a polycationic carrier can increase intracellularPAMP delivery and potency. PAMPs utilizing intracellular receptors(like TLR9, TLR3, and RIG-I) may benefit from a cationic carrier orparticulate formulation for attraction to cell surfaces and increased APCuptake, respectively. Two such TLR3 agonist candidates, BO-112 andHiltonol (polyI:C:LC), include polyI:C formulated with polycations. Inthe most recent update of an IT BO-112 clinical trial, patients exhibiteda modest overall response rate (ORR) and high percentage of AEs.Increased circulating immune cells and no detectable BO-112 in theblood suggests injection site retention [180]. BO-112 is an aqueouscomposition at pH 2.7-3.4 with glucose or mannitol in an optimalparticle size range of 45-85 nm and zeta potential between 40-45 mV[102]. Optimal size of particles for APC uptake and processing is ap-proximately 100 nm, similar to the size of viruses [109]. The molarratio between nitrogen in polyethylenimine (PEI) and phosphorous inDNA (N/P ratio) for the polyI:C/ polyethylenimine (PEI) complex isbetween 2.5-4.5 and the PEI MW is between 17.5-22.6 kDa [102]. ITHiltonol showed preliminary success in a single patient on both localand distal tumor sites; however systemic side effects or AEs were notreported [181]. Hiltonol is formulated with carboxymethylcellulose(CMC), a hydrophilic, negatively charged material, in an aqueous salinesolution. The molar ratio of PO4 groups to the ε amino group of thelysine in polyIC:LC is 1:1 which corresponds to an excess of ε aminogroups, which may contribute to further complexing with CMC [101].The polylysine ranges from 13-35 kDa, however there is no report ofcomplexes between polylysine and polyI:C.
The RIG-I agonist, MK-4621, is a synthetic RNA oligonucleotide. AnIT clinical trial was terminated due to excessively high incidence of AEs:100% grade 1-2 AEs and 48% grade 3-4 AEs [182]. To increase re-tention at the injection site and mitigate the side effects, negativelycharged MK-4621 was complexed with a positively charged carrier (PEIvariant JetPEI) [183]. A clinical trial is planned for the complexed MK-4621.
Emulsion formulations can also improve efficacy and retention of ITtherapeutics. The TLR4 agonist G100 is a glucopyranosyl Lipid A (GLA)derivative with a single phosphate group and six C14 acyl chains for-mulated in a squalene emulsion (also called GLA-SE) [105]. Theemulsion contains the excipients squalene, egg phosphatidylcholine(PC), DL-α-tocopherol and Poloxamer 188 [106]. The particle/dropletsize is 82.7-111 nm [105–108] and the zeta potential is -17 mV [109].Because TLR4 is located on the cell surface, intracellular uptake is notrequired. The formulation of GLA has critical effects on TLR activation[108]. GLA-SE resulted in greater immune activation than GLA for-mulated as an aqueous suspension in various mouse models and ahuman skin explant model. One trial studying G100 resulted in a 10%CR, 40% PR, and 50% PD, with an AE incidence greater than 80%[184]. Moreover, responders demonstrated increased inflammationwith infiltration of CD8+ and CD4+ T-cells following treatment.
Live attenuated bacteria have also been used in IT cancer im-munotherapy. IT administered BCG resulted in no better than stabledisease and all patients experienced AEs or SAEs [185]. BCG is a grampositive, rod shaped bacterium. In the case of the TICE® BCG vaccine,the average length of the bacterium is 2.36 μm and a width of 0.474 μmbut micro-aggregates of approximately 30-50 nm in diameter may form[113]. BCG are negatively charged but can be positively charged atlower pH’s as the isoelectric point (pI) depends on the method of pre-paration. BCG is recognized by TLR4 and TLR2 through its myco-bacterial components (cell wall skeleton and peptidoglycan) and byTLR9 (bacterial DNA) [186]. A phase I/II trial of an IT administered
BCG non-live vaccine, composed of the BCG cell wall skeleton andtrehalose dimycolate, resulted in at least one injected nodule re-sponding in 48% of melanoma and breast cancer patients [187]. ITClostridium novyi-NT trials are in progress but too early on to drawcomparisons [188]. More research is needed to evaluate transport ofbacterial candidates after IT injection.
Early clinical trials suggest unmodified TLR agonists lead to agreater AE incidence than those structurally modified or formulatedwith a cationic carrier. TLR agonists comprised of DNA or RNA motifsare negatively charged. Since extracellular space and cell membranesare also negatively charged, IT administration of these nucleic acidmaterials are not conducive to retention. Net positively charged for-mulations may improve retention at the injection site. Such interactionscould limit systemic exposure and mitigate the AEs commonly asso-ciated with PAMP immunotherapies. Further exploration of optimalphysiochemical properties for retention and efficacy could greatly en-hance future IT therapies incorporating PAMPs.
5.2. Cytokines
Cytokines play an important cell signaling role and are major reg-ulators of immunity. These small proteins can activate immune re-sponses and encourage cancer cell destruction [189,190]. Like PAMPs,to avoid systemic toxicity, cytokines should be localized at the tumor.
Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) is agrowth factor that stimulates hematopoietic stem cells to differentiateinto dendritic cells, granulocytes, and monocytes [191]. Recombinanthuman GM-CSF is a 14-35 kDa glycoprotein with 127 amino acids[117] composed of four bundles of α-helices and is non-spherical withdimensions of ca. 2x3x4 nm [118]. GM-CSF has been studied as animmunostimulatory adjuvant to induce anti-tumor immunity [192]although a recent report suggests GM-CSF may stimulate tumor growthand metastasis in certain cancers [191]. Severe AEs were reported inmalignant mesothelioma patients given intralesional infusions of 2.5-10mg/kg/day GM-CSF, which may be a result of systemic exposure [193].Conversely, lower dosage, daily injections of 15-50 μg or 400 μg GM-CSF given to melanoma patients produced more mild side effects, likelydue to lower systemic exposure [194,195]. The lower AEs seen in thesestudies may have resulted from a combination of the lower dosageconcentration, or a difference in the location and morphology of tumorsassociated with the cancer type (i.e. mesothelioma vs melanoma).
The IL-2 cytokine has also been widely explored in cancer. HumanIL-2 has a MW of 15.5 kDa, is comprised of 133 amino acids [119], andhas a hydrodynamic radius of ~3 nm [196]. Interestingly, IL-2 can beimmunostimulatory or suppressive by activating cytotoxic effector cellsor regulatory T (Treg) cells, respectively [119]. Typically, high doses ofIL-2 are immunostimulatory and generate an anti-tumor immune re-sponse, while low doses of IL-2 are used for immunosuppression. The T-cell expansion that ensues in the presence of IL-2 has the potential topromote an anti-tumor response that overcomes a senescent micro-environment established by tumors [197]. Trials with IL-2 commonlyreported systemic AEs [120,198,199]. Nevertheless, patients withmelanoma [120,199] responded better to IT IL-2 treatment comparedto patients with head and neck squamous cell carcinoma (HNSCC)[198], possibly due to higher neoantigen load in melanoma comparedto HNSCC [200]. Moreover, the addition of 6-7 kDa poly-ethyleneglycol (PEG) chains increases the drug’s solubility, extends half-life, andreduces off-target immunogenicity, which translated into better patientresponses [121,122]. Additional studies demonstrated that PEGylationlowered the drug’s affinity for receptors on Treg cells to a greater extentthan receptors on CD8+ T cells, which resulted in a more favorableCD8+ T-cell activation over Tregs [201].
The anti-tumor responses to IT IL-2 generally are limited to theinjected tumor, and do not extend to untreated metastases (i.e. no ab-scopal effect). To address the lack of systemic immunity with in-tratumoral IL-2 treatment, an intratumoral ipilimumab/IL-2
A. Huang, et al. Journal of Controlled Release 326 (2020) 203–221
213
combination was administered in patients with unresectable stage III/IV melanoma in a phase I study [157]. The hypothesis was that thecombination treatment would effectively activate tumor infiltratinglymphocytes and promote systemic immunity. A local response wasobserved in 67% (8 out of 12) of the patients and an abscopal responsewas observed in 88.9% (8 out of 9) patients. Treatment-related AEswere of grade 1 or 2. Some of the responders had increased frequency ofCD8+ T-cells expressing IFNγ, Tbet, granzyme-B and/or perforin, sug-gesting a systemic immune response.
Additional IT cytokine immunotherapies include IFNα, TNFα, andIL-12. Recombinant α-interferon administered IT in patients withprostate cancer showed a 30% complete response (CR) rate [202].Tumor Necrosis Factor-α (TNFα) has been intratumorally injectedwhile co-administering subcutaneous IFN-a2b for advanced prostatecancer as well [203]. Notably, TNFα leakage into the systemic circu-lation was observed 2 hours after injections and may have contributedto AEs. Recombinant human interleukin-12 (rhIL-12) tested in HNSCCwas detected in the plasma 30 minutes after IT injection with a half-lifeof 7.2 h [204]. Such systemic exposure can be harmful to patients, as aphase II study with a similar treatment regimen for 10 HNSCC patientsexhibited prevalent AEs [205].
Cytokines elicit signaling cascades by acting together with otherdirective signals, so cocktail approaches and combination therapieshave also been attempted, but with limited success. One such cocktailapproach involved a multi-cytokine solution consisting of IL-2, IL-1α,IL-1β, GM-CSF, IFNα, TNFα, TNFβ, IL-3, IL-4, IL-6, IL-8, IL-10, andmacrophage inflammatory protein 1α. IT or peritumoral injection inpatients with HNSCC in combination with intravenous cyclopho-sphamide, intraoral indomethacin, and oral zinc showed a 16.7% CRrate [206]. Tumors accumulated an elevated number of CD4+ T-cellsand natural killer cells. Notably, this high-powered cocktail led to 8.3%of patients developing sepsis and Wegener granulomatosis, suggestingsystemic exposure.
To limit the systemic cytokine exposure after IT injection, a tumor-targeting domain may be conjugated or engineered into the ther-apeutic. For instance, IL-4(38-37)-PE38KDEL is a chimeric proteincomposed of modified IL-4 and a truncated form of Pseudomonas exo-toxin (PE), which can target and bind to IL-4 receptor-positive glio-blastoma cells [123]. Likewise, IL13-PE38QQR (IL13PE) is a chimericprotein of IL-13 conjugated to truncated PE and binds to IL-13 receptorson malignant glioma cells [124]. The IL-2-based immunokine (dar-leukin) and the TNFα-based immunokine (fibromun) further in-corporate a diabody derived from the L19 antibody to introduce fi-bronectin binding functionality that capitalizes on overexpression intumors [207]. With the absence of the Fc region on the diabody frag-ment of the antibody, the molecule does not interact with FcRn and hasa more limited half-life than a full IgG molecule. Nonetheless, thesmaller size allows for better penetration and distribution in the tumor.In a Phase II trial in metastatic melanoma, the combination therapy ofdarleukin and fibromun (called daromun) resulted in AEs that werelimited to local injection site reactions and an overall response rate(ORR) of 55% [208].
Cytokines are by nature small (< 70 kDa) and water-soluble, whichpotentially confounds their retention within the TME [209]. Severalclinical approaches have sought to address these detriments, but thecontinued development of strategies for the IT administration of cyto-kines within the TME will undoubtedly optimize efficacy while mini-mizing AEs. These strategies should continue to seek modificationstrategies that do not impede receptor binding or penetration within thetumor.
5.3. Oncolytic viruses
Oncolytic viruses (OVs) (20 – 500 nm) kill tumor cells by cell lysis[210]. Subsequent viral replication and induction of an immunogenicresponse further compound their effects [211]. A variety of virus types
have been designed to be oncolytic for IT therapy, including adeno-virus, enterovirus, herpes simplex virus, parvovirus, measles (Rubeola)virus, reoviruses, and vaccinia virus classes[127,132,134,135,137,212–217].
OVs are typically modified or designed to improve affinity for tumorcells, while also limiting infection in healthy cells. One method toachieve tumor-selective replication is to delete viral genes that main-tain viral replication in cancer cells, but inhibit viral replication innormal cells [211]. Examples include the deletion of the E1B 55-kDagene (e.g. ONYX-015) or E1A gene in adenoviruses (e.g. DNX-2401),RL1 gene deletion in herpes simplex virus (HSV) type 1 (155 – 240 nmin diameter) (e.g. HSV-1716) [130,132], and the deletion of viral genesencoding vaccinia growth factor (VGF) and thymidine kinase (TK) inthe vaccina virus vvDD-CDSR [137].
Viruses can also be designed to specifically target tumor cells byexploiting alterations in cell surface receptors. For instance, in additionto the E1A gene deletion, DNX-2401 has an RGD-motif engineered intothe fiber H-loop [127]. This motif enhances tumor infectivity/cell entryby allowing the virus to utilize the αvβ3 and αvβ5 integrins enriched ontumor cells. The coxsackievirus a21 (CVA21) (~31 nm in diameter) canbind to intracellular adhesion molecule 1 (ICAM-1) and decay accel-eration factor (DAF) proteins that are highly expressed on certain tumorcells [128]. The live-attenuated measles virus Edmonston-Zagreb vac-cine strain (120-250 nm in diameter) [133] can bind to CD46 that areexpressed by some cancer cell lines, making these cells a preferredtarget [134].
The use of OVs as a monotherapy is generally well-tolerated withmild AEs such as injection site pain and flu-like symptoms. However,they have shown varying success, where a few treatments led to someclinical responses and others to no clinical responses with limited evi-dence of abscopal effects. A common lack of abscopal effects by OVsmay suggest poor immune activation, and that the observed tumordestruction may occur as a direct consequence of viral infection and thesubsequent adaptive immune response targeting viral antigens, ratherthan tumor neoantigens.
5.4. Cancer gene therapy
5.4.1. VirusesReplicative (oncolytic) and non-replicative (non-oncoyltic) viruses
can be used as vectors to deliver foreign DNA into cells with high genetransfer efficiency [218]. Several IT viral therapies have been designedto express factors such as GM-CSF (e.g. T-Vec, ONCOS-102, Pexa-Vec)[138,140], interferon (IFN)-γ (e.g. TG1042) [143,219], tumor necrosisfactor-α (TNFα) (e.g. TNFerade) [144,145,220], or IL-12 (e.g. Ad-RTS-hIL-12 or INXN-2001) [221]. Suicide genes have also been delivered,such as the bacterial gene called E. coli purine nucleoside phosphorylase(PNP), which converts fludarabine into the anti-cancer agent fluor-oadenine [146]. Further, the herpes simplex kinase thymidine kinase(HSV-TK) has been used to incorporate ganciclovir into a toxic phos-phorylated compound [147,222,223].
Talimogene laherparepvec (T-Vec/Imlygic®) was approved by theFDA and EMA in 2015 for treating melanoma lesions. This modifiedoncolytic herpes simplex virus-1 (155-240 nm) can selectively replicatein cells and destroy infected tumor cells [44]. T-Vec is ICP34.5-deficient(similar to HSV-1716) allowing selective replication in tumor cells[224]. Of note, a comparison between intratumoral T-Vec and sub-cutaneous GM-CSF in patients with unresectable stage IIIB/C/IV mel-anoma in a phase III trial showed a higher efficacy for T-Vec comparedto GM-CSF alone [225]. Specifically, compared to the GM-CSF treatedpatients, the T-Vec-treated patients had a higher median overall sur-vival (OS, 23.3 months vs 18.9 months), durable response rate (DRR,19.3% vs 1.4%) ORR (31.5% vs 6.4%), CR (16.9% vs 0.7%), partialresponse (PR, 14.6% vs 5.7%), and disease control rate (DCR) (76.3% vs56.7%). Common AEs with T-Vec treatment include fatigue, chills,pyrexia, nausea, and flu-like illness. However, T-Vec-treated patients
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had higher instances of grade 3 or 4 AEs compared to GM-CSF-treatedpatients (11.3% vs 4.7%), which include vomiting, cellulitis, dehydra-tion, deep vein thrombosis, and tumor pain.
TNFerade uses an interesting technique for the localized delivery ofTNFα. TNFerade is a replication-deficient adenovirus type 5 that carriesa transgene encoding human TNFα. However, a radiation-inducibleEgr-1 promoter gene was placed upstream to the TNFα cDNA, allowingfor control of the time and location of TNFα delivery through the use ofradiation therapy. Ad-RTS-hIL-12 is an adenoviral vector that was en-gineered for the controlled expression of IL-12. This involves the use ofthe RheoSwitch Therapeutic System®, which requires the oral activatorveledimex to induce IL-12 expression [221]. These inducible systemsallow the temporal regulation of gene product expression.
5.4.2. PlasmidsAlthough less common, DNA plasmids can also be used for gene
delivery. For instance, the IL-12 plasmid cDNA (pNGVL3-mIL12) wasgiven at 50 μg with saline (0.76 mL) in patients with cutaneous orsubcutaneous metastases in a phase I clinical study [148]. Since theefficacy of these therapies require their entry into cells, the negativelycharged nature of plasmid DNA would likely make it difficult for DNAto pass through the negatively charged cell membranes. Nevertheless,45.5% of the analyzed patients had SD and 54.5% of the analyzed pa-tients had PD. 41.7% of patients had a PR (i.e. lesion size decrease) inthe treated tumor, but non-treated lesions did not decrease in size. NoIL-12 or IFNγ was detected in patient serum. The treatment was well-tolerated with no toxicity higher than grade 1.
Electroporation was used to help deliver tavo, a 6215 bp plasmidthat encodes for the p35 and p40 subunits of the human IL-12 protein[149,226]. Clinical responses were similar between treatment the full-length IL-12 plasmid cDNA and tavo with electroporation; however, adirect comparison cannot be made due to their difference in plasmiddesign, study design, and dosage regime.
Another method to improve delivery of DNA into cells is throughincorporation with cationic lipids or cationic polymers, forming lipo-plexes or polyplexes, respectively. The EGFR antisense DNA is a plasmidof pNGVL1-U6-EGFRAS that was prepared in complex with DC-chol li-posomes [227]. The plasmid is likely about 9600 base pairs; the pNGVLvector (also called pUMVC) is 9287 bp, human U6 promoter is 241 bp,and EGFRAS is 39 bp [227–229]. Mixing BC-819 (a plasmid DNA thatencodes for the A fragment of diphtheria toxin under the control of a H19gene promoter), with PEI formed 80-90 nm polyplexes [151]. BC-819polyplexes were given as intravesical treatments in patients with bladdertumors in a phase IIb clinical trial. None of the 39 evaluated patients haddisease stage or grade progression [230]. 64% of evaluated patients wererecurrence free at 3 months, and complete tumor ablation was observedin 33.3% with no new lesions at 3 months. The recurrence-free rate was45% at 1 year and 40% at 2 years. Other than flu-like symptoms, AEswere mostly related to the lower urinary tract. Also, CYL-02 (a plasmidthat encodes for the DCK-UMK fusion protein, which phosphorylates andactivates the pro-drug gemcitabine) was prepared in 5% w/v glucosewith a PEI nitrogen to DNA phosphate (N/P) ratio of 8 to 10. No particlesize information was provided for CYL-02; however, it is likely around 45nm based on another reported polyplex with N/P of 8-10 that was madewith JetPEI, which appears to be the same JetPEI used to make CYL-02[155]. A phase I clinical trial of IT injections of CYL-02 in combinationwith gemcitabine IV infusion was conducted in pancreatic cancer pa-tients [154]. CYL-02 DNA and the expression of therapeutic messengerRNA was detected in the tumor after one month of treatment, demon-strating the successful DNA delivery to tumors and long-term gene ex-pression. No objective response was observed, but the treatment in-hibited tumor progression in 95% of patients two months after treatmentand 91% of the metastasis-free patients had no new tumor development.Further, the treatment did not prevent the progression of distant tumorsas metastatic tumors progressed in 71% patients. This combinationtreatment was well tolerated, and the toxicity profile was similar to
gemcitabine alone. The treatment was well tolerated in patients and AEsassociated with CYL-02 injection were of mostly grade 1 or 2.
5.5. Monoclonal antibodies
Monoclonal antibodies (mAbs) (150 kDa, ~10-15 nm) have shownpromising therapeutic efficacy as cancer treatment [158]. Im-munostimulatory mAbs can target antigens expressed on the surface oftumor cells and induce cytotoxic T lymphocyte (CTL) responses, whichresult in tumor cell death [231]. Immune checkpoint inhibitors (ICIs)are therapeutic mAbs that target the receptors of inhibitory signalingpathways to reverse immune suppression and reactivate immune-mediated antitumor responses [232]. Immune checkpoint inhibitors,such as antibodies targeting CTLA-4 and PD-1/PD-L1, have demon-strated broad activation of tumor-specific T-cells by blocking negative-feedback mechanisms of the immune system. The most common ad-ministration route of these mAbs is systemic, however, systemic de-livery of mAbs can induce immune-related AEs [233]. Therefore, ITadministration of mAbs has been suggested to retain mAbs in the tumormicroenvironment and reduce systemic exposure and associated in-flammatory side effects [234]. Local antibody administration hasshown accumulation in tumor-draining lymph nodes, which may assistin generating anticancer immunity or addressing metastases [235].
Ipilimumab, a human IgG1 that targets CTLA-4, was the first ap-proved immune checkpoint inhibitor for advanced melanoma and hassignificantly improved the overall survival rate associated with thisdisease [236]. Systemic ipilimumab administration is commonly asso-ciated with a low response rate and life-threatening toxicities, whichhas prompted the exploration of IT delivery. Current clinical trials of ITadministered ipilimumab are mostly in combination with therapiesincluding myeloid DCs, anti-PD-L1 mAbs, and IL-2, and these combi-nations showed improved tolerability and abscopal effects[157,237,238]. In one phase I study of IT ipilimumab combined with IL-2 for advanced melanoma, T-cells were activated within the tumor andin the draining lymph nodes, indicating IT administration enhanced thelocal anti-tumoral responses and also induced distal effects [157].
Co-stimulatory receptors such as CD40 and OX40 are other targetsin cancer therapy. These co-stimulatory receptors are mainly expressedon APCs, and when activated, the presentation of tumor antigens in-creases and cytokines are released to improve the activation of anti-tumor T cells [239]. The human IgG1 agonistic CD40 antibody (anti-CD40 mAb), ADC-1013, has been investigated via both IT and IV ad-ministration in advanced solid malignancies. ADC-1013 has been op-timized through the use of Fragment Induced Diversity (FIND) tech-nology to improve binding affinity [240]. This optimization made itpossible to achieve high efficacy with very low doses. A phase I trial forIT administered ADC-1013 in patients with advanced solid tumors hasshown safety and B-cell expansion after treatment, which could be re-lated to the antitumor efficacy [241,242]. Clinical trials of IT anti-OX40mAbs are limited, but some are investigating the combination of anti-OX40 mAb, BMS 986178, with TLR9 agonist SD-101 and radiation inpatients with advanced solid malignancies or lymphomas [243,244].
5.6. Small molecules
Compared to proteins, small molecules typically have the advantageof high potency and improved tissue penetration. Significant systemictoxicity has been a limiting factor for small molecule drugs and thus ITformulations have been investigated.
Small molecules are commonly modified into a prodrug or for-mulated as emulsions or colloids to improve retention after IT injection.Several delivery systems are under clinical trials including polymer-drug conjugates, liposomal carriers, and polymeric micelles [245]. Forexample, one of the most extensively studied and widely utilized che-motherapy drugs is cisplatin (MW = 300 Da). HNSCC patients weregiven IT administrations of cisplatin/epinephrine injectable gel in a
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phase III study [246]. 29% of patients had a CR or PR for at least 28days. AEs include injection-related pain, elevated blood pressure, ta-chycardia, and local cytotoxic effects (e.g. inflammation, bleeding, ul-ceration, etc). Two patients (1.67% of patients) experienced severepain.
Another widely utilized small-molecule treatment is PV-10. PV-10 isa 10% saline solution of Rose Bengal disodium (MW = 1018 Da), awater-soluble xanthene dye, which has been developed as an intrale-sional formulation. A phase I trial of IT injection of PV-10 in patientswith metastatic melanoma showed effective chemoablation for bothinjected lesions and un-injected lesions, as “bystander” responses (27%ORR for non-target lesions), suggesting the activation of systemic im-mune [247,248]. The treatment was well-tolerated; common AEs in-clude mild or moderate pain, inflammation, and pruritus at the treat-ment site. In the following phase II study, the observed response ratewas 51% among injected lesions, with a 26% CR rate [249].
Small molecule drugs enter tumors mainly through non-selectivediffusion and passive targeting, so an ideal form of these molecules islikely nonionized to fully enable conductive diffusion. The acidic mi-croenvironment of tumor tissue causes chemoresistance against weakly-basic drugs, which become protonated and positively charged upon en-tering the tumor and are less membrane permeable. The alkylating agentcisplatin (pH 3.5-5.5) and nucleotide analogue gemcitabine (pH 2.7-3.3)remain nonionized and have higher cytotoxicities at lower pH. The effectof surface charge on nanoparticles has been investigated on many nano-sized formulations as well. Positively charged particles retain in thetumor at higher concentrations compared to the surrounding tissue [250]and diffuse out at a slower rate in comparison to anionic particles [251].This observation is due to the electrostatic interactions with negativelycharged proteoglycans of the tumor vasculature. For highly chargedparticles like gemcitabine hydrochloride and PV-10, a disodium salt, theelectrostatic interactions might be a significant limitation to their mo-bility within the tumor, which could affect the efficacy of IT injections.
As small molecules are largely unhindered by the transport phe-nomena that dictate the distribution of other classes we have discussedin this review, chemical modifications can serve to selectively impedeegress from the TME. Non-specific binding of small molecules to tumorcells or the extracellular matrix components can enhance the retentionwithin the tumor. Ligand-receptor binding also delays clearance.Polymerization and complexation of small molecules enables their re-tention and depot-release within the TME. Tuning the charge propertiesof small molecules can aid intracellular penetration of these compoundsas well as retention in the tumor. IT delivery of small molecules isappealing because the lower specificity of these candidates’ mechanismcan be overcome by the physical retention of their presence at the TME.
6. Emerging trends in intratumoral therapies and futuredirections
IT cancer therapies currently in clinical trials encompass a wide rangeof molecular structures and formulations. Local administration and re-tention of IT therapies may amplify local therapeutic efficacy and limitside-effects compared to systemic cancer therapies. Tumor transportmechanisms that limit leakage from the tumor should reduce systemictoxicities. With advances in technology and surgical procedures, such asinterventional radiology, endoscopy, and laparoscopic surgery, thechallenge is not the act of administering IT therapies [4,252]. Althoughintratumoral administration helps deliver therapeutic agents to the vi-cinity of the target tumor cells, challenges such as the drug diffusibility,tumor-cell uptake, and drug degradation or clearance remain. To helpovercome some of these challenges, the physiochemical properties of thedrug or its formulation may be modified to promote tumor retention.
Property-enhancing strategies include altering the structure of themolecule and utilizing formulation strategies. Approaches such as re-ducing drug solubility, increasing molecular size (e.g. PEGylation),modulating charge, or including tumor-targeting or matrix-adhesive
domains can all be incorporated to sustain the therapeutic at the in-jected tumor. For example, since the TME is more acidic than normaltissues, increasing the isoelectric point a molecule (e.g. antibody ca-tionization or via addition of basic amino acids) may facilitate tumorretention [253–255].
Another promising approach to extend IT residence is through the useof nanoparticles (10-200 nm). Nanoparticles can serve as drug carriers,can sustain the release of the drug, and can be formulated for targeteddrug delivery [256,257]. Formulating drugs in polymeric complexes ornanoparticles can also extend the persistence of the drug at the site ofadministration. The highly cross-linked collagen and densely packed cellsin the tumor tissue limit particle diffusion [257]. Particles larger than theextracellular matrix mesh (~40 nm, but can be variable) exhibit limiteddiffusion in the extracellular matrix. Furthermore, particles with size <200nm were reported to be effective for cellular uptake [258]. For ex-ample, various particles or emulsions have also been studied as slow-release systems for injected antibodies in pre-clinical studies. Anti-CD40has been conjugated to immunostimulatory poly(γ-glutamic acid) na-noparticles to successfully improve localization of the mAb as the na-noparticle minimized systemic cytokine release [259]. Coupling anti-CD40 to polylactide nanoparticles, however, did not show an improve-ment of anti-tumor activity [260]. Other anti-CD40 formulations basedon mineral oil or dextran-based microparticles have shown capacity toactivate tumor-specific T-cell responses and significantly decrease AEscompared to systemic infusion [261]. However, the mineral oil-basedmicroparticles were preferred as the dextran-based microparticles causedoverly severe local inflammation. Categorically, such approacheshearken back to the success of G100 by Immune Design (Table 3).
As reviewed above, OVs, gene therapy, and bacterial therapies haveshown promise, suggesting particles in this size range also represent aviable approach. Persisting in the tumor space is not enough, however,the therapeutic must also access the target. Agents, such as CpG,PolyI:C, or plasmids, require uptake into cells to interact with theirintracellular targets. Cationic complexes, viruses, or bacterial therapiesmay enhance persistence in tumor tissue and also facilitate the deliveryof TLR agonists, DNA or RNA molecules into cells.
Intratumoral immune stimulation with limited systemic exposuremay widen the therapeutic index for immunostimulants. Combining ITtherapy with checkpoint inhibitors represents a logical synergy, butdrug-drug interactions raise possible concerns, which could be avoidedif IT treatment is predominantly retained in the tumor. Beyond ther-apeutic design, other challenges such as the administration regimen(i.e. dose, volume, frequency of injections, etc.), IT injection techni-ques, and the identification and selection of the cancer therapy (orcombination therapies) will continue to evolve as the field progressestoward stimulating more effective immune responses in tumors.
Declaration of Competing Interest
The authors report no conflicts of interest.
Acknowledgements
The authors would like to acknowledge the funding from a PilotGrant from The University of Kansas Cancer Center. AH was supportedby the Gretta Jean and Gerry D. Goetsch Scholarship. RL was supportedby a generous gift from the Joe and Jeanne Brandmeyer Family. JDGwas supported by the Postdoctoral Fellowship in Pharmaceutics fromthe PhRMA Foundation as well as the Madison and Lila Self GraduateFellowship at the University of Kansas.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.org/10.1016/j.jconrel.2020.06.029.
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