Journal of Controlled Release · 2021. 2. 19. · infiltrating lymphocytes [4,7,8]. Intuitively, the design of IT therapies is significantly different than that of systemic cancer

Contents lists available at ScienceDirect

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.

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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-

A. Huang, et al. Journal of Controlled Release 326 (2020) 203–221

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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.


205

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


[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


[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


[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]


206

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


207

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0522

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CT02

6449

67N

CT03

4455

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CT04

2708

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CT03

8650

82N

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72N

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0077

32N

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95N

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5218

70N

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7317

42N

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4109

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9279

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2661

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3223

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ated

with

carb

oxym

ethy

lcel

lulo

se(C

MC)

•Inan

aque

ous

salin

eso

lutio

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

)


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

]


)


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

]


)


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

ralv

ecto

ral

low

high

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sgen

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and

high

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plex

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low

phos

phor

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ion

ofG

CV,f

orm

ing

cyto

toxi

cG

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ipho

spha

te

NCT

0084

4623

[147

]

Plas

mid

IL-1

2pl

asm

idcD

NA

(pN

GVL

3-m

IL12

)•F

orm

ulat

edin

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48]

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gene

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(tav

o);

plas

mid

IL-1

2•6

215

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nof

IL-1

2N

CT00

3232

06N

CT01

5793

18N

CT01

4408

16N

CT02

3453

30

[149

]

EGFR

antis

ense

DN

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ated

~96

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mor

cells

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bits

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orpr

olife

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NCT

0000

9841

[150

]

BC-8

19(a

lso

calle

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TA-H

19)

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0bp

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0071

1997

[151

–153

]


)


211

Table3

(continued)

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gory

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lene

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ates

the

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

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ute

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ence

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mpl

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ende

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sein

wat

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=0.

821

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rage

part

icle

size

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amic

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357

nm

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cre

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nof

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toat

tach

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erce

lls.

NCT

0167

2450

[157

,158

]

AD

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13A

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D40

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bs•A

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onos

peci

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edto

indu

ceeff

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rT-c

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that

atta

ckth

etu

mor

.

NCT

0237

9741

[159

]

TTI-6

21M

odifi

edan

tibod

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i-CD

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here

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osis

,and

antit

umor

activ

ity.

NCT

0289

0368

[160

]

BMS

9861

78an

ti-O

X40

mA

b•A

hum

anIg

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sele

ctiv

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bind

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and

activ

ates

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0to

indu

cepr

olife

ratio

nof

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mph

ocyt

esth

atat

tack

tum

oras

soci

ated

antig

ens.

NCT

0383

1295

[161

]

Smal

lMol

ecul

eIN

T230

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spla

tin,c

hem

othe

rape

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rmul

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ting

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ule

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tinan

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nbla

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e.

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pene

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ion

enha

ncer

faci

litat

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sper

sion

ofth

etw

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ugs

thro

ugho

utin

ject

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mor

sand

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les

incr

ease

ddi

ffusi

onin

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cells

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30-6

thor

ough

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tura

tes

and

kills

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cted

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ditio

n,th

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(T-c

ellm

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ted)

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une

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that

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noto

nly

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utno

n-in

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seen

mic

ro-m

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NCT

0305

8289

[162

]

Cisp

latin

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neph

rine

inje

ctab

lege

lCi

spla

tin,c

hem

othe

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utic

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tain

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mg/

mL

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latin

,0.1

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mL

epin

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ine,

and

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llage

nas

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nca

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x

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atum

oral

inje

ctio

nof

cisp

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ject

able

gela

chie

ves

high

conc

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

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Para

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enes

ulfo

nam

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nific

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grow

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is

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ces

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som

alin

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ility

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chon

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lda

mag

e,an

din

hibi

tsA

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thes

is

NCT

0344

8146

[164

]

Gem

cita

bine

chem

othe

rape

utic

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cleo

side

prod

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0272

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0183

4170

[165

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PV-1

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diso

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inco

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NCT

0269

3067

NCT

0255

7321

[166

]


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


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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