Novel Miniaturized Drug Conjugate Leverages HSP90 Driven ... · 6/4/2020 · Lack of site-specific...

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Title: Novel Miniaturized Drug Conjugate Leverages HSP90 Driven Tumor Accumulation to

Overcome PI3K Inhibitor Delivery Challenges to Solid Tumors

Running Title: HSP90 Conjugate Overcomes PI3K Inhibitor Delivery Challenges

Authors and Affiliations: Samantha Perino1, Benoit Moreau2, Jessica Freda1, Amanda Cirello1, Brian H.

White2, James M. Quinn1, Kristina Kriksciukaite1, Ashwajith Someshwar1, Janel Romagnoli2, Megan

Robinson1, Sara Movassaghian1, Tyler Cipriani2, Richard Wooster2, Mark T. Bilodeau1, Kerry A. Whalen1

Authors affiliations: 1Tarveda Therapeutics Inc, 134 Coolidge Ave, Watertown MA U.S.A. 2Tarveda

Therapeutics former employees

Corresponding Author: Samantha Perino, Tarveda Therapeutics Inc, 134 Coolidge Ave, Watertown MA

02472. Phone 617-224-3551; email:[email protected]

Running Title: HSP90 Conjugate Overcomes PI3K Inhibitor Delivery Challenges

Keywords: Drug discovery technologies/Novel assay technology, drug targets/protein kinase &

phosphatase drug targets, Molecular pharmacology, Kinase inhibitors, small molecule therapeutics.

Disclosure of Potential Conflicts of Interest: All authors were or are employees and shareholders of

Tarveda Therapeutics Inc.

on November 16, 2020. © 2020 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 4, 2020; DOI: 10.1158/1535-7163.MCT-19-0964

http://mct.aacrjournals.org/

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Abstract: The Phosphatidylinositol 3 kinase (PI3K) pathway is considered a master regulator for cancer

due to its frequent activation, making it an attractive target for pharmacologic intervention. While

substantial efforts have been made to develop drugs targeting PI3K signaling, few drugs have been able

to achieve the inhibition necessary for effective tumor control at tolerated doses.

HSP90 is a chaperone protein that is overexpressed and activated in many tumors and as a

consequence, small molecule ligands of HSP90 are preferentially retained in tumors up to 20 times

longer than in normal tissue. We hypothesize that the generation of conjugates that use a HSP90

targeting ligand and a payload such as Copanlisib, may open the narrow therapeutic window of this and

other PI3K inhibitors.

In support of this hypothesis, we have generated a HSP90-PI3K drug conjugate, T-2143 and utilizing

xenograft models, demonstrate rapid and sustained tumor accumulation of the conjugate, deep

pathway inhibition, and superior efficacy than the PI3K inhibitor on its own. Selective delivery of T-2143

and the masking of the inhibitor active site was also able to mitigate a potentially dose limiting side

effect of Copanlisib, hyperglycemia. These data demonstrate that by leveraging the preferential

accumulation of HSP90 targeting ligands in tumors, we can selectively deliver a PI3K inhibitor leading to

efficacy in multiple tumor models without hyperglycemia in mice. These data highlight a novel drug

delivery strategy that allows for the potential opening of a narrow therapeutic window through specific

tumor delivery of anticancer payloads and reduction of toxicity.




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Introduction

The phosphatidylinositol-3-kinase (PI3K) pathway is one of the most frequently dysregulated signaling

cascades in cancer and is implicated in a wide range of tumor types [1]. The PI3K-AKT-mTOR pathway is

an intracellular signaling pathway that regulates cellular processes such as increased cell growth,

proliferation, and differentiation, which in combination with its common dysregulation in cancer, makes

it an attractive target for pharmacologic intervention [2]. Given the importance of this pathway in

cancer, more than 40 compounds that target the PI3K-AKT-mTOR pathway have been tested in clinical

trials involving patients with a wide range of tumor types. However, many of these agents have not

advanced beyond early stage clinical testing due to the inability to achieve deep pathway inhibition in

tumor tissue while avoiding dose-limiting toxicities in the patient [3-4]. Despite numerous PI3K-AKT-

mTOR inhibitors entering clinical trails only five pathway inhibitors, temsirolimus (mTOR), everolimus

(mTOR), idelalisib (PI3K), alpelisib (PI3K), and copanlisib (PI3K) have been approved by the FDA for

clinical use in the treatment of a number of different cancers, with the majority of approvals in liquid

tumors, demonstrating a clear need for novel approaches to achieving deep PI3K-AKT-mTOR pathway

inhibition while hyperglycemia in the solid tumor setting.

Lack of site-specific drug delivery, particularly delivery of cytotoxic agents to tumors, has led to the

emergence of tumor-selective drug delivery systems over the last thirty years. Tumor selective drug

delivery systems include the use of targeting moieties, such as monoclonal antibodies, peptides, and

synthetic polymers, chemically linked to an active cytotoxic agent to form a drug conjugate [5-6]. While

antibody drug conjugates (ADCs) have demonstrated efficacy in the treatment of solid tumors, with five

being currently marketed, they have potential deficiencies that include the slow speed of diffusion and

poor tumor penetration, potentially limiting effectiveness in the solid tumor setting [7]. The increased

time that an ADC is in circulation can lead to the prolonged release of payload in the bloodstream

causing potential systemic toxicities [8]. Given the liabilities of antibody-based therapies, miniaturized

drug conjugates have the potential to target tumor cells allowing for rapid accumulation of potent

payloads in tumor cells deep into the core of the tumor and slowly release the payload over time giving

sustained delivery of the drug to the tumor while being quickly cleared from the plasma, resulting in a

decrease in overall toxicity. PEN-866, currently in Phase 1/2a (NCT03221400) applies these fundamental

principles and is a synthetic small molecule drug conjugate comprised of a Heat Shock Protein 90

(HSP90) targeting moiety attached through a cleavable linker to SN-38, the active metabolite of the

topoisomerase I inhibitor, irinotecan (approved worldwide for the treatment of patients with colorectal

cancer and other cancers). HSP90 is a highly conserved and widely expressed molecular chaperone

protein that regulates a diverse range of cellular functions such as the folding, stability and degradation

of many proteins. As a result, HSP90 exerts marked effects on normal biology and disease processes [9-

10]. Although HSP90 is highly expressed in most cells, it has been shown to be upregulated and

activated in a variety of tumor cells compared to normal healthy tissue.

The activated form of HSP90 found in cancer tissues is present in a highly complexed state with co-

chaperone proteins with high ATPase activity, with these complexes functioning as a network to

enhance cellular survival, regardless of tissue of origin or genetic context. The consequence of this high

activation state is a propensity to selectively bind with high affinity to HSP90 inhibitors as compared to

the latent form of HSP90 found in normal tissue rendering HSP90 inhibitors selective for cancer cells [11-

12]. The selectivity of HSP90 inhibitors for the activated form of HSP90 present in the cancer cells allows

for preferential killing of cancer cells compared to normal cells [13-14]. While PEN-866 has been




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designed to limit HSP90 inhibition, it still demonstrates selective targeting of the activated cancer form

of HSP90, and is able to penetrate, accumulate and be retained in the tumor cells leading to superior

antitumor activity when compared to an equimolar SN-38 dose of irinotecan and to small cell lung

cancer (SCLC) standard of care agents, cisplatin and topotecan [15-16].

Here we report on the preclinical characterization of an HSP90-PI3K conjugate. By leveraging the

preferential accumulation of HSP90-targeting ligands in tumors, as seen with PEN-866, and by masking

of the PI3K inhibitor active site though conjugation to an HSP90 targeting ligand, our HSP90-PI3K

conjugate selectively accumulates and is retained in tumors leading to deep pathway inhibition over

time with a decrease in a commonly observed toxicity, hyperglycemia, for PI3K alpha inhibitors. These

proof-of-concept experiments include demonstration of extended intratumoral drug exposures,

extended inhibition of the PI3K-AKT-mTOR pathway, and a superior therapeutic window as compared to

the PI3K inhibitor alone. Together, these results demonstrate a superior tumor-selective drug delivery

system that can deliver payloads directly to tumors, achieve sustained and deep pathway inhibition

while mitigating hyperglycemia

Materials and Methods

Compounds

The investigational compounds T-2026, T-2143 and T-2212 were synthesized according to the

procedures described in the supplementary material and were isolated and utilized as their respective

trifluoroacetate salts. T-2026 is the PI3K inhibitor payload that was incorporated into the conjugates. T-

2143 is the HSP90-binding conjugate of T-2026 and T-2212 is the non-binding control analog of T-2143

where part of the HSP90 binding pharmacophore has been blocked with the addition of a methyl group.

Copanlisib as the dihydrochloride salt was purchased from MedKoo Biosciences, Inc. The doses used for

all of the compounds in all in vivo experiments represent free base equivalent doses.

HSP90 binding activity assay.

Assays were carried out at BPS Biosciences, California USA. Briefly, the reactions were conducted at

room temperature for 3 hours in a 100 µl mixture containing assay buffer, 5 nM FITC Labeled

Geldanamycin and the test compound. Each compound was run in duplicate, ten-point curve starting at

a concentration of 10 M. Fluorescence intensity was measured at an excitation of 485 nm and an

emission of 530 nm using a Tecan Infinite M1000 microplate reader. Fluorescence intensity is converted

to fluorescence polarization using the Tecan Magellan6 software. The fluorescence polarization data

were analyzed using the computer software, Graphpad Prism. The fluorescence polarization (FPt) in

absence of the compound in each data set was defined as 100% activity. In the absence of protein and

the compound, the value of fluorescent polarization (FPb) in each data set was defined as 0% activity.

The percent activity in the presence of the compound was calculated according to the following

equation: % activity = (FP-FPb)/(FPt-FPb)×100%, where FP= the fluorescence polarization in the presence

of the compound. For IC50 measurements, the values of % activity versus a series of compound

concentrations were then plotted using non-linear regression analysis of Sigmoidal dose-response curve

generated with the equation Y=B+(T-B)/1+10((LogEC50-X)×Hill Slope), where Y=percent activity, B=minimum

percent activity, T=maximum percent activity, X= logarithm of compound and Hill Slope=slope factor or

Hill coefficient. The IC50 value was determined by the concentration causing a half-maximal percent

activity.

Cell Culture




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All cells lines were purchased from the American Type Culture Collection (ATCC). The following cell lines

were maintained in the recommended media: NCI-H460 (ATCC HTB-177, RRID: CVCL_0459), HTC-116

(ATCC CCL-247, RRID: CVCL_0291), LS174T (ATCC CL-188, RRID: CVCL_1384), BT-474 (ATCC HTB-20,

RRID: CVCL_0179) and SKOV3 (ATCC HTB-77, RRID: CVCL_0532).

In vitro cellular proliferation

The following cell lines were seeded at 2500 cells/well in a 96-well plate: HCT116, LS174T and BT-474.

NCI-H460 cells were seeded at 500 cells/well. All cell lines were seeded in their respective medium.

Plated cells were incubated overnight before treatment. Cells were treated with a dose range of T-2212

and T-2143 which were dissolved in DMSO (Sigma D4540). The starting concentration was 10 µM for

each compound which was diluted by 3-fold for a total of ten data points. The number of viable cells was

determined by CellTiter-Glo 2.0 Cell Viability Assay (Promega G9243) through quantifying ATP following

the manufacturers protocol. All samples were run in duplicate. Inhibitory concentration of 50 percent

growth was determined using GraphPad statistical software, GraphPad Prism 6.

Cell Free Enzymatic Assay

The determinations of inhibitory concentration of 50 percent of PI3K3CA, PIK3CB, PIK3CD, and PIK3CG

activity with T-2143, copanlisib, or T-2026 were carried out at ProQinase GmbH using the Free Choice

Kinase Assay. All samples were run in duplicate.

In vivo studies

All studies were conducted in accordance with the Tarveda Therapeutics Institutional Animal Care and

Use Committee (IACUC). All mice were treated in accordance with the OLAW Public Health Service

Policy on Human Care and Use of Laboratory Animals and the ILAR Guide for the Care and Use of

Laboratory Animals. Female athymic nude mice (CrTac:NCr-Foxn1nu) were purchased from Taconic (IMSR

Cat# TAC:ncrnu, RRID: IMSR_TAC:ncrnu). Infectious disease testing on the cell lines was done by Charles

River Laboratories Wilmington, MA by PCR using the Mouse Essential Clear Panel, within 3 years of cell

line use. Cells were only used if they had been passaged a total of 25 times or less. Mice were 8-11

weeks old and weighed approximately 20 grams at the time of dosing. T-2143, T-2212, copanlisib, and T-

2026 were all dosed intravenously. Animals were euthanized once tumors reached a maximum of 2000

mm3 or at the end of study, whichever came first. At the end of each study, the animals were

euthanized via CO2 inhalation followed by cervical dislocation to ensure death.

Cells were harvested from tissue culture and made into a final suspension by mixing with matrigel (BD

Biosciences, Catalog #CB-40234) (1:1 ratio). All cells were implanted subcutaneously in the right flank of

mice. LS174T (H1047R) and NCI-H460 (E545K) cells were implanted at a concentration of 2.5 × 106 cells

per mouse, BT-474 (K11N) cells were implanted at a concentration of 5.0 × 106 cells per mouse. Tumor

growth was monitored throughout the study and measured twice weekly. Tumor volume was measured

in two dimensions using calipers and volume was calculated using the formula: (w2 × l)/2= mm3,

assuming 1 mg is equivalent to 1 mm3 of tumor volume. The health of the mice was monitored, and

noteworthy clinical observations were recorded. Acceptable toxicity was defined as group mean body

weight (BW) loss of less than 20% during the study and not more than one treatment related death

among ten treated animals. Mice were randomized into treatment groups of ten animals per group and

therapy began when tumor volumes were approximately 200 mm3 usually 7-14 days post implantation.

For mouse in vivo studies, T-2143 and T-2212 were formulated in 5% DMSO (Alfa Aesar, HPLC grade) and

95% of freshly prepared 10% Solutol (Kolliphor HS 15) in 5.2% Dextrose (USP grade, Sigma Aldrich).




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Copanlisib HCl (MedKoo Biosciences, Inc.) was prepared in 5% Mannitol (Pearlitol PF; Roquette) in water

for injection (HyPure, GE Hyclone). T-2026 dosing solution was prepared in 15% DMSO:Solutol (Alfa

Aesar, HPLC grade, Kolliphor HS 15) in 85% Dextrose (USP grade, Sigma Aldrich, Batch). All dosing

solutions were prepared fresh and transferred through 0.22 µm PTFE filters (Millex -LG, Merck Millipore)

in a sterile hood and the target doses were verified by established HPLC methods.

Pharmacokinetic Studies

Mice were dosed intravenously, three animals per each time-point were anesthetized using CO2 and

~1.0 mL of blood sample was collected via cardiac puncture using a needle (BD, Catalog# 14-826-88).

While animals were still anesthetized, the animals were euthanized via cervical dislocation. Blood was

transferred to K2EDTA sample collection tube (BD, Catalog #02-669-33), inverted four times and

centrifuged for 10 minutes at 10,000 x g, plasma harvested, transferred into labeled 96-well plate, and

stored at -80º C.

Pharmacodynamic Studies

Mice bearing LS174T and BT-474 tumors were treated with copanlisib at 10.6 mg/kg, 25 mg/kg of T-2143

or 25 mg/kg of T-2212 (N=3). Tumors were collected 1, 24, or 72 hours post dose. Xenograft tumors

were processed for lysate using Cell Lysis Buffer 10X (Cell Signaling 9803) diluted to 1X. Proteins were

separated by SDS-PAGE on NuPAGE 4-12% Bis-Tris Protein Gels, 1.5mm, 15-well (Thermo Fisher

NP0336BOX). Proteins were transferred from the gel to a nitrocellulose membrane using iBlot Transfer

Stack, nitrocellulose, regular size (Thermo Fisher IB301001). After the transfer, the membrane was

blocked in PBS Blocking Buffer (LI-COR 927-40000). This was followed by overnight incubation of 1:2000

Phospho-Akt (Ser473) (D9E) XP Rabbit mAb (Cell Signaling 4060, RRID:AB_2224726) and 1:1000 GAPDH

(D4C6R) Mouse mAb (Cell Signaling 97166, RRID:AB_2756824) at 4˚C. After the primary incubation, the

membrane was washed with Pierce 20X TBS Tween 20 Buffer (Thermo Fisher 28360) and incubated

further for one hour at room temperature with the secondary antibodies: 1:10000 IRDye 680RD Goat

anti-Rabbit IgG (LI-COR 926-68071) and 1:10000 IRDye 800CW Goat anti-Mouse IgG (LI-COR 926-32210).

Then the membrane was imaged on the LI-COR Odyssey Imaging System. Bands were quantified based

on their size and brightness using LI-COR Odyssey imaging software.

Efficacy Studies

All doses were scaled to the BW of the individual animals at a dose volume of 10 mL/kg. Treatment

regimens and dosages in each experiment are described in the Results and in the Figure Legends. Each

treatment group consisted of ten animals. Percent tumor growth inhibition (%TGI) was defined as the

difference between the mean tumor volume (MTV) of the vehicle and the MTV of the drug treated

group, expressed as a percentage of the MTV of the vehicle group. Statistical analysis was done using

one-way ANOVA and Tukey’s multiple comparisons in GraphPad statistical software, GraphPad Prism 6.

A single efficacy study is represented in the graphs presented in the paper, however, for both the BT-

474 and LS174T studies, separate graphs are shown to comparing T-2143 to either the negative control,

T-2122, or the components that collectively represent T-2143 copanlisib and ganetespib, respectively, as

shown in Figures 2 and 5.

Body Weight measurements

Animals were weighed on the days indicated in the graphs (Supplementary Figures S3a-c). For each

treatment group, weights were averaged and compared to the average weight of the group on first day




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of treatment to obtain the percent change in weight. Statistical analysis was done using one-way

ANOVA and Tukey’s multiple comparisons in GraphPad statistical software, GraphPad Prism 6.

Blood Glucose Study

Non tumor bearing mice were dosed intravenously with equimolar concentrations of the vehicle, 10.8

mg/kg copanlisib, 25 mg/kg T-2143 and 10.8 mg/kg T-2026. Each treatment group consisted of N=5.

Mice were monitored for any significant health changes. For the T-2143 and vehicle control groups tails

were pricked 1, 4, 6, and 24-hours post dosage. For all other groups blood was sampled 1-hour post

dosage and blood glucose readings were taken with the AlphaTRAK 2 Blood Glucose Monitoring System

(Zoetis CFGW210-M1673).

Tumor and Plasma Pharmacokinetic Analysis

In vivo sample preparation

Standard calibrations were prepared by serially diluting 3 mM methanol (Fisher Scientific, Cat. A456-4)

stock solutions of T-2143, T-2212 and T-2026 in pooled BALB/C female mouse Lithium Heparin plasma

(Bioreclamation IVT, Cat. MSE02PLLHXNN). Mouse tumors were weighed and homogenized in PBS

(Fisher Scientific, Cat. 14040133) containing 100 nM of dichlovous (Sigma, Cat. 45441) using a handheld

tissue homogenizer (Omni). A protein precipitation procedure was performed with 100 µL of blank

plasma standards, homogenate and sampled mouse plasma, crashed with 300 µL of acetonitrile (Fisher

Scientific, Cat. A998SK-4) containing 0.1% formic acid (Fisher Scientific, Cat. A117-50) and 100 ng/mL of

glyburide (Sigma, Cat. G2539) into a 96-well plate. The samples were capped, vortexed to mix, then

centrifuged at 3500 x g for 5 minutes. 100 µL of the supernatant was then transferred to a new 96-well

plate before LC-MS/MS analysis.

LC-MS/MS for Quantification

An LC-MS/MS method was developed for investigational molecules; T-2143, T-2212 and T-2026. Leap

technologies HTS PAL autosampler was used to inject 10 µL of sample volume to ABSciex 4000 series

tandem mass spectrometer coupled with a Shimadzu LC10AD VP LC system (Columbia, MD). An ACE 3

C18 3 µM, 50 X 2.1 mm column (PN ACE-111-0502) was used to chromatographically separate analytes.

Mobile phase consisted of acetonitrile containing 0.1% formic acid (A) and water containing 0.2M of

ammonium acetate (Fisher Scientific, A637-500) and 0.1% formic acid(B). A gradient elution was

employed, starting at 5% A, holding for 0.5 minutes, increasing to 95% A over 3 minutes, holding for 1.5

minutes then re-equilibrating at 5% A for 1.1 minutes. Mass spectrometric detection was performed

using electrospray ion source operating in positive mode. The mass spectrometer was operated in

multiple reaction monitoring mode, monitoring m/z for analytes, T-2143, T-2212, T-2026 and glyburide

(internal standard). Analysist software version 1.6.2 (ABSciex) was used to fit T-2143, T-2212 and T-2026

standard curves with a linear 1/x weighted fit.

Pharmacokinetic Analysis

Non-compartmental pharmacokinetic analysis was performed using Phoenix WinNonlin version 8.1

(Certara).

Results

HSP90 Binding is Essential for Miniaturized Drug Conjugate Activity




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Our HSP90-PI3K conjugates are comprised of an HSP90 targeting moiety, cleavable linker, and an anti-

cancer therapeutic payload. Linkers and payloads of the Pentarin platform are designed and optimized

in such a way as to mask the payload’s active site through linker attachment to the payload, rendering

the payload inactive until intracellular cleavage releases the active payload within the tumor cell. The

pharmacophore of the T-2143 is based off of the HSP90 inhibitor, ganetespib. For our proof of concept

experiments the PI3K inhibitor copanlisib was chosen as the model payload. Copanlisib was modified by

converting the terminal morpholine to a piperazine to produce T-2026. This incorporated an amine that

could be used for the purposes of linking. The piperazine analog of copanlisib was shown to be highly

potent across PI3K sub-types, similar to copanlisib (Table 1). The conjugate of this piperazine was

constructed with a carboxyesterase cleavable linker to the HSP90 binding pharmacophore as shown for

the structure of T-2143 (Figure 1a). It was found that linking to the payload in this way diminished

inhibition of PI3K sub-types (Table 1). A non-binding control of T-2143, T-2212, was generated through

blocking the HSP90 binding pharmacophore with a single methyl group addition to one of the resorcinol

hydroxyl groups. The binding affinity to HSP90 of the molecules was determined using a competitive

binding assay, where the competitive molecule was a fluorescently labeled version of the potent HSP90

binding molecule, geldanamycin. The addition of a methyl group proved to be sufficient to completely

block the binding affinity as compared to T-2143 with an IC50 of 84 nM and Kd of 1.4 nM as compared to

no appreciable inhibition of activity by T-2212 up to concentrations of 10 M (Figure 1b). Further, the Kd

of T-2143 is equivalent to ganetespib, with a binding Kd of 0.5 nM. This conservative molecular

modification has very little impact on the overall properties of T-2212 and has been shown to not have

an impact on the mouse pharmacokinetic profile relative to T-2143 (Table 2; Supplemental Figure S1).

PIK3CA mutations occur in about 15% to 30% of breast, endometrial, and colon cancers. A panel of cell

lines harboring common gain of function activating PIK3CA mutations, and reported to be sensitive to

pathway inhibition, were chosen for HSP90-PI3K conjugate testing. The in vitro antiproliferative

activities of T-2143 and T-2212 were tested in comparison to the PI3K inhibitor alone, copanlisib [17-22].

In the cell lines selected, potent cell kill activity similar to copanlisib could only be achieved with T-2143,

not T-2212. Furthermore, in three out of the five cell lines tested fifty percent cell growth inhibition

could not be achieved with T-2212, whereas T-2143 and copanlisib demonstrated a potent effect on cell

viability, showing that HSP90 targeting is essential for delivery of the PI3K inhibitor payload from our

miniaturized drug conjugate (Table 3).

To further investigate the dependence of HSP90 targeting for tumor specific payload delivery of our

HSP90-PI3K conjugate, tumor pharmacokinetic and efficacy studies were carried out in LS174T colon

cancer xenograft-bearing mice comparing T-2143 against T-2212. Initial experiments defined the

maximum tolerated dose (MTD) of T-2143 and T-2212 at 25 mg/kg given once weekly. In support of the

MTD data, the body weights for all efficacy studies are shown in Supplementary Figure S3 (a-c) and

demonstrate tolerability of the compounds at doses up to 25 mg/kg given once weekly. The validity of T-

2212 as an appropriate control was further evaluated through plasma pharmacokinetic studies in mice,

described in more detail below. Briefly, the plasma concentration versus time curves showed a high

degree of similarity for T-2143 and T-2212 and resulted in closely matched half-lives and areas under the

curve (AUC), suggesting that any differences in in vivo activity would not be a result of differences in

plasma pharmacokinetic profiles, but due to differences in HSP90 binding ability (Table 2 Figure S1).

Following a single bolus injection of T-2143 or T-2212, at their maximum tolerated dose (MTD) 25mg/kg,

LS174T tumors and plasma were harvest 24, 48, and 72 hours post dose. Tumors and were analyzed for




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total HSP90-PI3K conjugate levels. In agreement with the in vitro data that suggests the targeting is

important for activity of the drug, T-2143 tumor levels were significantly higher than T-2212 tumors

levels with approximately 6-fold higher concentration at both the 24- and 48-hour time points (Figure

2a). T-2143 and T-2212 were also tested for differences in effects on tumor growth inhibition (TGI). Mice

bearing LS174T or BT474 tumors were treated with T-2143 or T-2212 and evaluated for their effects on

TGI after two weekly MTD doses. In the LS174T model, T-2143 treatment led to a TGI of 87% while T-

2212 treatment resulted in a minimal TGI of 29%, demonstrating a statistically significant increase in

tumor growth inhibition with T-2143 as compared to non-targeted control (P = 0.006) (Figure 2b) Results

in the BT474 model demonstrated similar results with T-2143 treatment leading to a 72% in TGI, while T-

2212 was only able to achieve a TGI of 16% (Figure 2c).Together, these data demonstrate that HSP90

binding is essential for successful tumor delivery of the PI3K inhibitor payload from our HSP90-PI3K

miniaturized drug conjugate.

Tumor Retention of HSP90-PI3K Conjugate Leads to Deep and Durable PI3K Pathway Inhibition

The pharmacokinetic profile of T-2143 was evaluated following administration of a single intravenous

dose in non-tumor bearing mice. T-2143 exhibited a low volume of distribution, a plasma clearance rate

of 2.62 mL/kg/min, and a half-life of 7.4 hours, demonstrating that T-2143 is relatively quickly cleared

from circulation reducing the chances for normal tissue exposure and toxicity (Figure 3). Levels of

released payload, T-2026, from T-2143 were also measured in the plasma. Very minimal amounts of T-

2026 were detectable in the plasma with a Cmax of 0.3 µM while T-2143 was able to achieve a Cmax of 121

µM, demonstrating that while T-2143 is in circulation, minimal amounts of payload are released

decreasing the chances for toxicity from payload exposure. As discussed above, the plasma half-life of T-

2212 was similar to T-2143 and the similarities extended to the other pharmacokinetic parameters

(Table 2). Next, pharmacodynamic experiments were conducted to investigate conjugate accumulation

and release the payload in tumor over time despite being cleared from the plasma.

PIK3CA mutations and amplifications are known to be drivers of AKT pathway activation, with AKT being

central to the activity of the PI3K/AKT/mTOR pathway [23-26]. As a result of AKT’s importance to the

pathway we investigated the ability of T-2143 to effectively target the tumor, be retained, and deliver

the PI3K inhibitor payload over time. Pharmacodynamic analysis was performed in PI3K mutated BT-474

breast cancer xenograft-bearing mice where T-2143 was administered as a single intravenous bolus

injection of T-2143 and tumors were collected at 1, 24, 48, and 72 hours post dose. Tumors were split

then analyzed for T-2143 tumor retention and pAKT(S473) levels over time. T-2143 was rapidly taken up

by the tumor with 1.2 µM measured at the 1-hour time point. The highest tumor levels were measured

24 hours post dose with 2.3 µM of T-2143 measured at this time point. T-2143 was well retained in the

tumor with 0.55 µM still measured 72-hour post dose. Tumor payload levels increased over time with

2.1 µM still detectable at the 72-hour time point (Figure 3b). Tumor accumulation and sustained release

of the payload translated into durable PI3K pathway inhibition. While at the 1-hour time point pAKT

levels only decreased 33% relative to the vehicle control, at 24 hours post dose a 75% reduction was

observed, which was sustained out to 72 hours with an 83% reduction measured at this time point

(Figure 3c). Together these data suggest that although the conjugate is rapidly cleared from the plasma,

T-2143 can effectively target and accumulate in tumors and that accumulation of T-2143 leads to

sustained release of the payload, and PI3K pathway suppression over time.




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In order to inform the schedule of efficacy dosing, more extended pharmacodynamic and

pharmacokinetic experiments were carried out in the LS174T xenograft model. Mice were administered

a single dose of T-2143 or its vehicle control and tumors were collected at 1, 24, 48, 72, and 168 hours

post dose. As in the BT-474 model, T-2143 rapidly accumulated and was retained in LS174T tumors with

0.431 µM of T-2143 and 1.45 µM of the payload detected at the 72-hour time point, which translated

into an 82% reduction in pAKT levels relative to the payload (Figure 4). By the 7-day (168 hour) time

point, only 0.09 µM of T-2143 was detectable in the tumor and the pAKT signal had returned to levels

similar to that of the vehicle, suggesting that T-2143 could be dosed on a once a week schedule to

achieve deep pathway inhibition.

Masking of Payload Active Site Increases Therapeutic Window

Hyperglycemia is a known and potentially dose limiting side effect of PI3K inhibitors due to their

interaction with the insulin-glucose regulatory axis [22]. While this effect is usually transient due to the

compensatory insulin release from the pancreas, it has been shown that this insulin feedback can

reactivate PI3K-mTOR signaling in tumors, compromising the inhibitor’s effectiveness [30]. Our HSP90

miniaturized drug conjugates are designed with the payload binding site blocked by the linker to prevent

binding to the target until the payload is released. Payload activity is blocked until the HSP90 ligand

targets the conjugate to tumor cells and the payload is released through linker cleavage preferentially in

the cancer cell. In addition, HSP90 conjugates are also designed to have short plasma circulation

allowing for rapid accumulation and penetration into the tumor while potentially limiting the overall

exposure to normal tissue. By incorporating tumor specific delivery and masking of the T-2143 payload

while in circulation we hypothesized that with our conjugate we could mitigate an increase in glucose,

resulting in a larger therapeutic window.

In a cell free PIK3CA enzyme assay, the HSP90-PI3K conjugate T-2143 is 116-fold less active than

copanlisib but retains potent cell kill activity demonstrating the ability to mask the payload’s active site

until it is delivered directly to the tumor cells (Table 1). To test this in vivo, mice were treated with a

single intravenous bolus dose of 25 mg/kg T-2143, an equimolar dose of copanlisib to the payload

amount of T-2143 (10.8 mg/kg), or the T-2143 vehicle alone. One-hour post compound administration,

copanlisib treatment led to four-fold increase in blood glucose levels in comparison to the vehicle

control. The average blood glucose level in the vehicle control group was 128 mg/dL while the copanlisib

group increased to 553 mg/dL and the T-2143 group average level stayed at 124 mg/dL (Figure 5). The

effect of T-2143 on blood glucose levels was additionally measured at 4, 6, and 24 hours post dose

administration. No increase in blood glucose levels relative to the vehicle control was detected with T-

2143 administration, demonstrating the ability of the HSP90-PI3K conjugate to reduce target specific

toxicity, hyperglycemia, through payload active site masking, potentially increasing the therapeutic

window (Supplemental 2a).

Superior Tumor Growth Inhibition Observed with HSP90-PI3K Conjugate in Comparison to Copanlisib

Alone.

Antitumor activity of T-2143 was evaluated in comparison to copanlisib alone in mouse xenograft

models of human cancer all reported by ATCC to have mutations in the PIK3CA gene that result in gain

of function activity of the pathway. The three models, LS174T (H1047R), BT-474 (K11N), and NCI-H460

(E545K), were treated with T-2143, copanlisib, ganetespib, the combination of copanlisib and

ganetespib, or a vehicle control. Ganetespib was chosen to represent the effects of the HSP90 targeting




11

moiety alone since it is structurally similar to the ligand used in T-2143 and the molecules have similar

binding affinities as described above [27]. Copanlisib and ganetespib doses were chosen on an

equimolar basis to either the amount of payload or ligand used to create T-2143. All treatments were

dosed on a once per week basis and given as a single bolus intravenous injection. In all three models

tested T-2143 treatment was well tolerated with no significant body weight loss observed

(Supplementary Figure S3 a-c). In the NCI-H460 non-small cell lung cancer xenograft model, T-2143 was

the only treatment able to produce statistically significant tumor growth inhibition as compared to

vehicle with a TGI of 48% (P=0.043). Copanlisib alone was only able to achieve a TGI of 15%, while the

combination of copanlisib achieved a TGI of 8% (Figure 6a). In the BT-474 breast cancer xenograft

model, T-2143 treatment resulted in a TGI of 74%, while copanlisib alone, or the combination of

copanlisib and ganetespib, was only able to achieve a TGI of 22% as compared to vehicle alone.

Furthermore, there was a statistically significant difference between the treatment of T-2143 when

compared to the activity of the combination treatment (P=0.02), demonstrating a superior efficacious

response in this model over the payload or combination treatment (Figure 6b). Results in the LS174T

colon cancer model were similar to that of the BT-474 model, where T-2143 was not only able to

achieve superior anti-tumor activity with a TGI of 87% compared to vehicle, but it was statistically

significant from the treatment of copanlisib alone (TGI 31%, P = 0.009) or the combination treatment

(TGI, 33%, P = 0.01) (Figure 6c). The superior efficacy of T-2143 compared to copanlisib is also

consistent with the ability of T-2143 to inhibit the signaling more potently and more sustainably as

compared to companlisib (Supplemental Figure S4). Together these results demonstrate the ability of T-

2143 to effectively target the tumor and release the payload overtime resulting in superior tumor

growth inhibition in comparison to the payload alone.

Discussion

Lack of tumor-specific delivery of anticancer agents presents a number of challenges to their ability to

be efficacious. Poor tumor specificity can lead to high toxicity and lack of sufficient target engagement,

all resulting in a low therapeutic window. While ADCs have provided a means of site-specific delivery of

anticancer agents, they have traditionally only been used to deliver well characterized cytotoxic agents

to cancer cells and have potential limitations in treating solid tumors [28-29]. In cancer, HSP90 is

expressed in an activated and highly complexed state, and it has been demonstrated that HSP90

inhibitors have a propensity to selectively bind with high affinity to the activated from as compared to

the latent form of HSP90 found in normal tissue rendering HSP90 inhibitors selective for cancer cells [11-

12]. This tumor selective delivery has also been demonstrated for the HSP90 conjugate, PEN-866, which

has potent binding affinity and tumor selectivity, without significantly inhibiting HSP90 activity [15].

Here we have demonstrated a way to leverage preferential HSP90 binding and selectively deliver anti-

cancer payloads beyond cytotoxic agents, through a set of proof-of-concept experiments.

The PI3K/AKT/mTOR signaling pathway is one of the most important intracellular pathways and is

dysregulated in a wide spectrum of human cancers. Although considered a master regulator of cancer,

only five pathway inhibitors, temsirolimus (mTOR), everolimus (mTOR), idelalisib (PI3K), alpelisib (PI3K),

and copanlisib (PI3K) have been approved to date for clinical use with the majority of approvals in liquid

tumors. Based on the clear need for novel approaches to achieving deep PI3K-AKT-mTOR pathway

inhibition while decreasing toxicity in the solid tumor setting, we utilized the Pentarin platform to

generate a HSP90 conjugate containing an inhibitor of the PI3K-AKT-mTOR pathway, T-2143. Given the




12

FDA approval status and pre-clinical activity of copanlisib, this drug was chosen as a model payload to

demonstrate our ability to selectively deliver payloads from this class directly to tumor cells [19].

T-2143 was designed to circumvent the limitations of currently available PI3K inhibitors. The

pharmacokinetic and pharmacodynamic profile of T-2143 demonstrated the ability to selectively target

and rapidly penetrate into solid tumors, have a significant effect on pathway inhibition, and mitigate a

known dose limiting toxicity of PI3K inhibitors that inhibit the alpha PI3K isoform and suggests the

potential to mitigate toxicities of drugs that inhibit this isoform [30]. The short plasma circulation time of

T-2143 limits overall exposure, however, tumor specific delivery of T-2143 was not compromised. As

shown in the experiments presented here, T-2143 was able to rapidly accumulate in tumors and achieve

deep PI3K/AKT/mTOR pathway inhibition as evidenced by the inhibition of phospho-AKT. Due to the

sustained release of the payload over time, T-2143 is able to achieve deeper pathway inhibition in

comparison to copanlisib alone which has been demonstrated to inhibit the pathway early but not in a

sustainable fashion as evidenced by the signal rebounding over time [19].

Here, we demonstrated that through optimized linker chemistry, the copanlisib active site of T-2143 was

masked while the conjugate remains in circulation, with selective cleavage and release of the PI3K

payload occurring within the tumor. Hyperglycemia is a known and potentially dose limiting side effect

of PI3K inhibitors due to their interaction with the insulin-glucose regulatory axis [22]. The masking of

the payload in T-2143 allowed for the mitigation of a spike in blood glucose in contrast to copanlisib

which led to a significant increase in blood glucose levels. Together, these data demonstrate that as a

result of the thoughtful design of T-2143, we were able to achieve deep tumor penetration, sustainable

pathway inhibition, and minimize toxic side effects seen with other alpha targeting- pathway inhibitors

thereby increasing the payloads therapeutic window.

The data presented here also demonstrated that the targeting moiety of T-2143 is necessary for tumor

accumulation. The HSP90 binding ability of T-2143 leads to accumulation of the payload in vivo, when

compared to the non-binding control, demonstrating that the HSP90 binding capacity of T-2143 is

necessary for intratumoral accumulation of the payload. Further evidence was provided by

demonstrating that not only was the HSP90 binding necessary for tumor accumulation, but that it was

sufficient to deliver the PI3K payload and achieve deep pathway inhibition over time. This was validated

by persistent drug activity and suppression of PI3K/AKT/mTOR pathway, which translated into

remarkable effects on tumor growth inhibition in comparison to copanlisib alone. In all models tested T-

2143 was able to achieve tumor growth inhibition greater than copanlisib alone, demonstrating that site

directed delivery and sustained release of the payload translates into greater activity in vivo.

Furthermore, each of the three models tested are reported to have a different PIK3CA mutational

status, showing that T-2143 can be effective regardless of the PIK3CA mutational background. Tumor

growth inhibition achieved with T-2143 was also superior to treatment with a combination of

ganetespib and copanlisib, providing evidence that our improved anti-tumor activity is unlikely a result

of the dosing of both of the payload and HSP90 targeting moiety but rather a direct result of sustained

intratumoral release of the payload from HSP90-PI3K conjugate accumulation. Together the data

demonstrates that our miniature drug conjugates are designed to leverage the preferential expression

of HSP90 in cancer, deliver payloads directly to tumors, and achieve deep pathway inhibition overtime

greater than the payload alone.




13

In summary, we have presented proof-of-concept experiments demonstrating a unique way of

leveraging the selective over-expression and activation of HSP90 in solid tumors to deliver payloads,

beyond traditional cytotoxic agents used in other tumor selective delivery systems. Using this platform,

we have the potential to target payloads to selectively accumulate to solid tumors and destroy the

tumor cells while minimizing exposure of healthy tissue to the payloads, thereby increasing the payloads

therapeutic window.

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Table 1: Activity of T-2143, Copanlisib, and T-2026 in Cell Free Enzymatic Assay

Isoform

Inhibition of PI3K IC50 (nM)

T-2143

Copanlisib

T-2026

PIK3CA 81 0.7 0.3

PIK3CB 666 13 9.6

PIK3CD 167 0.5 1.1

PIK3CG 337 4.7 2.2

Table 1: Conjugation of Payload to HSP90 Targeting Moiety Results in Masking of the

Payload’s Active Site. The ability of T-2143 and copanlisib to inhibit four isoforms of PIK3CA

were evaluated in a cell free enzymatic assay.




16

Table 2: T-2143 vs T-2212 Mouse Plasma Pharmacokinetic Profile

Parameter

Unit

T-2143

T-2212

Dose mg/kg 25 25

t1/2 hour 7.4 6.1

Cmax μM 121 122

AUC24h μM*h 133 114

Vz mL/kg 1691 1663

Vss mL/kg 1282 1270

CL mL/kg/min 2.6 3.2

Table 2: T-2143 and T-2212 Exhibit Similar Plasma Pharmacokinetic Profiles in Mice.

Mice were treated with a single IV bolus of 25 mg/kg of T-2143 or T-2212. Mice were sacrificed

and plasma collected at 0.83, 0.5, 2, 4, and 24 hours post dose. Plasma samples were analyzed

for T-2143 or T-2212 levels. T1/2 is elimination half-life, Cmax is maximum plasma drug

concentration, AUC24 is area under the plasma concentration curve from the time zero to time t,

VZ is volume of distribution during terminal phase, Vss is volume of distribution at steady state,

CL is total body clearance of drug from plasma.

Table 3: T-2143 Inhibits in Vitro Cellular Proliferation

Cell line

Inhibition of Proliferation IC50(nM)

T-2143

T-2212

Copanlisib

NCI-H460 10 90 39

LS174T 26 NA 109

HCT-116 27 NA 799

BT-474 44 NA 155

SKOV3 11 48 21

Table 3: T-2143 Inhibits In Vitro Cellular Proliferation Similar to Copanlisib, while the

non-binding control, T-2212, does not. Cells were treated with T-2143, T-2212, or copanlisib

for 72 hours and then measured for proliferation (N=3). *NA=50% inhibition not achieved at

highest concentration tested of 10µM.




17

Figure 1: Chemical Structure and binding affinity of the HSP90-PI3K conjugate T-2143

and its non-HSP90 binding control T-2212

(a) T-2143 was constructed by linking the piperazine analog of copanlisib with a carboxyesterase

cleavable linker to the HSP90 binding pharmacophore. T-2212, the non-binding control of T-

2143, the HSP90 binding pharmacophore was blocked with a single methyl group addition to

one of the resorcinol hydroxyl groups to produce T-2212. T-2143 synthesis route summarized in

supplemental data.(b) The binding affinity of T-2143, T-2212 or positive control compound,

Geldanamycin to HSP90 N-terminal protein was determined using an in vitro competitive

binding assay competition of fluorescently labeled geldanamycin for binding to purified

recombinant HSP90α.

Figure 2: T-2143, but not T-2212, is retained in the PI3K mutant Colon Xenograft Model

LS174T, and Translates into Significant Anti-Tumor Activity

(a) Mice were treated with a single IV bolus of 25 mg/kg of T-2143 or T-2212. Mice were

sacrificed and tumors collected at 24, 48, or 72 hours post dose. Tumors were analyzed for T-

2143 or T-2212 levels. Error bars represent SEM (N=3/ time point). (b) Animals bearing

LS174T xenograft tumors were given T-2143(P < 0.0001) and the negative control, T-2212 (not

significant) on Day 1 and Day 7. Error bars represent SEM and statistical analysis done by one-

way ANOVA (n = 10 per group).

Figure 3: T-2143 is Cleared from Circulation and with minimal payload release, while

Tumor Accumulation and Sustained Payload Release Results in Deep PI3K Pathway

Inhibition in the PI3K mutant Breast Cancer Xenograft Model, BT-474

(a) Non-tumor bearing mice were treated with a single IV bolus of 25 mg/kg of T-2143. Mice

were sacrificed and plasma collected at 0.83, 0.5, 2, 4, and 24 hours post dose. Plasma was

analyzed for T-2143 levels and amount of released payload, T-2026. Error bars represent SEM

(N=3/ time point). (b) Mice bearing BT-474 tumors were administered a single 25mg/kg dose of

T-2143 or its Vehicle control. Tumors were collected at 1, 24, 48, and 72 hours post dose, and

analyzed for T-2143 levels and released payload levels. Error bars represent SEM (N=3/ time

point) (c) Tumors were also analyzed for effects of T-2143 on pAKT (S473) levels. Error bars

represent SEM (N=3/ time point).

Figure 4: T-2143 Tumor Accumulation and Sustained Payload Release Allows for Once a

Week Dosing Schedule

(a) Mice bearing LS174T tumors were administered a single IV dose of a vehicle control or

25mg/kg of T-2143. Tumors were collected at 1, 24, 48, 72, and 168 hours post dose, and

analyzed for T-2143 levels and released payload levels overtime. Error bars represent SEM (N =

5/time point) (b) Tumors were also analyzed for effects of T-2143 on pAKT (S473) levels. Error

bars represent SEM (N=5/ time point).




18

Figure 5: Masking of Payload’s Active Site Mitigates Known Dose Limiting Toxicity

Observed with PI3K Inhibitors.

Non-tumor bearing mice were administered a single IV dose of 25 mg/kg T-2143 or a 10.8

mg/kg copanlisib. One-hour post dose blood was collected, and glucose levels measured using a

hand-held glucometer. Error bars represent SEM (N=5/group). The payload for T-2143, T-2026,

and was shown to have a similar effect to copanlisib(data in supplemental S2a). T-2143 was also

tested at 4, 6, and 24 hours post dose (data in supplemental S2b).

Figure 6: Superior Tumor Growth Inhibition Observed with T-2143 in Comparison to

Copanlisib Alone

Mice bearing either NCI-H460, BT-474, or LS174T tumors were treated with vehicle control, 25

mg/kg T-2143, 10.8 mg/kg copanlisib, 8.3 mg/kg ganetespib, or the combination of copanlisib

and ganetespib. Compounds were administered on Day 0 and Day 7. All statistics were

calculated using one-way ANOVA (a) In the NCI-H460 model T-2143 TGI was statistically

significant from the vehicle control (P=0.04) all other treatments were not statistically

significant. (b) In the BT-474 model T-2143 treatment was statistically different from that of

copanlisib (P = 0.02) and the combination treatment (P = 0.02). (c) In LS174T model T-2143

treatment was statistically different from that of copanlisib (P=0.009) and the combination

treatment (P = 0.01). Error bars represent SEM (n = 10 per group). BW data are summarized in

Supplementary Figures S3a-c.




Figure 1a:

-3 -2 -1 0 1 2

0

50

100

150

Concentration {log(M)}

Perc

en

t A

cti

vit

y

Geldanamycin

T-2143

T-2212

Figure 1b:




Figure 2a:

0 5 1 0

0

1 0 0 0

2 0 0 0

D a y

Av

era

ge

Tu

mo

r V

olu

me

(mm

3)

V e h ic le C o n tro l

T -2 1 4 3

T -2 2 1 2

Figure 2b:

0 5 10 15

0

500

1000

1500

2000

2500

Day

Avera

ge T

um

or

Vo

lum

e

(mm

3)

Vehicle Control

T-2143 (25mpk)

T-2212 (25mpk)

Figure 2c:

24 48 72 24 48 72 24 48 72

0

1

2

3

4

Time Post Dose (hours)

Co

nce

ntr

ati

on

(

M) T-2143

T-2212

Copanlisib




Figure 3a: Figure 3b:

Figure 3c:




Figure 5:





Figure 6c:




Published OnlineFirst June 4, 2020.Mol Cancer Ther Samantha Perino, Benoit Moreau, Jessica Freda, et al. Challenges to Solid TumorsTumor Accumulation to Overcome PI3K Inhibitor Delivery Novel Miniaturized Drug Conjugate Leverages HSP90 Driven

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