RN927C, a site-specific Trop-2 antibody-drug …mct.aacrjournals.org/content/molcanther/early/2016/08/27/1535-7163...Cell lines were tested for Trop-2 expression using immunofluorescence

1

RN927C, a site-specific Trop-2 antibody-drug-conjugate (ADC) with enhanced stability, is

highly efficacious in preclinical solid tumor models

Pavel Strop1,5,*, Thomas-Toan Tran1,6,*, Magdalena Dorywalska1, Kathy Delaria1,8, Russell

Dushin2, Oi Kwan Wong1, Wei-Hsien Ho1, Dahui Zhou2, Aidong Wu3, Eugenia Kraynov3, Laura

Aschenbrenner4, Bora Han4,9, Christopher J. O’Donnell2, Jaume Pons1,7, Arvind Rajpal1,5, Dave

L. Shelton1 and Shu-Hui Liu1

1Oncology-Rinat R&D, Pfizer Inc., 230 E Grand Ave, South San Francisco, CA 94080 USA

2Worldwide Medicinal Chemistry, Pfizer Inc., 445 Eastern Point Rd, Groton, CT 06340 USA

3Pharmacokinetics, Dynamics & Metabolism, 4Drug Safety R&D, Pfizer Inc., 10646 Science

Center Dr, San Diego, CA 92121 USA

5Current Address: Bristol-Myers Squibb, 700 Bay Rd Ste A, Redwood City, CA 94063 USA

6Current Address: NGM Biopharmaceuticals, Inc. 630 Gateway Blvd, South San Francisco, CA

94080 USA

7Current Address: Alexo Therapeutics, 951 Gateway Blvd Ste 201, South San Francisco, CA

94080 USA

8Current Address: Grifols Diagnostic Solutions, 6455 Christie Ave B-334C, Emeryville, CA

9Current Address: Adheren Inc. 5858 Horton St Ste 255, Emeryville, CA 94608 USA

*Pavel Strop and Thomas-Toan Tran contributed equally to this article

Corresponding author: Shu-Hui Liu, Email: [email protected] TEL: 650-615-7462

Running title: Site-specific Trop-2 ADC with enhanced stability

Key Words: Trop-2, ADC, site-specific

The authors disclose no potential conflicts of interest

on May 14, 2018. © 2016 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 August 31, 2016; DOI: 10.1158/1535-7163.MCT-16-0431

http://mct.aacrjournals.org/

2

Abstract

Trop-2, also known as TACSTD2, EGP-1, GA733-1 and M1S1, is frequently expressed

on a variety of human carcinomas and its expression is often associated with poor prognosis of

the diseases. However, it is also present on the epithelium of several normal tissues. A

comprehensively designed Trop-2-targeting ADC, balancing both efficacy and toxicity, is

therefore necessary to achieve clinical utility. To this end, we developed a cleavable Trop-2-

ADC (RN927C) using a site-specific transglutaminase-mediated conjugation method and a

proprietary MTI (microtubule inhibitor) linker-payload, PF-06380101. Robust in vitro

cytotoxicity of RN927C was observed on a panel of Trop-2 expressing tumor cell lines, with IC50

generally in the subnanomolar range. As expected for an MTI-containing ADC, RN927C readily

induced mitotic arrest of treated cells in vitro and in vivo, followed by subsequent cell death.

The in vivo efficacy of RN927C was tested in multiple cell line and patient-derived xenograft

tumor models including pancreatic, lung, ovarian and triple negative breast tumor types. Single

dose administration of RN927C at 0.75 - 3 mg/kg was generally sufficient to induce sustained

regression of Trop-2 expressing tumors and showed superior efficacy over standard treatment

with paclitaxel or gemcitabine. Administration of RN927C in non-human primate toxicity

studies resulted in target mediated effects in skin and oral mucosa, consistent with Trop-2

expression in these epithelial tissues with minimal, non-dose limiting off-target toxicities. Based

on the combined efficacy and safety results, RN927C is postulated to have a favorable

therapeutic index for treatment of solid tumors.

Introduction




3

Antibody-drug conjugates (ADCs) were developed to improve the therapeutic indices of

cytotoxic anti-cancer agents. The approach makes use of an immunoconjugate in which a

cytotoxic agent is chemically or enzymatically linked to an antibody that selectively binds to an

internalizing tumor-associated antigen (1-3). This strategy allows specific delivery of the

cytotoxic agent to the tumor site while minimizing the exposure to normal tissues. Trop-2

(Trophoblast cell-surface antigen 2), also referred to as M1S1, GA733-1 (gastric antigen 733-1),

EGP-1 (epithelial glycoprotein-1), or TACSTD2 (tumor-associated calcium signal transducer 2),

is a type I transmembrane cell surface glycoprotein originally identified in human placental

trophoblasts (4) and subsequently found to be highly expressed in most human carcinomas.

Although the biological role of Trop-2 is still under investigation (5), various studies have shown

that overexpression of Trop-2 correlates with increased cancer growth (6, 7), tumor

aggressiveness, metastasis, and poor prognosis in various human carcinomas (8-14). Studies

have also shown that Trop-2 contributes to tumor pathogenesis at least in part by activating the

ERK1/2 MAPK pathway which has important implications in cancer cell proliferation,

migration, invasion, and survival (15). Regulated proteolysis of Trop-2 is suggested to drive

epithelial hyperplasia and stem cell self-renewal via β-catenin signaling (16). Other studies,

however, have indicated that loss of Trop-2 can also contribute to tumorigenesis depending on

the cell type and context (17-19).

Immunohistochemistry (IHC) analysis on human tumor samples has demonstrated

expression of Trop-2 protein in multiple tumor types including pancreatic (8), ovarian (9),

endometrial (10), non-small cell lung (11), colon (12), gastric (13) and oral cancers (14). The

association of Trop-2 expression with advanced disease and/or clinical outcome in multiple

tumor types, and the relatively restricted expression in normal adult tissues, underscore the




4

potential benefit of targeting Trop-2 to fill an unmet need in cancer treatment. An anti-Trop-2

SN38 conjugate, hRS7-CL2A-SN-38 (IMMU-132), consisting of a humanized Trop-2 antibody

conjugated to SN38, the active component of irinotecan, has been shown to be efficacious in

several epithelial cancer cell line xenograft models (20-22). IMMU-132 (sacituzumab

govitecan) is currently being tested in clinical trials for solid tumors and has reported

encouraging therapeutic activity in patients with difficult to treat cancers (23). The safety profile

of IMMU-132 is similar to irinotecan with neutropenia and diarrhea being the most commonly

observed side effects (23).

ADCs synthesized by conjugating to reduced cysteine sulfhydryl groups or lysine side

chain amines yield heterogeneous conjugates that have been associated with aggregation,

increased toxicity and inconsistent pharmacokinetics (24-26). One approach that can potentially

overcome these limitations is to employ site-specific conjugation of antibodies and many

experimental approaches have been developed including conjugation through engineered

cysteine residues, non-natural amino acids, antibody carbohydrates, aldehyde tags, and utilizing

enzymes such as sortases and phosphopantetheinyl transferases (2, 27-36). A site-specifically

conjugated anti-CD33 ADC, SGN-CD33A, has shown encouraging activities in targeting drug-

resistant acute myeloid leukemia (AML) (37). We have previously disclosed the unique qualities

of conjugating antibodies through an enzymatic method employing microbial transglutaminase

(30). Here we describe the design and utility of a new Trop-2-ADC, RN927C, comprised of a

humanized anti-Trop-2 hIgG1 antibody, conjugated with the AcLys-VC-PABC-PF-06380101

linker payload at the C-terminus of the antibody heavy chain via an enzymatic process. This

conjugate contains a valine-citruline cleavable linker and PF-06380101 (38), which is a potent

anti-mitotic agent that inhibits tubulin polymerization. Upon binding to the extracellular portion




5

of Trop-2 on the cell surface, RN927C is internalized and traffics to lysosomes. After being

processed by the lysosomal proteases, the payload PF-06380101 is released and induces cell

cycle arrest resulting in cell death. Indeed, RN927C showed potent in vitro cell killing activity

in tumor cell lines with wide range of Trop-2 expression levels. In vivo testing of RN927C in

cell line and PDX models representing several solid tumor types confirmed robust activity often

inducing tumor regression with a single dose. The AcLys-VC-PABC linker in RN927C is

shown to be stable in the bloodstream from preclinical mouse PK studies and is expected to

provide an enhanced off-target safety profile (30, 39). In addition, our site-specific conjugation

technology produces highly homogeneous conjugates (30, 40) and has the potential to minimize

unwanted PK behavior and toxicity often associated with highly loaded ADCs (24, 27).

Materials and Methods

Generation of RN927C

The antibody portion of RN927C is derived from immunization of Balb/c mice using

recombinant Trop-2 extracellular domain fused with human Fc. The mouse antibody was then

humanized by CDR grafting/affinity maturation and cloned into human IgG1/k constant domains

to create the parental antibody. The C-terminal lysine on the heavy chain of the humanized anti-

Trop-2 antibody was replaced with the LLQGA tag using standard molecular biology techniques.

The DNA encoding this antibody was cloned into an in-house expression plasmid, transiently

expressed in HEK293 cells, and then purified with Protein-A MabSelect SuRe columns (GE

Healthcare). For AcLys-VC-PABC-PF-06380101 conjugation, the antibody concentration was

adjusted to 5 mg/mL in buffer containing 25 mM Tris-HCl, pH 8.0, 150 mM sodium chloride.

Linker-payload (40) was added in a 10-fold molar excess over antibody, the conjugation reaction




6

was initiated by addition of 2% (w/v) bacterial transglutaminase (Ajinomoto Activa TI, Japan)

and incubated with gentle shaking at 37°C for 16 hours. The reaction mixture was adjusted to

0.75 M ammonium sulfate, 25 mM potassium phosphate, pH 7 (Buffer A), and the material was

applied to a Butyl HP column (Butyl HP, GE Healthcare), washed with 5 column volumes of

Buffer A, and eluted with a linear gradient over 20 column volumes into 25 mM potassium

phosphate, pH 7. Fractions containing the conjugate were pooled, dialyzed into 1x PBS,

concentrated using a 10 kDa Amicon Ultra centrifugal filter unit (Millipore), and 0.2 µm sterile

filtered. The final product DAR (Drug-Antibody-Ratio) was 2.0 as determined by HIC and mass

spectrometry analysis.

Cell lines and Reagents

A431, Fadu, OVCAR3, BxPC3, Calu-3, NCI-H292, NCI-H1650, HCC-827, MDA-MB-468,

Colo205, SKBR3 and SW620 were all purchased from American Type Culture Collection

(ATCC) in 2008. Cell lines were tested for Trop-2 expression using immunofluorescence and

FACS. No further authentication was performed. Gemcitabine (GEMZAR) was purchased from

Eli Lily and company. Paclitaxel was purchased from Sigma-Aldrich. Receptor quantitation

was performed using Quantum Simply Cellular kits (Bangs Laboratories, Inc.) according to

manufacturer’s instructions.

Cytotoxicity assays

Human tumor cells from ATCC were cultured in DMEM + 10% FBS (fetal bovine serum)

(A431, Fadu, OVCAR3, SKBR3, MDA-MB-468 and SW620) or RPMI + 10% FBS (BxPC3,

NCI-H1650, NCI-H292, Calu-3, HCC-827 and Colo205). Cells were seeded at 2000 cells/well

(A431, Fadu, MDA-MB-468, SW620, BxPC3, NCI-H1650, NCI-H292, Calu-3, HCC-827 and




7

Colo205) or 3000 cells/well (OVCAR3 and SKBR3) in growth media on white 96 well plates on

day 0. RN927C or negative control ADC were added to each well on day 1 in the dilution range

of 0.0006 - 40 μg/mL final concentration (equivalent to 0.004 - 267 nM). Free PF-06380101(38)

was tested in the final concentration range of 0.0006-40 nM. CelltiterGlo viability assays

(Promega) were performed 4 days after treatment. Luminescence signals were detected on the

M5 plate reader (Molecular Devices). All the readings were normalized to % of value derived

from control untreated wells. IC50s were calculated by logistic non-linear regression (GraphPad

Prism 6.03) and reported as the concentration of antibody (nM) that reduced cell viability by

50%.

Internalization assay

HCC-827 cells were cultured until nearly confluent, harvested and re-suspended in 5 ml of cold

binding buffer (DMEM + 10% FBS + 10 mM HEPES) at 1.6 million cells/ml. Primary antibody

(RN927C or parental Ab) was labeled with Dylight 800 antibody labeling kit per manufacturer’s

protocol (Thermo Scientific). The labeled antibody was then added to the cells to a final

concentration of 2 μg/ml and incubated at 4oC for 1 hour. To initiate internalization, cells were

washed and re-suspended in 5 ml pre-warmed binding buffer supplemented with 2 μg/ml

unlabeled parental Ab and incubated in a 37oC water bath. At each duplicate time point, 0.25 ml

sample was removed and internalization was stopped by the addition of 4 volumes of quench

buffer (cold 1 x PBS + 0.2% sodium azide) and placed on ice. Subsequently, cells were treated

with 1x trypsin-EDTA (Mediatech) supplemented with 5 mg/ml papain (Sigma-Aldrich) and

incubated in a 37oC water bath for 25 min to remove un-internalized antibodies on the surface.

After wash cells were re-suspended in 200 μl of fix buffer (1xPBS + 1.5% paraformaldehyde +




8

1:4000 Draq5 (Biostatus) and centrifuged onto a 96-well poly-D-lysine coated plate (BD

biosciences). For time point zero, a second set of samples was collected without the

trypsin/papain step to measure the maximal amount of surface-bound fluorescence signals. The

fluorescence signals were acquired by Odyssey CLx Infrared Imaging System (LI-COR

Biotechnology) and analyzed using Odyssey software version 3.0.30. For each time point,

relative fluorescence signal, Fr(t), was determined from the fluorescence antibody signal divided

by the Draq5 DNA signal. Subsequently, the normalized amount of internalized antibody at each

time, Y(t), was calculated from Fr(t) divided by Fmax, the maximum surface-bound fluorescence

signal at time zero, and plotted as a function of time. The resulting graph was fitted to the

equation Y(t) = (Bmax * t)/(T½ + t) using GraphPad Prism 6.03 where t is time, Bmax is the

maximal amount of internalized antibody and T½ is the time at which half maximum

internalization occurs.

Immunofluorescence

Trop-2 expression: BxPC3 and SW620 cells were seeded on 12 mm coverslips put into 24 well

plates in growth media until 70-80% confluent. Mouse Ab from which RN927C was derived

was used at 2 μg /mL in RPMI + 10 % FBS + 20 mM HEPES and incubated at 4°C for 1 hour.

The cells were washed twice with 1 x PBS and then fixed in 4% formaldehyde solution for 10

minutes at room temperature. Afterwards the cells were washed 2x with PBS and then incubated

in 400 μL non-permeabilizing blocking buffer (1 x PBS + 5 % Donkey serum) for 1 hour. Trop-2

expression was then detected with Cy3-conjugated donkey anti-mouse 2nd Ab (1:300, Jackson

ImmunoResearch Laboratories). Colocalization with lysosomal marker: cells were grown on

coverslips as described previously. After removing the growth media, 10 μg/mL of RN927C in




9

RPMI + 10 % FBS + 20 mM HEPES were added to the cells and incubated at 4°C for 1 hour.

The antibody solution was then removed and the cells were washed once with 1 x PBS. 0.5 mL

growth media was then added and the cells were moved to 37°C incubator for 2 hours. The cells

were washed twice with 1 x PBS, fixed and then incubated in 400 μL blocking buffer (1 x PBS +

0.3 % TX-100 + 5 % Donkey serum) for 1 hour. Lysosomal compartments were labeled with

anti-LAMP2 (Abcam) diluted 1:100 in 300 μL blocking buffer for 1 hour at room temperature

followed by 2nd Ab detection (1:300 for Cy3-donkey anti-mouse and 1:200 for Dylight488 goat

anti-human, both from Jackson ImmunoResearch Laboratories). The coverslips were mounted

on slides with a drop of Vectashield with DAPI (Vector Laboratories) to stain the nuclei. Images

were observed and acquired by a scanning confocal microscope LAS AF from Leica.

Phosphorylated histone H3 staining: cells were grown on chamber slides (Thermo Scientific)

until 50-70% confluent and then treated overnight with 0.1, 1 and 10 μg/mL of RN927C. Anti-

phospho-histone H3 rabbit antibody (Cell Signaling Technology) was used at 1:1600 dilution for

staining and detection was done using Alex488-F(ab’)2 donkey anti-rabbit IgG (Jackson

ImmnuoResearch Laboratories) as 2nd Ab. Images were observed and acquired by a Nicon

Eclipse E800 microscope (Nicon Instruments).

In vivo studies:

All animal studies were conducted in an AAALAC accredited facility under IACUC

(Institutional Animal Care and Use Committee) approved protocols. Three animals were used at

each time point in the PK study and five animals per cohort were used in all the efficacy studies.

For BxPC3 xenograft model used in both PK and efficacy studies, 2 million cells were implanted

subcutaneously into 5-6 weeks old female CB17/SCID mice (The Jackson Laboratory) until the




10

tumor sizes reached 250-300 mm3 before treatment started. For SW620 model, 3 million tumor

cells were implanted subcutaneously into 5-7 weeks old female NCr nu/nu mice (Taconic) until

the tumor sizes reached ~300 mm3. Pan 0123 (pancreatic ductal carcinoma from peritoneal

biopsy), Pan 0135 (pancreatic adenosquamous carcinoma) and Pan0146 (metastatic pancreatic

carcinoma from liver biopsy), and LG0476 (Squamous non-small cell lung carcinoma) PDX

models were acquired from The Jackson Laboratory, CTG-1017 (triple negative breast cancer)

model was acquired from Champion Oncology, and the Ova196756 (ovarian adenocarcinoma

metastasized to colon) model was established in house from surgical tumor specimen propagated

first in NSG mice (The Jackson Laboratory). For these models 1-2 mm3 of tumor fragments

were implanted subcutaneously into the lateral flanks of female CB17/SCID mice from Taconic.

Animals were randomized by tumor sizes once they reached ~300 mm3 or otherwise indicated,

and RN927C and other reagents were administered through bolus tail vein injection. Tumor

volume was calculated with the following formula: Tumor volume = (length x width 2) / 2.

Animals were humanely sacrificed before their tumor volumes reached 2000 mm3. All tumor

models except SW620 were tested positive for Trop-2 expression by immunohistochemistry

(data not shown).

Pharmacokinetic analysis:

Serum samples were analyzed using an enzyme-linked immunosorbent assays (ELISA)

developed on GyroLab@ immunoassay platform. For the total antibody assay, RN927C or

unconjugated Ab were captured using biotinylated polyclonal goat anti-human IgG (H+L)

antibody (Southern Biotech), and detected with a polyclonal goat anti-human IgG (H+L)

(Bethyl) labeled with Alexa Fluor 647. For the ADC assay, RN927C was captured with

biotinylated polyclonal goat anti-human IgG (H+L) antibody (Southern Biotech). The detection




11

of the captured RN927C was done with a polyclonal antibody generated in-house that recognizes

PF-06380101. The instrument response was used to construct a standard curve and calculate

concentration of study samples and QCs. For both antibody and ADC assays, the lower limit of

quantitation was 50 ng/mL. Frozen tumor samples were thawed on ice and homogenized

(FastPrep®-24 tissue homogenizer) in ice-cold lysis buffer (Invitrogen). The resulting

homogenate was centrifuged at 12,000 rpm for 20 minutes at 4 °C to separate the supernatant

containing RN927C. The samples were analyzed for total antibody and ADC using the same

analytical procedures as for serum. Pharmacokinetic data analysis was performed by the non-

compartmental method using Phoenix software v. 6.3 (Pharsight, Prinseton, NJ).

Immunohistochemistry: Formalin-fixed, paraffin embedded tumor sections were processed and

stained with anti-phospho-H3 (Ser10) rabbit pAb (#9701, Cell Signaling Technology) according

to manufacturer’s instruction followed by detection with EnVision HRP-labeled polymer anti-

rabbit 2nd Ab (DAKO).

Results

RN927C is a homogeneous ADC compound

The chemical composition of RN927C is shown in Figure 1A. RN927C is generated by

conjugating AcLys-VC-PABC-PF-06380101 (38, 40) to the transglutaminase tag (LLQGA)

located at the C-terminus of the antibody heavy chain (see Materials and Methods). The AcLys

linker was chosen to improve the stability of the conjugate in circulation (40). The conjugation

reaction yielded a product with DAR (Drug-Antibody-Ratio) of 1.9 and after a single HIC

purification step a homogeneous conjugate with DAR 2.0 was achieved (Fig. 1B). The process




12

is readily scalable, and high degree of reproducibility was observed among multiple gram scale

production lots as measured by cytotoxicity on Trop-2-expressing BxPC3 cells (Fig. 1C).

RN927C internalizes efficiently and traffics to the lysosomal compartment

The uptake of antibody drug conjugates into the target cells and its subsequent release,

usually by lysosomal proteases, is important for cytotoxic drug delivery. The internalization

kinetics of RN927C was measured and compared to the parental unconjugated Ab in Trop-2

expression NSCLC cell line HCC-827. Both RN927C and the unconjugated parental Ab

internalized efficiently, with t1/2 at 23.4 and 31.6 minutes, respectively (Fig. 2A). The

localization of RN927C was further examined by confocal microscopy, and significant amount

of RN927C was found to co-localize with the lysosomal marker LAMP-2 after two hour

incubation (Fig. 2B).

RN927C induces mitotic arrest in Trop-2-expressing cells

The presumed mechanism through which RN927C elicits cytotoxicity is by intracellular

release of the PF-06380101 payload, which results in the disruption of microtubule

polymerization leading to mitotic arrest, apoptosis and cell death. To confirm this mechanism,

Trop-2 positive BxPC3 and Trop-2 negative SW620 cells (Fig. 3A) were incubated with various

concentrations of RN927C overnight and processed for immunofluorescence (Fig. 3B and C).

Cells in mitotic phase were indicated by positive staining of phosphorylated histone H3 (41).

RN927C induced a dramatic increase of positive phosphorylated histone H3 staining in BxPC3

cells treated with RN927C at the lowest experimental concentration of 0.1 μg/mL (Fig. 3B),

indicating many treated cells were trapped in the mitotic phase. Cells started to detach from the

slides after overnight incubation of RN927C at 10 μg/mL, resulting in loss of cells. In contrast,




13

the untreated cells display the typical low percentage of mitotic cells (Fig. 3B). RN927C

incubation did not show any increase of phosphorylated histone H3 staining on Trop-2 negative

SW620 cells at 10 μg/mL (Fig. 3C), indicating that the activity of RN927C to induce mitotic

arrest in these cells is dependent on Trop-2 expression. RN927C - induced increase in

phosphorylated H3 staining is also observed in vivo on a pancreatic PDX model Pan0146, with

the peak staining observed 3-5 days after RN927C dosing (Supplementary Fig. S1).

RN927C exerts potent in vitro tumor cell killing activity

Next we examined the in vitro cytotoxicity of RN927C on a variety of tumor cell lines.

Tumor cell lines from multiple tumor types including skin, pancreas, lung, head and neck, breast,

ovary and colon were exposed to RN927C and negative control conjugate at concentrations

ranging from 0.04 to 267 nM for 4 days. Most cell lines were readily killed by RN927C with

IC50 below 1 nM. Negative control conjugate was not active against any of the cell lines, and

RN927C did not show any cytotoxicity toward Trop-2 negative SW620 cells (Table 1). The lack

of killing of Trop-2 negative SW620 cells is not due to intrinsic insensitivity to the PF-06380101

as free payload is active against SW620 cells with IC50 of 0.305 nM, comparable of that of the

Trop-2 expressing BxPC3 cells at 0.116 nM or Fadu cells at 0.683 nM (Table 1). These results

indicated that RN927C is efficacious in killing cells derived from multiple tumor types and the

killing requires Trop-2 expression.

RN927C linker is stable in vivo

To evaluate the pharmacokinetic characteristics of RN927C in vivo, single 1.5 mg/kg dose of

RN927C was injected i.v. into BxPC3 tumor-bearing animals with tumor sizes ~250mm3. Both

blood and tumor samples were collected at the following time points (0, 0.083, 2, 6, 24, 72, 120,




14

168, 240 and 336 hours). Total antibody (including both conjugated and unconjugated

antibodies) and ADC concentration were measured as described in the Materials and Methods.

Note that the immunoassay-based measurement cannot distinguish between ADC of DAR1 and

DAR2 therefore certain degree of payload loss can still occur and not be shown by the assay.

ADC and total antibody concentration in serum were similar throughout the time course,

indicating only a slow release of the payload in circulation (Fig. 4A). The serum AUC0-336 for

total Ab is 2488 (μg*hr/mL) and for ADC is 2154 (μg*hr/mL), suggesting that most of the Ab

remaining in circulation still contains payload PF-06380101. There is a slightly faster loss of

payload within the tumors, possibly due to increased internalization and processing of RN927C

occurring in Trop-2 positive tumor cells (Fig. 4B). AUC0-336 percentage of ADC from the total

Ab population in serum is 87% (Fig. 4A), showing good serum linker stability of RN927C in

vivo.

RN927C is highly efficacious in multiple tumor xenograft models

The in vivo efficacy of RN927C was tested on multiple tumor models. In a panel of

pancreatic tumor xenograft models including cell line BxPC3 and PDX models Pan0123,

Pan0135 and Pan0146, one or two doses (every two or three weeks) of RN927C treatment

resulted in sustained tumor growth inhibition/regression at doses between 0.75 to 1.5 mg/kg (Fig.

5A-D). A negative control conjugate consisting of a non-binding antibody conjugated in the

same manner as RN927C showed no effect on these tumor models at 3 mg/kg, the highest dose

tested in these studies. Additionally, in the Pan0146 model, two doses of RN927C at ≥ 0.75

mg/kg were more efficacious than gemcitabine treatment given at 75 mg/kg twice weekly for 6

doses (Fig. 5D).




15

RN927C is also highly efficacious in other solid tumor types tested (Fig. 5E-G). Robust

anti-tumor activity was observed in an ovarian PDX model Ova196756 with single dose

treatment of RN927C at doses > 1.50 mg/kg resulting in long-term tumor regression and tumor

eradication (Fig. 5E). In a lung PDX model LG0476, 1.5 mg/kg single injection of RN927C is

more efficacious in inducing tumor regression than gemcitabine treatment given at 75 mg/kg

twice weekly for 8 doses. Four weekly doses of paclitaxel at 20 mg/kg also only achieved partial

tumor growth inhibition in the same study (Fig. 5F).

In a TNB (triple negative) breast cancer PDX model CTG-1017, single injection of

RN927C induces tumor regression of large tumors (~830 mm3) for more than 60 days. Tumors

that re-grew were treated again 63 days after the first RN927C injection and tumor regression

was again achieved for long duration (Fig. 5G). The anti-tumor effect of RN927C is Trop-2-

dependent as an SW620 colon cancer model devoid of Trop-2 expression showed no response to

RN927C compared to the control conjugate, indicating target expression is required for efficacy

(Fig. 5H). On the other hand standard of care irinotecan resulted in significant tumor growth

inhibition in SW620 model after 4 doses treatment at 40 mg/kg (Fig. 5H).

Exploratory toxicology

The non-clinical safety profile of RN927C has been characterized in rats (up to 30

mg/kg) and non-human primates (up to 6 mg/kg) in repeat-dose studies (two doses; on day 1 and

day 15). The key safety signals observed in cynomolgus monkeys were found in tissues that

express Trop-2. Reversible findings included increased mitoses and single cell necrosis in

multiple epithelial tissues in the skin, injection sites, upper alimentary canal (oral mucosa and

esophagus) and vagina. These were likely related to the activity of the PF-06380101 payload in




16

the Trop-2 expressing tissues. On the other hand, in rats, organ toxicities were more pronounced

in the hematopoietic system and lymphoid tissues, presumably due to the ability to test higher

doses in rats due to lack of cross-reactivity of RN927C to tissues that express rat Trop-2.

Discussion

In this paper we described a novel anti-Trop-2 ADC, RN927C, composed of a humanized

anti-Trop-2 antibody with a site-specifically conjugated novel MTI-payload, PF-06380101 (30,

38, 40), resulting in improved ADC stability and durable anti-tumor responses after a single dose

of therapy. Trop-2 as a tumor antigen has the advantage of high prevalence in many solid tumor

types. It also internalizes efficiently with t1/2 ~ 30 min (Fig. 2), generally considered a

prerequisite for efficient ADC delivery into the cells. We and others have found Trop-2 to be

highly expressed on multiple epithelial tumor types including pancreatic, breast, ovarian,

NSCLC, prostate, gastric and oral cancers (5) . However, it is also expressed on a number of

normal epithelial tissues such as skin and oral mucosa. For example, human keratinocytes were

found to express Trop-2 at ~ 50,000 copies / cell based on receptor quantitation (data not shown).

In general, it is determined that there is 2-10 fold increase of Trop-2 expression in tumors (++ to

+++) compared to target-positive normal tissues (+ to ++). The design of an anti-Trop-2 ADC

thus required a careful balance between efficacy and safety. Several factors were taken into

consideration: first different antibody affinities were explored and a carrier Ab of medium

affinity to Trop-2 (KD ~ 14 nM at 37 degree) was chosen to favor binding of higher Trop-2

expressing tumor tissue over normal tissues. In the cytotoxicity assays this medium affinity Ab

was similarly active to Abs with 10 fold higher affinity on cell lines with high Trop-2 expression




17

(+++), but showed lower killing activity in low Trop-2 expressing cells (+) that have similar

target levels as normal tissues (data not shown). Secondly, use of a transglutaminase-mediated

site-specific conjugation technology to enable production of a nearly homogeneous Trop-2

ADCs carrying two payloads (DAR2) (30). The majority of ADCs that have been used clinically

have been manufactured through cysteine disulfide bond or lysine based conjugations, producing

mixtures of ADC species with various DARs with an average around 4. By decreasing the

payload to antibody ratio, making a homogeneous ADC, and eliminating high DAR species we

aim to reduce the toxicity to lower Trop-2 expressing tissue. The generally high Trop-2

expression on tumor cells and efficient internalization of the ADC should compensate for the

reduced drug loading (Fig. 2). Indeed, most of the tumor cell lines we tested are sensitive to

RN927C with IC50 in the sub-nanomolar range (Table 1). RN927C is highly efficacious in vivo,

with tumor regression generally achieved with single injection of RN927C at a dose of > 1.5

mg/kg with some sensitive models responding to a dose of 0.75 mg/kg (Fig. 5). Efficacy is seen

in pancreatic, ovarian, lung and breast cancer models and is Trop-2 expression dependent. We

also found that RN927C is generally more efficacious than standard of care in these models. For

instance, in the pancreatic cancer PDX model Pan0146, RN927C treatment at >1 mg/kg Q2W for

two doses induces tumor regression, while multiple twice a week gemcitabine dosing only

resulted in tumor growth inhibition (Fig. 5D). Favorable comparison is also observed in NSCLC

PDX model LG0476 where a single dose of RN927C treatment outperforms multiple doses of

gemcitabine or paclitaxel (Fig. 5F).

Many ADCs in the clinic are limited by the off-target toxicity, most notably

hematological toxicity and neurotoxicity. Off-target toxicities can arise from both non-target-

specific uptake or premature release of payloads. Loss of payloads from ADCs in circulation can




18

result in reduced efficacy and a toxicity profile resembling that of the free payload. Several

ADC programs utilizing a cleavable linker and MMAE payload reported neutropenia and

peripheral neuropathy as dose-limiting toxicities (42). IMMU-132, an ADC targeting Trop-2

with payload SN38 (active metabolite of irinotecan) also concluded their safety profile to be

similar to irinotecan (hematological and gastrointestinal toxicity), with no/little toxicities related

to Trop-2 expressing normal tissues (23). Additionally, rapid loss of payloads likely results in

accumulation of conjugate-free antibodies, as antibody half-lives are usually longer than the

intact ADCs. Cardillo et al., reported in a mouse PK study that the AUCs for the intact IMMU-

132 versus the carrier antibody hRS7, were 1516 (h*μg/mL) and 13112 (h*μg/mL), respectively

(21). The persistent presence of unconjugated antibody could have complicated effects to the

ADC efficacy. While some antibodies can elicit anti-tumor effect and possibly aid to the

response such as in the case of T-DM1 for HER2 positive breast cancer, the unconjugated

antibody is also a potential competitor for target binding and could negatively ADC efficacy. In

fact, we have observed a strong inhibitory effect from unconjugated Trop-2 antibodies on the in

vivo efficacy of RN927C, and this effect is most profound within the first three days

(Supplementary Fig. S2).

To reduce the off-target toxicities resulted from de-conjugation of free payloads or linker

cleavage observed with ADCs made with conventional cysteine disulfide bond or lysine based

conjugations, we utilized stable isopeptide linkage and incorporated an AcLys-VC-PABC linker

that was shown to be more stable in circulation (30, 39, 40). We have indeed observed

improved stability of our Trop-2 ADC in vivo, evidenced by the small difference between ADC

and total Ab pharmacokinetic profiles (Fig. 4). Preclinical exploratory safety studies of RN927C

conducted in non-human primates with doses up to 6 mg/kg for two doses showed mostly on




19

target epithelial toxicities (rash and mucositis) that were fully recoverable, consistent with Trop-

2 expression on skin and oral mucosa. Notably there were no adverse hematological findings

observed in monkeys, indicating reduction of off-target toxicity presumably due to the stable

nature of our linker-payload. With a more stable compound we have observed a potential

correlation between Trop-2 expression and efficacy. Trop-2- negative tumor models such as

SW620 typically being insensitive to RN927C treatment, and tumors with high and

homogeneous Trop-2 expression responding well with sustained efficacy (Fig. 5). It is likely

that a companion diagnostic test for patient selection based on Trop-2 expression level would

enhance clinical activity of a Trop-2 targeting ADCs. In summary, we have developed a

homogeneous site-specific Trop-2 ADC with enhanced stability, and the preclinical efficacy and

safety data support clinical testing of RN927C in multiple solid tumor types.

Acknowledgements

The authors would like to thank Jessica Yu and Jeanette Dilley for their hybridoma work, Kevin

Lindquist, Christine Bee, and Yasmina Abdiche for Biosensor analysis; Bryant Chau, Colleen

Brown, Ishita Barman and Michael Chin for protein and antibody production; Jyothirmayee

Kudaravalli, Ratika Chopra, and Jing-Tyan Ma for antibody characterization and technical

assistance; Victor Lui and Santiago Farias for Mass Spec analytical support, Rachel DeVay for

confocal microscopy assistance, and Birte Nolting for technical development in scale up. The

authors wish to acknowledge the contributions of Andreas Maderna and Matthew Doroski for

their discovery of the PF-06380101 payload, and Michael Green and Ramalakshmi

Chandrasekaran for preparation of key intermediates used in the synthesis of AcLys-VC-PF-

06380101.




20

References:

1. Sievers EL, Senter PD. Antibody-drug conjugates in cancer therapy. Annual review of medicine. 2013;64:15-29. 2. Panowksi S, Bhakta S, Raab H, Polakis P, Junutula JR. Site-specific antibody drug conjugates for cancer therapy. mAbs. 2014;6:34-45. 3. Polakis P. Antibody Drug Conjugates for Cancer Therapy. Pharmacological reviews. 2016;68:3-19. 4. Lipinski M, Parks DR, Rouse RV, Herzenberg LA. Human trophoblast cell-surface antigens defined by monoclonal antibodies. Proceedings of the National Academy of Sciences of the United States of America. 1981;78:5147-50. 5. Shvartsur A, Bonavida B. Trop2 and its overexpression in cancers: regulation and clinical/therapeutic implications. Genes & cancer. 2015;6:84-105. 6. Wang J, Day R, Dong Y, Weintraub SJ, Michel L. Identification of Trop-2 as an oncogene and an attractive therapeutic target in colon cancers. Molecular cancer therapeutics. 2008;7:280-5. 7. Trerotola M, Jernigan DL, Liu Q, Siddiqui J, Fatatis A, Languino LR. Trop-2 promotes prostate cancer metastasis by modulating beta(1) integrin functions. Cancer research. 2013;73:3155-67. 8. Fong D, Moser P, Krammel C, Gostner JM, Margreiter R, Mitterer M, et al. High expression of TROP2 correlates with poor prognosis in pancreatic cancer. British journal of cancer. 2008;99:1290-5. 9. Bignotti E, Todeschini P, Calza S, Falchetti M, Ravanini M, Tassi RA, et al. Trop-2 overexpression as an independent marker for poor overall survival in ovarian carcinoma patients. European journal of cancer. 2010;46:944-53. 10. Bignotti E, Zanotti L, Calza S, Falchetti M, Lonardi S, Ravaggi A, et al. Trop-2 protein overexpression is an independent marker for predicting disease recurrence in endometrioid endometrial carcinoma. BMC clinical pathology. 2012;12:22. 11. Kobayashi H, Minami Y, Anami Y, Kondou Y, Iijima T, Kano J, et al. Expression of the GA733 gene family and its relationship to prognosis in pulmonary adenocarcinoma. Virchows Archiv : an international journal of pathology. 2010;457:69-76. 12. Ohmachi T, Tanaka F, Mimori K, Inoue H, Yanaga K, Mori M. Clinical significance of TROP2 expression in colorectal cancer. Clin Cancer Res. 2006;12:3057-63. 13. Muhlmann G, Spizzo G, Gostner J, Zitt M, Maier H, Moser P, et al. TROP2 expression as prognostic marker for gastric carcinoma. Journal of clinical pathology. 2009;62:152-8. 14. Fong D, Spizzo G, Gostner JM, Gastl G, Moser P, Krammel C, et al. TROP2: a novel prognostic marker in squamous cell carcinoma of the oral cavity. Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc. 2008;21:186-91. 15. Cubas R, Zhang S, Li M, Chen C, Yao Q. Trop2 expression contributes to tumor pathogenesis by activating the ERK MAPK pathway. Molecular cancer. 2010;9:253. 16. Stoyanova T, Goldstein AS, Cai H, Drake JM, Huang J, Witte ON. Regulated proteolysis of Trop2 drives epithelial hyperplasia and stem cell self-renewal via beta-catenin signaling. Genes & development. 2012;26:2271-85. 17. Wang J, Zhang K, Grabowska D, Li A, Dong Y, Day R, et al. Loss of Trop2 promotes carcinogenesis and features of epithelial to mesenchymal transition in squamous cell carcinoma. Molecular cancer research : MCR. 2011;9:1686-95. 18. Lin JC, Wu YY, Wu JY, Lin TC, Wu CT, Chang YL, et al. TROP2 is epigenetically inactivated and modulates IGF-1R signalling in lung adenocarcinoma. EMBO molecular medicine. 2012;4:472-85.




21

19. Zhang K, Jones L, Lim S, Maher CA, Adkins D, Lewis J, et al. Loss of Trop2 causes ErbB3 activation through a neuregulin-1-dependent mechanism in the mesenchymal subtype of HNSCC. Oncotarget. 2014;5:9281-94. 20. Cardillo TM, Govindan SV, Sharkey RM, Trisal P, Goldenberg DM. Humanized anti-Trop-2 IgG-SN-38 conjugate for effective treatment of diverse epithelial cancers: preclinical studies in human cancer xenograft models and monkeys. Clin Cancer Res. 2011;17:3157-69. 21. Cardillo TM, Govindan SV, Sharkey RM, Trisal P, Arrojo R, Liu D, et al. Sacituzumab Govitecan (IMMU-132), an Anti-Trop-2/SN-38 Antibody-Drug Conjugate: Characterization and Efficacy in Pancreatic, Gastric, and Other Cancers. Bioconjugate chemistry. 2015;26:919-31. 22. Goldenberg DM, Cardillo TM, Govindan SV, Rossi EA, Sharkey RM. Trop-2 is a novel target for solid cancer therapy with sacituzumab govitecan (IMMU-132), an antibody-drug conjugate (ADC). Oncotarget. 2015;6:22496-512. 23. Starodub AN, Ocean AJ, Shah MA, Guarino MJ, Picozzi VJ, Jr., Vahdat LT, et al. First-in-Human Trial of a Novel Anti-Trop-2 Antibody-SN-38 Conjugate, Sacituzumab Govitecan, for the Treatment of Diverse Metastatic Solid Tumors. Clin Cancer Res. 2015;21:3870-8. 24. Hamblett KJ, Senter PD, Chace DF, Sun MM, Lenox J, Cerveny CG, et al. Effects of drug loading on the antitumor activity of a monoclonal antibody drug conjugate. Clin Cancer Res. 2004;10:7063-70. 25. Wang L, Amphlett G, Blattler WA, Lambert JM, Zhang W. Structural characterization of the maytansinoid-monoclonal antibody immunoconjugate, huN901-DM1, by mass spectrometry. Protein science : a publication of the Protein Society. 2005;14:2436-46. 26. Boswell CA, Mundo EE, Zhang C, Bumbaca D, Valle NR, Kozak KR, et al. Impact of drug conjugation on pharmacokinetics and tissue distribution of anti-STEAP1 antibody-drug conjugates in rats. Bioconjugate chemistry. 2011;22:1994-2004. 27. Junutula JR, Raab H, Clark S, Bhakta S, Leipold DD, Weir S, et al. Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index. Nature biotechnology. 2008;26:925-32. 28. Shen BQ, Xu K, Liu L, Raab H, Bhakta S, Kenrick M, et al. Conjugation site modulates the in vivo stability and therapeutic activity of antibody-drug conjugates. Nature biotechnology. 2012;30:184-9. 29. Axup JY, Bajjuri KM, Ritland M, Hutchins BM, Kim CH, Kazane SA, et al. Synthesis of site-specific antibody-drug conjugates using unnatural amino acids. Proceedings of the National Academy of Sciences of the United States of America. 2012;109:16101-6. 30. Strop P, Liu SH, Dorywalska M, Delaria K, Dushin RG, Tran TT, et al. Location matters: site of conjugation modulates stability and pharmacokinetics of antibody drug conjugates. Chemistry & biology. 2013;20:161-7. 31. Okeley NM, Toki BE, Zhang X, Jeffrey SC, Burke PJ, Alley SC, et al. Metabolic engineering of monoclonal antibody carbohydrates for antibody-drug conjugation. Bioconjugate chemistry. 2013;24:1650-5. 32. Zimmerman ES, Heibeck TH, Gill A, Li X, Murray CJ, Madlansacay MR, et al. Production of site-specific antibody-drug conjugates using optimized non-natural amino acids in a cell-free expression system. Bioconjugate chemistry. 2014;25:351-61. 33. Drake PM, Albers AE, Baker J, Banas S, Barfield RM, Bhat AS, et al. Aldehyde tag coupled with HIPS chemistry enables the production of ADCs conjugated site-specifically to different antibody regions with distinct in vivo efficacy and PK outcomes. Bioconjugate chemistry. 2014;25:1331-41. 34. Beerli RR, Hell T, Merkel AS, Grawunder U. Sortase Enzyme-Mediated Generation of Site-Specifically Conjugated Antibody Drug Conjugates with High In Vitro and In Vivo Potency. PloS one. 2015;10:e0131177. 35. Grunewald J, Klock HE, Cellitti SE, Bursulaya B, McMullan D, Jones DH, et al. Efficient Preparation of Site-Specific Antibody-Drug Conjugates Using Phosphopantetheinyl Transferases. Bioconjugate chemistry. 2015;26:2554-62.




22

36. Lyon RP, Bovee TD, Doronina SO, Burke PJ, Hunter JH, Neff-LaFord HD, et al. Reducing hydrophobicity of homogeneous antibody-drug conjugates improves pharmacokinetics and therapeutic index. Nature biotechnology. 2015;33:733-5. 37. Kung Sutherland MS, Walter RB, Jeffrey SC, Burke PJ, Yu C, Kostner H, et al. SGN-CD33A: a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML. Blood. 2013;122:1455-63. 38. Maderna A, Doroski M, Subramanyam C, Porte A, Leverett CA, Vetelino BC, et al. Discovery of cytotoxic dolastatin 10 analogues with N-terminal modifications. Journal of medicinal chemistry. 2014;57:10527-43. 39. Dorywalska M, Strop P, Melton-Witt JA, Hasa-Moreno A, Farias SE, Galindo Casas M, et al. Effect of attachment site on stability of cleavable antibody drug conjugates. Bioconjugate chemistry. 2015;26:650-9. 40. Dorywalska M, Dushin R, Moine L, Farias SE, Zhou D, Navaratnam T, et al. Molecular basis of valine-citrulline-PABC linker instability in site-specific ADCs and its mitigation by linker design. Molecular cancer therapeutics. 2016. 41. Goto H, Tomono Y, Ajiro K, Kosako H, Fujita M, Sakurai M, et al. Identification of a novel phosphorylation site on histone H3 coupled with mitotic chromosome condensation. The Journal of biological chemistry. 1999;274:25543-9. 42. Burris H. Antibody drug conjugates: promise beyond breast cancer. SOWG plenary session, San Francisco. 2014.




23

Table 1

cell name Tumor type Trop-2

expression

(receptor #)

RN927C

IC50 (nM) Neg ctrl-ADC

IC50 (nM) PF-06380101

IC50 (nM)

A431 Epidermoid +++

(700,000) 0.202 + 0.050 248.7

Fadu Pharynx squamous +++

(387,000) 0.507 + 0.219 >267 0.683 + 0.066

BxPC3 Pancreas ++/+++

(137,000) 0.674 + 0.286 >267 0.116 + 0.034

HCC-827 Lung ++/+++ 0.779 + 0.328 >267

OVCAR3 Ovary +++ 0.560 168.5

RL95-2 Endometrium ++/+++ 0.150 156.5

Calu-3 Lung ++ 0.533 n/d

NCI-H292 Lung ++ 0.633 n/d

NCI-H1650 Lung ++

(100,000) 1.920 >267

MDA-MB-468 Mammary ++ 0.773 >267

SKBR3 Mammary +/++ 0.420 200.5

Colo205 Colorectal +/++

(50,000) 50.6 + 22.5 n/d

SW620 Colorectal - >267 >267 0.305 + 0.046

Table 1. Cell lines from multiple tumor types are sensitive to RN927C. Cytotoxicity assay

of RN927C on a panel of tumor cells from different cancer types were performed as described in




24

Material and Methods. Trop-2 expression (number of + symbols) levels were empirically

assigned based on staining intensity of RN927C parent Ab by FACS (data not shown), and

corresponded to the following receptor copy number ranges (+: < 10-50,000; ++: 50-100,000;

+++: > 100,000). For A431, Fadu, BxPC3, NCI-H1650 and Colo205 cells the actual receptor

numbers were listed in parentheses. For A431, Fadu, BxPC3, HCC-827, and Colo205 cells the

inhibitory concentration of 50% (IC50) was calculated by logistic nonlinear regression and is

reported as the mean± stdev in nM of antibody concentration from multiple experiments. For

SW620 no average can be calculated as most IC50 values from various experiments exceeded the

top concentration of 267 nM. Only single experimental value was listed for OVCAR3, RL95-2,

Calu-3, NCI-H292, NCI-H1650, MDA-MB-468, and SKBR3 cells. Free payload PF-06380101

was tested on tumor cell lines BxPC3, SW620, and Fadu tumor cells. Note that Trop-2 negative

cell SW620 is sensitive to free payload killing.




25

Figure Legends

Figure 1. Structure, homogeneity and manufacturing reproducibility of RN927C. A. The

chemical structure of the RN927C linker payload including the engineered transglutaminase tag

LLQGA. B. The HIC (hydrophobic interaction chromatography) profile of RN927C after

purification. RN927C was conjugated and purified as described in Material and Methods. The

resulting conjugate appeared as a single peak with payload loading equivalent of DAR 2 on the

HIC column. C. Cytotoxicity assay of three batches of RN927C showed reproducible killing

activity of Trop-2 expressing BxPC3 cells.

Figure 2. Internalization kinetics and intracellular trafficking of RN927C. A. The

internalization kinetics of parental unconjugated Ab and RN927C on Trop-2 expressing NSCLC

HCC-827 cells were measured as described in Material and Methods in duplicates. The t1/2

(time at which half maximum internalization occurs) was determined to be 31.6 minutes for the

unconjugated parent Ab and 23.4 minutes for RN927C. B. The internalized RN927C is shown to

be co-localized with lysosomal marker LAMP-2. RN927C (green) was seen on the cell surface

with confocal microscopy after one hour incubation at 4 degree (0 hr), while LAMP2 marked the

lysosomes (red). The nuclei were stained with DAPI (blue). After 2 hr incubation at 37 degree,

significant amount of RN927C colocalized with LAMP-2 (yellow). Scale bar, 10 μm.

Figure 3. Mitotic arrest induced by RN927C in Trop2 expressing cells. A. BxPC3 and

SW620 cells were stained with mouse parental Ab for RN927C and then detected with Cy3-

conjugated donkey anti-mouse 2nd Ab (red). BxPC3 expressed high level of Trop-2 on the cell

surface while SW620 showed no staining. Scale bar, 20 μm. B. Trop-2 positive BxPC3 cells

were seeded on tissue culture slides and treated with RN927C overnight at concentration




26

indicated. The cells were then washed and fixed and stained with anti-phosphorylated histone

H3 (pH3) Ab and detected by Alexa488-conjugated donkey anti-rabbit 2nd Ab (green). In

control untreated cells (control) only a few cells were pH3 positive (green, compared to nuclear

staining by DAPI in blue), reflecting a normal low number of cells in mitosis in unsynchronized

population. Treatment of RN927C for as low as 0.1 μg/mL drastically increases the percentage

of pH3 positive cells after overnight incubation, indicating that many cells in the stage of mitotic

arrest. Scale bar, 50 μm. C. In contrast, Trop-2 negative SW620 cells were insensitive to

RN927C treatment at 10 μg/mL (right panel) and the pH3 staining signal did not change

compare to control (untreated). Scale bar, 50 μm.

Figure 4. RN927C is relatively stable in tumor bearing mice. 1.5 mg/kg of RN927C was

administered i.v. to BxPC3 tumor bearing mice and the concentrations of total Ab and ADC

were determined at time points indicated in both the serum samples (A) and tumor specimens (B)

as described in Material and Methods. ADC exposure (AUC0-336) was 87% to total Ab in the

serum (A) and 73% in the tumor (B).

Figure 5. RN927C induces tumor regression in multiple tumor models. A. Single dose

treatment of RN927C at 0.75, 1.5 and 3.0 mg/kg on pancreatic cell line BxPC3 xenograft model.

Tumor regression was observed at ≥1.5 mg/kg. B. Single dose treatment of RN927C at 0.75, 1.5,

and 3.0 mg/kg in pancreatic PDX Pan0123 xenograft model. Tumor regression was observed at

all doses. C. RN927C was administered to Pancreatic PDX model Pan0135 at 0.75, 1.5, and

3.0 mg/kg every two weeks for two treatments. Prolong regression was observed in all doses.

D. RN927C was given to pancreatic PDX model Pan0146 at 0.75 – 1.50 every two weeks for

two doses. Regression was seen at ≥ 1.0 mg/kg. Contrarily Gemcitabine treatment (75 mg/kg)




27

at twice weekly dosing for 6 doses resulted in only partial growth inhibition. E. Single dose

treatment of RN927C at 0.75, 1.5, and 3.0 mg/kg in ovarian Ova196756 PDX xenograft model.

Tumor regression was observed at all doses but more persistent at doses ≥1.5 mg/kg. F. Single

dose treatment of RN927C at 1.5 mg/kg resulted in sustained tumor regression in Lung LG0476

PDX xenograft model. Negative control conjugate at 1.5mg/kg showed no effect. Gemcitabine

at 75 mg/kg twice weekly dosing for 8 doses induces tumor regression but tumor re-growth

occurred sooner than the RN927C treated group. Paclitaxel at weekly dosing of 20 mg/kg for 4

doses resulted in partial tumor growth inhibition. G. In TNB (triple negative breast) cancer

PDX model CTG-1017, single injection of RN927C induces tumor regression of large tumors

(~830 mm3) for more than 60 days. Tumors that re-grew were treated again 63 days after the

first RN927C injection and tumor regression was again achieved for long duration. H. Single

dose treatment of RN927C at 6.0 mg/kg has no anti-tumor effect in Trop-2 negative colon cancer

cell line SW620 xenograft model. Irinotecan at 40 mg/kg twice weekly dosing of 4 doses

resulted in significant tumor growth inhibition. All tumor models continued to grow when

treated with non-binding negative control conjugates at the highest doses in all studies.




A

C

Figure 1

B




Figure 2

0 hr 2 hr

A

B




BxPC3_mIgG

SW620_mIgG

BxPC3_aTrop2

SW620_aTrop2

A

B

C

Figure 3

untreated

RN927C 1 mg/mL RN927C 10 mg/mL

RN927C 0.1 mg/mL

untreated RN927C 10 mg/mL

Trop-2 expression on tumor cells

Phosphorylated histone H3 staining on BxPC3 cells

Phosphorylated histone H3 staining on SW620 cells




A

B

Figure 4




39 46 53 60 67 74 81 88 95 1021091161230

500

1000

1500

3.00 mg/kg neg ctrl conjugate

0.75 mg/kg RN927C

1.50 mg/kg RN927C

3.00 mg/kg RN927C

Pan0135

Days Post Tumor Implantation

Tu

mo

r V

olu

me (

mm

3)

(Me

an

SE

M)

Figure 5

A B

C D

G H

E F




Published OnlineFirst August 31, 2016.Mol Cancer Ther Pavel Strop, Thomas-Toan Tran, Magdalena Dorywalska, et al. tumor modelswith enhanced stability, is highly efficacious in preclinical solid RN927C, a site-specific Trop-2 antibody-drug-conjugate (ADC)

Updated version

10.1158/1535-7163.MCT-16-0431doi:

Access the most recent version of this article at:

Material

Supplementary

http://mct.aacrjournals.org/content/suppl/2016/08/27/1535-7163.MCT-16-0431.DC1

Access the most recent supplemental material at:

Manuscript

Authoredited. Author manuscripts have been peer reviewed and accepted for publication but have not yet been

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://mct.aacrjournals.org/content/early/2016/08/27/1535-7163.MCT-16-0431To request permission to re-use all or part of this article, use this link



http://mct.aacrjournals.org/lookup/doi/10.1158/1535-7163.MCT-16-0431

http://mct.aacrjournals.org/content/suppl/2016/08/27/1535-7163.MCT-16-0431.DC1

http://mct.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://mct.aacrjournals.org/content/early/2016/08/27/1535-7163.MCT-16-0431


Documents

RN927C, a site-specific Trop-2 antibody-drug …mct.aacrjournals.org/content/molcanther/early/2016/08/27/1535-7163...Cell lines were tested for Trop-2 expression using immunofluorescence