Produced by Cell-Free-Protein Synthesis · 1 Targeted Drug Delivery with an Integrin-Binding Knottin-Fc-MMAF Conjugate Produced by Cell-Free-Protein Synthesis Nicolas V. Currier1,*,

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Targeted Drug Delivery with an Integrin-Binding Knottin-Fc-MMAF Conjugate Produced by Cell-Free-Protein Synthesis

Nicolas V. Currier1,*, Shelley E. Ackerman2,*, James R. Kintzing2, Rishard Chen3, Maria

Filsinger Interrante2, Alexander Steiner3, Aaron K. Sato3 and Jennifer R. Cochran2,4

1Divsion of Pediatric Hematology/Oncology, Stanford Medical School, 2Department of

Bioengineering, Stanford University, 3 Sutro Biopharma, Inc., South San Francisco, CA, 4Department of Chemical Engineering, Stanford University

*These authors contributed equally To whom correspondence should be addressed: [email protected] Shriram Center for Bioengineering and Chemical Engineering 443 Via Ortega, Room 356 Stanford, CA 94305-4125 Phone: (650) 724-7808 Fax: N/A Running title: Knottin-Fc-Drug Conjugate as an Anti-Tumor Agent Potential conflicts of interest Aaron K. Sato, Alexander Steiner and Rishard Chen are employees of Sutro Biopharma Inc. Nicolas V Currier is now an employee of Biogen Inc. Jennifer R Cochran is listed as a co-inventor on intellectual property owned by Stanford University related to this work. Financials This work was funded in part by the Stanford Bio-X Interdisciplinary Initiatives Program, and seed funding from the Stanford Cancer Institute (all authors). The authors wish to acknowledge the following fellowship support: Anne T. and Robert M. Bass Endowed Fellowship in Pediatric Cancer and Blood Diseases (N.V. Currier), Stanford Bio-X Bowes Fellowship (S.E. Ackerman), NSF Graduate Fellowship (J.R. Kintzing), and the Stanford Bioengineering REU program (M.F. Interrante).

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ABSTRACT Antibody-drug conjugates (ADCs) have generated significant interest as targeted therapeutics for cancer treatment, demonstrating improved clinical efficacy and safety compared to systemic chemotherapy. To extend this concept to other tumor targeting proteins, we conjugated the tubulin inhibitor monomethyl-auristatin-F (MMAF) to 2.5F-Fc, a fusion protein composed of a human Fc domain and a cystine knot (knottin) miniprotein engineered to bind with high affinity to tumor-associated integrin receptors. The broad expression of integrins (including αvβ3, αvβ5, and α5β1) on tumor cells and their vasculature makes 2.5F-Fc an attractive tumor targeting protein for drug delivery. We show that 2.5F-Fc can be expressed by cell-free protein synthesis, during which a non-natural amino acid is introduced into the Fc domain and subsequently used for site-specific conjugation of MMAF through a non-cleavable linker. The resulting knottin-Fc-drug conjugate (KFDC), termed 2.5F-Fc-MMAF, had approximately 2 drugs attached per KFDC. 2.5F-Fc-MMAF inhibited proliferation in human glioblastoma (U87MG), ovarian (A2780), and breast (MB-468) cancer cells to a greater extent than 2.5F-Fc or MMAF alone or added in combination. As a single agent, 2.5F-Fc-MMAF was effective at inducing regression and prolonged survival in U87MG tumor xenograft models when administered at 10 mg/kg two times per week. In comparison, tumors treated with 2.5F-Fc or MMAF were non-responsive, and treatment with a non-targeted control, CTRL-Fc-MMAF, showed a modest, but not significant therapeutic effect. These studies provide proof-of-concept for further development of KFDCs as alternatives to ADCs for tumor targeting and drug delivery applications.




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INTRODUCTION Antibody-drug conjugates (ADC) have risen to the forefront of cancer therapy in

recent years due to their ability to combine the tumor targeting selectivity of monoclonal

antibodies with the delivery of potent cytotoxic agents (1). This excitement has been led

by recent FDA-approvals of ADCs including Adcetris (Seattle Genetics) and Kadcyla

(Immunogen/Roche) (2). Adcetris (brentuximab vedotin), which targets the cell surface

protein CD30, is approved for relapsed or post-stem cell transplant Hodgkin lymphoma

and systemic anaplastic large cell lymphoma (3). Kadcyla (ado-trastuzumab emtansine)

targets the HER2 receptor, which is present on 20-30% of breast tumors (4). Numerous

other ADCs, composed of a variety of tumor targeting mAbs and cytotoxic payloads, are

currently under development and are progressing forward in clinical trials (2). To further

improve their therapeutic properties, development efforts have been focused on

optimization of the drug warhead or the linker moiety that connects the drug to the

antibody (5, 6). Less attention has been paid to exploration of the tumor-targeting agent

aside from utilizing mAbs against different cell surface receptors.

We previously created EETI 2.5F (7), a unique engineered integrin-targeting agent

based on a cystine knot miniprotein derived from the Ecballium elaterium trypsin inhibitor

II (EETI) (8). Cystine-knot miniproteins, (also known as knottins), are composed of 30-50

amino acid residues interconnected through a core of at least three disulfide bonds that

confer high thermal, chemical and proteolytic stability (9, 10). We showed that compared

to other integrin-targeting agents, 2.5F binds with high affinity to α5β1 integrin and

integrins containing an αv subunit (e.g. αvβ3 and αvβ5) (7). As a molecular imaging agent,

radiolabeled or dye-labeled 2.5F generated robust tumor-specific signals with minimal off-

target binding in mouse tumor models (11-13). Similar results were observed with a

version of 2.5F fused to a murine antibody Fc domain (11), suggesting that a knottin-Fc

fusion might be well suited as a “pseudo-antibody” for integrin-mediated drug delivery to

tumors.

Here, we describe knottin-Fc drug conjugates (KFDCs) as an alternative to ADCs.

In proof-of-concept studies, the integrin-binding peptide 2.5F was fused to a human Fc

domain and tested as a vehicle for chemotherapeutic drug conjugation and targeted

delivery to tumors. Compared to a small peptide, 2.5F-Fc offers the potential benefits of




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bivalent antigen binding, extended serum half-life due to larger size and FcRn-mediated

recycling, and the ability to leverage conjugation strategies developed for antibody drug

conjugates. This work spans several facets of KFDC development and characterization,

including: 1) production of functional knottin-Fc fusions using cell-free protein synthesis,

2) introduction of a non-natural amino acid containing an azido functional group into the

knottin-Fc fusions, 3) site-specific conjugation of a model cytotoxic drug (MMAF) and

non-cleavable linker to this azido group using copper-free click chemistry, and 4)

characterization of the resulting knottin-Fc-drug fusions in tumor cell culture and animal

models compared to control compounds.

MATERIALS AND METHODS Protein constructs and cell lines EETI 2.5F (GCPRPRGDNPPLTCSQDSDCLAGCVCGPNGFCG) or non-integrin

binding control sequence CTRL (GCVTGRDGSPASSCSQDSDCLAGCVCGPNGFCG)

(7) were fused to a standard human IgG1 Fc sequence with a single amino acid

substitution that was replaced with an amber codon at the F404 position. The sequences

were codon-optimized for production in E. coli (DNA 2.0, Inc.) and cloned into a plasmid

to be used in a cell free protein synthesis reaction. U87MG and MB-468 cell lines were

obtained from American Type Culture Collection (ATCC), and A2780 cells were obtained

from Sigma-Aldrich. All cell lines were obtained from cell banks in 2015 (except A2780

cells, obtained in 2014), within 6 months of experimentation. Cell lines obtained from

ATCC were tested by the manufacturer for sterility (aerobic and anaerobic cultures),

human pathogens (HIV, HepB, HPV, EBV, mycoplasma and CMV) and authenticity

(cytochrome C oxidase I (COI) and short tandem repeat (STR) analysis) as per

manufacturer’s certificate of analysis. A2780 cells, obtained from Sigma-Aldrich, were

tested by the Culture Collections division of Public Health England for sterility (aerobic

and anaerobic cultures), human pathogens (mycoplasma) and authenticity (mitochondrial

DNA sequencing and STR analysis). All media components were purchased from Life

Technologies except where indicated. U87MG cells were grown in Dulbecco’s Modified

Eagle Medium (DMEM) (cat# 11995-073) with 10% fetal bovine serum (FBS) (cat#

26140079) and 1% penicillin/streptomycin (cat# 15140-122). MB-468 cells were grown in




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DMEM and Ham’s F-12 medium (DMEM/F12) (Gibco/Thermo Fisher; cat# 11320-033)

with 10% FBS and 1% penicillin/streptomycin. A2780 cells were grown in Roswell Park

Memorial Institute medium (RPMI) 1640 + L-glutamine (cat# 11875-093), with 10% FBS

and 1% penicillin/streptomycin.

Cell free protein synthesis and purification Knottin-Fc fusion constructs were produced as previously described using cell free protein

synthesis (14) with modifications described below. E. coli strain SBDG224 was grown in

a continuous fermentation batch and converted into extract. The extract was treated with

75 µM iodoacetamide for 30 min at RT (20°C). Final concentrations of components in the

reaction mixture were 30% extract, 2 mM para-azidomethyl-L-phenylalanine (pAMF, a

non-natural amino acid), 0.5 µM pAMFRS (an aminoacyl tRNA synthetase), 2 mM GSSG,

2 mM amino acids (except 0.5 mM for tyrosine and phenylalanine), and 10 µg/mL

plasmid. The reaction was incubated in a bioreactor for 16-18 hours at 30°C with 30% air

saturation, 0.2 vvm sparged air flow, and no pH control. The protein purification process

consisted of three major steps: clarification, chromatography and formulation. In the

clarification step, the harvest was centrifuged and filtered through a 0.45 µm disposable

filter unit. Then, the desired protein was captured using GE Healthcare’s MabSelect SuRe

resin, which functions like Protein A medium. A step gradient with 0.1 M citric acid at pH

3.5 was used to elute the desired protein. Subsequently, the eluate was concentrated and

dialyzed into HyClone 1X PBS (Fisher Scientific). SDS-PAGE on purified proteins was

carried out on NuPAGE Novex 4-12% Bis-Tris gels (ThermoFisher Scientific) with 1x

MES buffer. To reduce samples, a final concentration of 1mM dithiothreitol (DTT) was

used. Size exclusion chromatography (SEC) was performed on a Sepax Zenix-C SEC-300

column (Sepax Technologies). The SEC buffer was 100 mM potassium phosphate,

150mM sodium chloride, pH 6.5. Protein synthesis yields were approximately 300 mg/L

from the cell free reaction, with about 100 mg/L recovery after purification. Note that all

Fc fusion proteins used in this work contain the pAMF non-natural amino acid at position

404, based on Eu numbering of the Fc domain (15).

MMAF conjugation and characterization




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Purified knottin-Fc variants containing pAMF were conjugated to the cytotoxic agent

MMAF through copper free click chemistry, using a constrained cyclooctyne reagent. In

brief, dibenzocyclooctyl (DBCO)-PEG4-MMAF (ACME Bioscience; Palo Alto, CA)

(Figure 1D) was dissolved in DMSO to a final concentration of 5 mM. The compound

was added to the purified protein sample in PBS buffer at a drug to protein molar ratio of

6 to 1. The mixture was incubated at room temperature (20°C) for 17 hr. Excess free

drug was removed by Zeba plates (ThermoFisher Scientific) equilibrated in PBS.

To determine drug loading, 1 mg/mL of knottin-Fc-MMAF in PBS was diluted 1:4 (v:v)

into 7.2 M Guanidine-HCl, 0.3 M sodium acetate, pH 5.3, and reduced by adding TCEP

(Thermo Scientific, Waltham, MA) at final concentration of 10 mM. The mixture was

incubated at 37°C for 15 min in an Eppendorf Thermomix R while shaking at 300 rpm. A

total of 2 µg of sample was injected into an Agilent ZORBAX C8 column (1.8 µm, 300 Å,

100x2.1 mm from Agilent, Palo Alto, CA), connected to an U-HPLC system containing a

binary gradient pump, temperature-controlled column compartment, autosampler and a

diode array detector. The RP-UHPLC assay was performed at 0.5 mL/min at 80°C using

0.1% trifluoroacetic acid (TFA) in water (mobile phase A, MPA) and 0.1% TFA in

acetonitrile (ACN) (mobile phase B, MPB), and monitored at absorbance of 280 nm. The

13-min method consists of a 3 min isocratic hold at 30% MPB, a linear gradient with a

4%/min increment from 30% to 50%, a 1-min wash using 95% MPB, and a 3-min re-

equilibration at 30% MPB. Shoulders in the HPLC peaks are minor impurities or

conformations. Drug protein ratio (DPR) was determined using the following equation and

was found to be ~1.9 for 2.5F-Fc-MMAF and CTRL-Fc-MMAF:

= 2 ×

Flow cytometric analysis of cell surface integrin expression Cells were detached from culture plates using cell dissociation buffer (phosphate buffered

saline (PBS: 1.0581 mM KH2PO4, 154.0041 mM NaCl, and 5.6002 mM Na2HPO4)

containing 0.05% EDTA, Invitrogen). Detached cells were subsequently washed with

PBS. 4x104 cells per staining reaction were re-suspended in 50 µl of PBS/BSA (0.1%

bovine serum albumin in PBS) containing a 1:100 dilution of AlexaFluor-488 anti-αVβ3




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integrin (R&D Systems), 1:100 dilution of AlexaFluor-488 anti-αVβ5 integrin (Millipore),

1:25 dilution of FITC anti-α5 integrin (BioLegend), or 1:200 dilution of goat anti-mouse

IgG-FITC (Life Technologies) as a control. Cells were incubated on ice with primary

antibodies for 75 min. Cells were pelleted and then washed with 1 mL of PBS/BSA. To

enhance the signal and increase the dynamic range of the assay, staining reactions

containing anti-αVβ5 integrin were then incubated on ice with a 1:200 dilution goat anti-

mouse IgG-FITC secondary antibody for 75 min and washed again with PBS/BSA. All

samples were analyzed using flow cytometry (Guava EasyCyte 8HT flow cytometer,

EMD Millipore) and the resulting data were evaluated using FlowJo software (TreeStar,

Inc.). Relative fluorescence for each sample was measured as a ratio of sample signal/

mean secondary only control signal. Error bars represent the standard deviation of

experiments performed in triplicate.

To quantify the integrin expression in terms of receptors/cell, the Quantum Simply

Cellular (QSC) Kit anti-Mouse IgG (Bangs Laboratories, Inc.) was used. Beads were

incubated with the same concentration of antibody as used on each cell line for 30 min at

room temperature. For αVβ5 integrin analysis, beads were washed with 1 mL PBS/BSA

following primary incubation, then incubated for 75 minutes at room temperature in

secondary antibody. Cells were then pelleted and washed with 1 mL PBS/BSA 3 times.

Beads were analyzed on the same day, using the same cytometer and settings as the cells.

Software provided with the kit was used to create a regression between fluorescence signal

and antibodies/cell that enabled quantification of the fluorescence signal from each of the

cell lines as # receptors/cell (Supplementary Figure 1). As a secondary antibody was used

to boost signal for αVβ5 integrin, the reported receptors/cell for that integrin are semi-

quantitative, as 1:1 binding of secondary to primary antibody is not definitive.

Competition cell binding assays To measure the relative binding affinities of knottin-Fc fusion proteins, competition

binding assays were performed as previously described with some modifications (16).

AlexaFluor 488 conjugated EETI 2.5F (AF488-2.5F) was used as a competitor to compare

the relative binding of 2.5F-Fc, 2.5F-Fc-MMAF, and CTRL-Fc-MMAF. Briefly, 4x104

cells were detached with cell dissociation buffer, washed with IBB (25 mM Tris pH 7.4,




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150 mM NaCl, 2 mM CaCl2, 1 mM MgCl2, 1 mM MnCl2, and 0.1% bovine serum

albumin), and incubated with 2 nM AF488-2.5F and varying concentrations of knottin-Fc

fusions in 1 mL of IBB at 4 °C for 2 hrs. The cell-bound fluorescence remaining after

washing twice with 1 ml of PBS/BSA was determined by flow cytometry, as described

above. Adjusted fluorescence values were calculated as the geometric mean of the

fluorescence signal for the negative control (cells only) subtracted from the geometric

mean of fluorescence signal of the sample. From this, the percent bound was calculated as

the adjusted fluorescence of each sample divided by the adjusted fluorescence of the

positive control for each cell line x 100. Half-maximal inhibitory concentration (IC50)

values were determined by nonlinear regression analysis using Prism (GraphPad). Error

bars represent the standard deviation of experiments performed in triplicate.

Cell Proliferation Cells were seeded in a 96-well plate at a density of 2,000 cells per well and grown

overnight at 37 °C, 5% CO2 in the media described for each cell line above. Cells were

subsequently treated with 100 μL of fresh media, containing varying concentrations of

knottin-Fc fusion proteins or linker-modified MMAF, and incubated for 5 d at 37 °C, 5%

CO2. Cell proliferation was measured using the Cell Counting Kit-8 (CCK-8; Dojindo), by

adding the water-soluble tetrazolium salt, WST-8, to each well in an amount equal to 10%

of the culture volume. After incubation for 1 h at 37 °C, absorbance at 450 nm was

measured with a Synergy H4 microtiter plate reader (BioTek Instruments). Cell viability

was expressed as a percentage of absorbance relative to the control of untreated cells. We

processed the data for these experiments by first subtracting a background value from each

well based on absorbance of CCK-8 + media (no cells). Cell viability is then expressed as

(sample – background) / (control – background) x 100. Error bars represent the standard

deviation of experiments performed in triplicate.

Animal experiments Animal procedures were carried out according to a protocol approved by the Stanford

University Administrative Panels on Laboratory Animal Care (APLAC #22942). For

tumor cell implantation, 6 week-old female Nu/Nu mice (Charles River Laboratory) were




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anesthetized with 2.5% isoflurane by inhalation with a flow rate of 1L/min. A volume of

100 µL of 50/50 PBS/matrigel (Corning #356231), containing 5x106 U87MG cells, was

injected subcutaneously into the flank. Tumors were allowed to grow for 6 days until

reaching a size of approximately 30-50 mm2 in tumor area. At day 6 post-inoculation, all

mice were weighed and tumors measured. Mice were binned into experimental groups to

ensure equivalent average tumor size and average weight across each group. All

compounds tested were administered via intraperitoneal injection in 100 µL PBS, with

dosing frequency and concentration dependent on therapeutic group as indicated. Tumors

were measured three times weekly using digital calipers and animal weight was recorded

on each dosing day to monitor mice for weight loss as a measure of compound toxicity.

Tumor area was calculated using Area = X * Y, where X is the longest axis of the tumor

and Y is the axis perpendicular. Euthanasia criteria were defined as 20% body weight loss

or tumor size greater than 100 mm2 in area.

Statistical Analysis Survival results were plotted using the Kaplan-Meier method. A log-rank (Mantel-Cox)

test was conducted to compare the survival curve of each treatment group to that of the

untreated control. The resulting p-values were adjusted by the Bonferroni method to

control the false positive rate. Tumor size across groups on Day 15 were analyzed using a

one way ANOVA. Each treatment group was compared to the untreated control using the

Dunnett method. Statistical analyses were performed using GraphPad Prism (GraphPad

Software, Inc).

RESULTS

KFDCs produced by cell-free protein synthesis and conjugation of MMAF through a non-natural amino acid

EETI 2.5F was genetically fused to a human IgG1 Fc domain to create a construct

termed 2.5F-Fc, which was produced by cell-free protein synthesis using an E. coli based

extract (14). A non-natural amino acid was selectively incorporated into the Fc domain by

replacing position F404 with an amber stop codon that allowed incorporation of para-

azidomethyl-L-phenylalanine (pAMF) using a modified aminoacyl tRNA synthetase




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(14)(Figure 1A). 2.5F-Fc proteins were expressed at high levels using cell-free protein

synthesis (typical reaction yields ~300 mg/ml), and following purification were analyzed

by SDS-PAGE and gel filtration chromatography (Figure 1B, C).

To create a KFDC, the pAMF azido functional group in the Fc domain was reacted

with a MMAF derivative containing a dibenzocyclooctyl (DBCO) group using strain-

promoted cycloaddition copper-free click chemistry (Figure 1D). Copper-free click

chemistry was chosen to minimize oxidation of the protein by reactive oxygen species and

to prevent potential cytotoxicity from residual copper in the sample. A flexible PEG linker

was used to bridge the MMAF molecule and the 2.5F-Fc fusion. This non-cleavable linker

minimizes off-target bystander effects by delivering intact protein-drug conjugate to cells

for receptor-mediated internalization and processing, and thus allowed us to demonstrate

the ability of the integrin-targeting 2.5F-Fc to deliver MMAF to tumor cells without

potential complications of extracellular drug cleavage. Reversed-phase HPLC (RP-HPLC)

analysis confirmed a drug-to-protein ratio of approximately 2, as expected based on the

incorporation of one unnatural amino acid per Fc chain (Figure 1E). A knottin-Fc-MMAF

conjugate containing a scrambled integrin binding sequence (7, 13) (CTRL-Fc-MMAF)

was prepared as a negative control and also possessed a drug-to-protein ratio of

approximately 2.

Tumor cell lines express variable levels of cell surface integrin receptors Three biologically diverse tumor cell lines, glioblastoma (U87MG), breast (MB-

468), and ovarian (A2780) carcinomas, were tested for the presence of cell surface

integrin receptors αvβ3, αvβ5, and α5β1 by antibody staining and detection using flow

cytometry. High levels of αvβ3 integrin were present on U87MG cells, whereas MB-468

and A2780 cell lines expressed only minimal levels of this integrin (Figure 2A). In

comparison, high levels of αvβ5 integrin were found on MB-468 cells, with lower levels

of this integrin present on both U87MG and A2780 cells (Figure 2B). U87MG cells

expressed the highest levels of the α5 integrin subunit, followed by lower levels of

expression on A2780 cells, and minimal expression on MB-468 cells (Figure 2C). The α5

integrin subunit is only known to pair with the β1 subunit (17), thus measurement of α5 is

indicative of α5β1 heterodimer expression. The variable expression of integrin receptors




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observed across these cell lines in our studies and others (18, 19), highlights a potential

benefit of the broad integrin-binding specificity of EETI 2.5F for tumor targeting

applications.

2.5F-Fc-MMAF binds with low nM affinity to human tumor cells

Competition binding assays were performed to compare the relative binding affinities

of 2.5F-Fc, 2.5F-Fc-MMAF, and CTRL-Fc-MMAF to U87MG cells. Cells were incubated

with varying concentrations of knottin-Fc proteins and a constant amount of AF488-labeled

2.5F peptide competitor at 4 °C to prevent internalization. Fluorescent binding signals were

measured using flow cytometry, and half-maximal inhibitory concentration (IC50) values were

determined by nonlinear regression analysis. 2.5F-Fc and 2.5F-Fc-MMAF had similar IC50

values (6.9 ± 1.1 nM versus 8.3 ± 1.3 nM, respectively) indicating that MMAF conjugation

has negligible impact on integrin-binding affinity (Figure 3A). CTRL-Fc-MMAF did not

compete AF488-2.5F binding to U87MG cells at concentrations up to 200 nM, demonstrating

lack of measurable integrin binding from this negative control protein (Supplementary Figure

2). The relative binding affinity of 2.5F-Fc-MMAF to A2780 and MB-468 cell lines was also

measured, with IC50 values of 1.1 ± 1.2 nM and 1.2 ± 1.2 nM, respectively (Figures 3B,C).

2.5F-Fc-MMAF inhibits proliferation of human tumor cells

Proliferation of U87MG, A2780, and MB-468 cell lines was assayed after treatment

with 2.5F-Fc-MMAF, and compared with the effects of 2.5F-Fc or linker-modified

MMAF added separately, or in combination (Figure 4). In all cell lines tested, 2.5F-Fc

treatment alone exhibits moderate inhibition of cell proliferation at the highest

concentrations tested (up to 1 µM), as measured by cell dehydrogenase activity, which

produces a formazan dye that can be detected by spectrophotometry. The cytotoxicity of

2.5F-Fc at high concentrations, particularly in A2780 cells, and to a lesser degree MB468

cells, is likely due to the ability of integrin-targeting molecules to disrupt cell adhesion.

Similar results were seen when tumor cells were treated with high concentrations of 2.5F

peptide in a previous study, in contrast with U87MG cells which are more resistant to

detachment-induced apoptosis (16). Linker-modified MMAF treatment alone inhibits cell

proliferation to some degree; a similar level of inhibition was observed upon co-




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administration of 2.5F-Fc plus linked-modified MMAF, indicating the lack of synergistic

effects from combination treatment. Similar trends were observed across all three cell

lines tested. The highest level of inhibition of cell proliferation occurred upon treatment

with 2.5F-Fc-MMAF, with the greatest effects observed in U87MG cells, potentially due

to the high integrin expression levels on these cells compared to A2780 and MB468 cells.

IC50 values for 2.5F-Fc-MMAF treatment were: U87MG = 9.2 ± 1.1 nM, A2780 = 26.1 ±

1.1 nM, and MB-468 = 54.1 ± 1.0 nM. Cells treated with CTRL-Fc-MMAF exhibit less

inhibition of proliferation compared to 2.5F-Fc-MMAF or linker-modified MMAF alone

(Supplementary Figure 3). These results demonstrate integrin-targeting specificity of drug

delivery mediated by 2.5F-Fc-MMAF, and suggest that conjugation of MMAF to CTRL-

Fc, which does not bind integrins, reduces non-specific activity of free drug.

2.5F-Fc-MMAF functions as an effective anti-tumor agent in U87MG xenografts

Next, 2.5F-Fc-MMAF was tested for its ability to inhibit tumor growth in U87MG

xenograft models. U87MG cells were chosen for these studies as they showed the greatest

response to inhibition of cell proliferation upon treatment with 2.5F-Fc-MMAF. In a

preliminary study, Nu/Nu mice (n=5 per treatment group) were inoculated with U87MG

cells in their right flank and tumors were allowed to establish for six days, with an average

size of 35 mm2 before initiation of therapy. We first evaluated different dosing amounts

and schedules of 2.5F-Fc-MMAF administered via intraperitoneal (IP) injection: 10 mg/kg

or 5 mg/kg administered twice or three times per week, or 1 mg/kg administered three

times per week, for a period of three weeks. A dose responsive effect on tumor regression

was seen, with the greatest effects observed in the 10 mg/kg treatment groups

(Supplementary Figure 4). The 10 mg/kg dosing groups both exhibited 80% survival at

day 50, as compared to 0% in the control mice (p=0.0189). No significant difference in

tumor regression (Supplementary Figure 4A) or survival benefit (Supplementary Figure

4B) was seen between two or three times a week dosing at either 5 mg/kg (p>0.99) or 10

mg/kg (p>0.99).

Based on these initial results, we performed a more extensive study, testing the in

vivo efficacy of 2.5F-Fc-MMAF compared with its individual components (2.5F-Fc or

linker-modified MMAF), and the non-binding CTRL-Fc-MMAF, all administered at equal




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dosing frequency and molar equivalent dosing. As above, Nu/Nu mice were inoculated

with U87MG cells in their flank and allowed to establish for six days, this time with an

average tumor size of 47.5 mm2 before initiation of therapy. Mice were assigned to one of

five groups (n= 8-9): 1) untreated control, 2) 2.5F-Fc-MMAF, 3) CTRL-Fc-MMAF, 4)

2.5F-Fc (all administered at 10 mg/kg), or 5) linker-modified MMAF (0.24 mg/kg;

corresponding to a molar equivalent of MMAF). Compounds were administered via IP

injection twice per week for a total of 3 weeks of dosing. The treatment appeared to be

well tolerated, with no significant weight loss or adverse events observed in any of the

treatment groups (Supplementary Figure 5). Significant tumor regression was again seen

with 2.5F-Fc-MMAF treatment; however, we hypothesize that the larger average tumor

size at the start of dosing (47.5 vs. 35 mm2) may have affected the durability of the

response compared to the pilot experiment. Day 15 was chosen for statistical comparison,

as it was the final day of the study with all animals included. When comparing tumor size

in treated groups compared to the untreated control at Day 15 post tumor inoculation, only

2.5F-Fc-MMAF administration yielded a significant regression in growth (p<0.0001). In

contrast, no significant effect on tumor regression or survival benefit was seen in the

groups dosed with 2.5F-Fc or MMAF alone, with results similar to the untreated controls

(Figure 5). The effect of CTRL-Fc-MMAF on tumor size at Day 15 was not significant

(p=0.7044). A moderate but not statistically significant effect (p=0.0772) on survival was

observed in the group dosed with CTRL-Fc-MMAF compared to untreated controls. It is

possible that prolonged serum half-life or Fc-mediated cell trafficking of MMAF could

result from its chemical conjugation to the CTRL-Fc fusion protein. Similar moderate

effects have been observed with MMAF-conjugated control antibodies in other studies (4,

20).

DISCUSSION Members of the integrin receptor subfamily have been found to be present at high

levels on a variety of tumors and tumor-associated vasculature (21, 22). These receptors,

which include integrins that contain an αv subunit (such as αvβ3 and αvβ5) and α5β1

integrin, play a key role in tumor invasion, metastasis, angiogenesis, and survival

highlighting them as promising targets for therapeutic intervention (21, 23-28). Integrin




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antagonists targeting αvβ3, αvβ3/αvβ5, or α5β1, in the form of peptidomimetics, small

molecules or monoclonal antibodies, have been the subject of intense oncology drug

development efforts for decades (29-35), although none are yet FDA approved. These

integrin-targeting agents were developed with the goal of inhibiting angiogenesis or

blocking cell signaling, adhesion, or migration (21), but have failed to perform well in

clinical trials (32, 34). A promising alternative is to instead use integrins for targeted

delivery of cytotoxins to tumors.

EETI 2.5F is unique in its ability to target a broad spectrum of tumor-associated

integrins, a feature that has not been achieved with any antibody to date, which instead

only bind αv-containing integrins or α5β1 integrin. Targeting multiple integrin receptors is

potentially beneficial for reducing drug resistance and tumor growth, as cancer cells can

change their integrin repertoire in response to drug treatment (36). Integrins, particularly

α6β4, α6β1, αvβ5, α2β1, and α3β1 (21), are found to be present on epithelial tissues, and

are also upregulated in wound healing. Our previous studies have shown that 2.5F and

2.5F-Fc, which bind with high affinity to both mouse and human integrins, exhibit high

selectivity for tumor versus healthy tissue in contrast to other integrin targeting agents (11,

13). It is possible that multi-specific receptor targeting and the low level of αvβ3 and α5β1

integrins present on adult epithelial tissue compared to tumors imparts selectivity for

diseased tissue, a concept that has been described in the literature for multi-specific

proteins (37).

First-generation ADCs were produced by randomly conjugating drugs to

antibodies through the sulfhydryl or amino group side chains of cysteine or lysine residues

(38). Kadcyla and Adcetris are produced by drug conjugation to surface-exposed lysines

or partial disulfide reduction and conjugation to free cysteines, respectively (39, 40).

These conjugation approaches lead to heterogeneous ADC products with varied numbers

of drugs conjugated across different locations within the antibody. Heterogeneous ADCs

have been shown in some cases to possess suboptimal activity and pharmacokinetic

properties compared to homogeneous ADC molecules (41-43). Site-specific drug

conjugation to create homogeneous ADCs has been carried out through introduction of

unnatural amino acids that allow orthogonal chemistries (44-46). Such non-natural amino

acids have been introduced into recombinantly produced antibodies using cell-based (44,




15

47) or cell-free (14) methods, introduced cysteine residues (41), lysine analog

incorporation (48, 49), or enzymatic conjugation using formylglycine converting enzyme

or trans-glutaminase (39). We demonstrate that functional knottin-Fc fusions can be

produced by cell-free protein synthesis, followed by subsequent site-specific chemical

attachment of a linker-warhead through an introduced non-natural amino acid, to produce

homogeneous drug conjugates.

We found that 2.5F-Fc-MMAF exhibits lower potency in vivo in comparison to

other recently reported ADCs (50, 51). This level of activity is consistent with the modest

potency observed in our in vitro studies, with IC50 values in the range of 10-50 nM (Figure

4). Lower potency could result from a number of factors including inefficient

internalization that limits the amount of drug delivered inside of the cell, suboptimal

trafficking that hinders localization and release of the conjugate within the lysosomal

compartments, or perhaps intrinsically low potency of the large pAMF-DBCO-PEG4-

MMAF moiety that is likely the active drug compound released. Thus, while 2.5F-Fc-

MMAF provides proof-of-concept data validating tumor targeting and drug delivery using

a knottin-drug conjugate, further optimization will be required for development of viable

clinical candidates based on this strategy. Integrin-targeted ADCs were previously

evaluated with CNTO 95, an anti-αv integrin antibody (52), conjugated to maytansinoids

(53). CNTO 95-drug conjugates (loading of ~3.5 drugs per antibody) showed potent anti-

tumor effects in lung and colon xenograft tumor models. This results of this study are

difficult to directly compare with 2.5F-Fc-MMAF since drug loading, linkers, warhead,

and tumor models are different; however, provide further evidence that integrins are

promising targets for drug delivery.

The three components of an ADC (mAb, linker, and drug) together play a

concerted role on the efficacy of the overall conjugate; thus optimization of each

component has been the subject of many development efforts. In our studies, attachment

of MMAF with a non-cleavable linker was chosen to validate the concept of a knottin-Fc-

drug conjugate as: 1) MMAF has been used in numerous previous studies describing novel

therapeutic molecules (50, 51), and 2) MMAF does not need to be cleaved from its linker

to be active, allowing us to perform initial studies without complications of premature

linker cleavage. An important next goal is to extend this approach to improve potency by




16

testing parameters including alternative warheads and cleavable linkers. ADCs under

clinical development mostly utilize three groups of cytotoxins including auristatins,

maytansines and calicheamicins (2), with other potent small molecules gaining traction,

such as pyrrolobenzodiazepine dimers (54). In addition, most ADCs in the clinical

pipeline contain linkers that get processed intracellularly though disulfide bond exchange

or protease cleavage following internalization (55). Cleavable linkers do not require

processing in the lysosome for activity and can help promote bystander activity through

efflux of hydrophobic drugs, which can be useful for increased efficacy against solid

tumors that exhibit heterogeneous target expression. Finally, the number and location of

attached cytotoxins (43, 56), as well as the choice of target and internalization rate (2, 5),

have also been critical features underlying ADC efficacy that can be optimized in next-

generation knottin-Fc drug conjugates.

While current efforts to advance ADC technologies have focused on novel drug

conjugation methods, linker optimization, alternate warheads, or varying drug loading

ratios, less work has been carried out on the tumor targeting agent itself beyond

antibodies. Several peptide-drug conjugates are in development, such as GNR1005, a 19

amino-acid peptide called Angiopep2 conjugated to paclitaxel (57), and Zoptarelin

doxorubicin, (AEZS-108), a small peptide agonist of the luteinizing hormone-releasing

hormone (LHRH) receptor linked to doxorubicin (58) (others reviewed in: (59)). Recent

studies suggest that antibody targeting is limited by poor and heterogeneous tumor

targeting driven by their large size (60). The 2.5F-Fc-MMAF protein is approximately 60

kDa in size, about 60% smaller than a traditional ADC, and thus presents a new tool to

explore the effects of protein size on tumor penetration in future studies (61). Our work

also highlights the potential for development of other tumor targeting drug conjugates

based on so-called ‘alternative scaffolds’, including, but not limited to anticalins,

affibodies, fibronectin domains, and designed ankyrin repeat proteins (62, 63). In this way,

combining the array of available tumor-targeting proteins with emerging linker, warhead,

and conjugation technologies provides numerous opportunities for the development of

next generation cancer therapeutics.

Acknowledgements:




17

The authors acknowledge Wherly Hoffman for assistance with statistical analyses, and

thank Sutro team members Nina Carlos, Xiaofan Li, Cuong Tran, Sammie Lai, Gang Yin,

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FIGURE LEGENDS Figure 1. Knottin-Fc synthesis and drug conjugation. A, Schematic of cell free protein synthesis of knottin-Fc and incorporation of non-natural amino acid (nnAA, red circle) at position F404 of the Fc domain. B, SDS-PAGE analysis of purified knottin-Fc proteins, analyzed under non-reducing (-DTT) and reducing conditions (+DTT). C, Size exclusion chromatography traces of purified knottin-Fc proteins, monitored at λ=280 nm. D, Schematic of copper-free click chemistry used to conjugate DBCO-PEG4-MMAF to the introduced non-natural amino acid pAMF using a non-cleavable linker. E, Reverse phase HPLC traces of unconjugated and conjugated 2.5F-Fc and CTRL-Fc. The peak areas were integrated to determine the drug-to-protein ratio, in this case with a DAR=1.9. Figure 2. Expression profiling of A, αvβ3; B, αvβ5; and C, α5β1 integrins present on tumor cell lines. Integrin expression and quantification of surface receptors/ cell was measured by flow cytometry following staining with primary antibodies (and secondary antibody for αvβ5) as described in the methods. Figure 3. Competition binding to tumor cell surface integrins. Varying concentrations of 2.5F-Fc or 2.5F-Fc-MMAF were incubated with AF488-labeled 2.5F peptide monomer and allowed to compete for binding to integrin receptors expressed on the surface of A, U87MG; B, A2780; or C, MB-468 cells. The fraction of AF488-2.5F bound to the cell surface is plotted versus the concentration of knottin-Fc. Data shown is the average of triplicate values and error bars represent standard deviation. Figure 4. Inhibition of tumor cell proliferation. A, U87MG; B, A2780; and C, MB-468 were incubated with varying concentrations of 2.5F-Fc, MMAF, 2.5F-Fc-MMAF, or the combination of 2.5F-Fc + MMAF. Cell viability at 120 hr was assessed by measuring the absorbance resulting from CCK8 reagent at λ=450 nm and is reported as the percent inhibition relative to untreated cells. In these experiments MMAF = DBCO-PEG4-MMAF. All IC50 values are expressed as the standard deviation of the mean for triplicate measurements. Figure 5. Growth inhibition of subcutaneous U87MG tumors implanted into Nu/Nu mice. Tumor response to twice weekly treatment with 10 mg/kg of 2.5F-Fc-MMAF, CTRL-Fc-MMAF, 2.5F-Fc, or molar equivalent (0.24 mg/kg) of MMAF was compared to untreated control. Tumor areas for each group are shown in A, and Kaplan-Meier survival curves are shown in B. Arrows indicate days of compound administration over a three week period. In these experiments MMAF = DBCO-PEG4-MMAF and day is defined as day post tumor inoculation. ***p<0.0004 (2.5F-Fc-MMAF vs. untreated).



















Published OnlineFirst March 29, 2016.Mol Cancer Ther Nicolas V. Currier, Shelley E. Ackerman, James R. Kintzing, et al. SynthesisKnottin-Fc-MMAF Conjugate Produced by Cell-Free-Protein Targeted Drug Delivery with an Integrin-Binding

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Produced by Cell-Free-Protein Synthesis · 1 Targeted Drug Delivery with an Integrin-Binding Knottin-Fc-MMAF Conjugate Produced by Cell-Free-Protein Synthesis Nicolas V. Currier1,*,