Site-specific PEGylation of anti-mesothelin recombinant ... · 12/21/2019 · pegylation was...

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Site-specific PEGylation of anti-mesothelin recombinant

immunotoxins increases half-life and anti-tumor activity

Authors: Zeliang Zheng1, Ryuhei Okada

2, Hisataka Kobayashi

2, Tadanobu Nagaya

2, Junxia

Wei1, Qi Zhou

1, Fred Lee

1, Tapan K Bera

1, Yun Gao

3, William Kuhlman

3, Chin-Hsien Tai

1, and

Ira Pastan1*

Author Affiliations:

1Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute,

National Institutes of Health, Bethesda, MD 20892-4264

2Laboratory of Molecular Theranostics, Center for Cancer Research, National Cancer Institute,

National Institutes of Health, Bethesda, MD 20892-4264

3Selecta Biosciences, 480 Arsenal Way, Watertown, MA 02472

*Correspondence: Dr. Ira Pastan, Laboratory of Molecular Biology, National Cancer Institute,

37 Convent Drive, Room 5106, Bethesda, MD 20892-4264

e-mail: [email protected]; Tel: (240) 760-6470; Fax: (240) 541-4501

Running Title: Site specific PEGylation improves immunotoxin activity

Keywords: Mesothelin, mesothelioma, cancer therapy, immunotherapy, pegylation

Conflict of Interest Statement:

I.P. is an inventor on several patents on immunotoxins that have all been assigned to the NIH.

on April 4, 2021. © 2019 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 December 23, 2019; DOI: 10.1158/1535-7163.MCT-19-0890

http://mct.aacrjournals.org/

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Abstract

Recombinant Immunotoxins (RITs) are chimeric proteins containing an Fv that binds to tumor

cells, fused to a fragment of Pseudomonas exotoxin (PE) that kills the cell. Their efficacy is

limited by their short half-life in the circulation. Chemical modification with polyethylene glycol

(PEG) is a well-established method to extend the half-lives of biologics. Our goal was to

engineer RITs with an increase in half-life and high cytotoxic activity. We introduced single

cysteines at different locations in five anti-mesothelin RITs and employed site-specific

PEGylation to conjugate them to 20kD PEG. Because our previous PEGylation method using β-

mercapto-ethanol reduction, gave poor yields of PEG-modified protein, we employed a new

method using TCEP to reduce the protein, and could PEGylate RITs at ~90% efficiency. The

new proteins retained 19-65% of cytotoxic activity. Although all proteins are modified with the

same PEG, the radius of hydration varies from 5.2 to 7.1 showing PEG location has a large effect

on protein shape. The RIT with the smallest radius of hydration has the highest cytotoxic

activity. The PEGylated RITs have a 10-30-fold increase in half-life which is related to the

increase in hydrodynamic size. Biodistribution experiments indicate that the long half-life is due

to delayed uptake by the kidney. Anti-tumor experiments show that several PEG-RITs are much

more active than unmodified RIT and the PEG location greatly affects anti-tumor activity. We

conclude that PEGylation is a useful approach to improve the half-life and anti-tumor activity of

RITs.




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Introduction

Small therapeutic proteins have limited efficacy because they are rapidly removed from

circulation (1). Two well-known mechanisms of removal are renal filtration and hepatic

elimination. Increasing the hydrodynamic volume is a widely adopted strategy to increase half-

life. Other strategies include fusion with albumin, albumin binding domain, Fc domain of an

antibody, or N-terminal glycosylation (1). Most of these strategies take advantage of the

naturally occurring FcRn-mediated, pH-dependent recycling of IgG and albumin that is

responsible for their exceptionally long half-life.

RITs are small chimeric proteins consisting of the Fv portion of an antibody that serves

as the targeting moiety, fused to a bacterial toxin that kills the target cells (2). Our lab studies

RITs that target mesothelin, a cell-surface glycoprotein that is robustly expressed in many

common solid tumors (3). The bacterial toxin is Pseudomonas exotoxin A (PE). To make RITs

domain I, the cell-targeting domain of PE is replaced by an anti-mesothelin Fv or Fab and

unnecessary portions of domain II are deleted. The size of the RITs ranges from 50 to 72kD, and

they have half-lives of 10 to 20 minutes in mice. Recently we reported that the addition of an

albumin binding domain to a RIT targeting mesothelin greatly increased half-life and anti-tumor

activity (4). This finding encouraged us to explore other approaches to increase half-life that do

not require the addition of a foreign protein domain, which could increase immunogenicity.

PEGylation is an approach in which polyethylene glycol (PEG) is conjugated to a

macromolecule. It is a well-established method for half-life extension and does this by increasing

the hydrodynamic volume and reducing renal filtration (5). Currently, there are 14 FDA

approved PEGylated drugs on the market and 20 more are in clinical trials. They range from

proteins to liposomes, indicating the versatile nature of PEGylation (6). Our initial effort at




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PEGylation was to modify human transforming growth factor- fused to the 38kD PE38; this

resulted in a protein with enhanced plasma half-life and anti-tumor activity; however, the

pegylation was lysine-specific that resulted in heterogenous populations of PEG-modified

immunotoxin because there are multiple lysines presented (7). We subsequently showed that

PEGylation of the Fv portion of LMB-2, a RIT composed of an Fv that binds to CD25 fused to

the PE38 also increased half-life, reduced toxicity and immunogenicity (8). The drawback of this

study was that the method involved reduction of the protein with β-mercaptoethanol resulting in

variable and poor yields and purity. Therefore, we could only modify a single position in the

immunotoxin. Our goal in this study was to make multiple PEG-modified immunotoxins, each

with a single PEG site-specifically conjugated at a unique position in order to find a site that

retains high cytotoxic activity, a long half-life in the circulation and high anti-tumor activity. In

order to do this, we needed to develop a new method of PEGylation using TCEP to reduce the

protein (9).

The mechanism by which RITs kill mesothelin expressing cancer cells consists of several

steps. First the RIT binds to the cell through recognition of mesothelin, followed by

internalization through endocytosis. Then the toxin is cleaved from the Fv by the furin protease

and undergoes retrograde transport through the Golgi to the endoplasmic reticulum (ER). Next

the toxin is released into the cytosol where it ADP ribosylates and inactivates elongation factor 2

(EF2); this halts protein synthesis causing cell death (3). Given the complex network of

interactions, it is important to identify positions on the RIT where PEG can be placed that do not

decrease the activity of the RIT by sterically hindering mesothelin binding, furin cleavage,

retrograde transport to the ER, transfer to the cytosol from the ER and ADP-ribosylation of EF2

(8).




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To overcome these challenges, we adopted approaches that would give us the best chance

to produce active protein: First we used a single-chain Fv as the targeting moiety, because it only

contains buried disulfide bonds, which cannot be PEGylated. Second, we exploited structural

modeling of the mesothelin-RIT-EF2 complex to locate cysteines at positions distant from the

mesothelin and EF2-binding sites. Third, we placed cysteines on spatially and functionally

distinct domains. These were located on the Fv, next to the furin cleavage site, and on domain III

of the toxin. Fourth, we employed the highly efficient maleimide-based site-specific conjugation

to prevent random PEGylation.

Materials and Methods

Materials: methoxy-PEG-maleimide (molecular weight 20 kD) was obtained from JenKem

Technology. Other materials were obtained from standard sources.

Bacterial Strains and Plasmids: Escherichia coli DH5 (High Efficiency) was obtained from

New England Biolabs for the propagation of plasmids. E. coli BL21(DE3), which carries T7

RNA polymerase gene under the control of an inducible promoter on a prophage, was used as a

host to express RIT. Plasmids that express RIT are under the control of T7 promoter and contain

a single-chain Fv that is genetically fused to a 24kD bacterial toxin PE by a flexible GS linker

and a furin cleavage site.

Construction, Expression, and Purification of RIT: Double-stranded gene fragment (gBlock,

Integrated DNA Technologies) that contains the designated cysteine was ligated onto our

standard laboratory RIT production vector as described before (10) using Gibson Assembly




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Master Mix (New England Biolabs) according to the manufacturer’s protocol. The correct

assembly of plasmids was confirmed by cutting with the appropriate restriction enzymes and

sequencing analysis. The plasmid was transformed into E. coli. BL21(DE3) and RIT was

induced with 1mM IPTG at OD.2.5 for 2 hours. All RITs were expressed and purified as

inclusion body according to the protocol described before (11). Briefly, inclusion body was

dissolved in GTE buffer (6M guanidine-HCl, 100mM Tris-HCl, 2mM EDTA) for 19 hours,

followed by refolding in 1000ml 100mM Tris-HCl, 1mM EDTA, 0.5M arginine and 0.5M

NDSB-201, pH 10.0 for 31 hours and dialysis against 50 liters of 30mM Tris-HCl, 0.1M urea for

19 hours. The refolded RITs were purified through Q-sepharose and MonoQ ion exchange

columns (GE Healthcare)(10).

PEGylation of RIT: RITs were PEGylated in collaboration with Selecta Bioscience. 4mg RIT

was buffer exchanged into 20mM potassium phosphate, pH 8.0, 2mM EDTA using Vivaspin 20

(5kD MWCO, GE healthcare) until the pH of the flow through matches that of the buffer. A 3-

molar excess of tris(2-carboxyethyl)phosphine (TCEP) was added to the final 2mg/ml RIT and

incubated at room temperature for 1-hour to reduce cysteine. Then a 10-molar excess of

methoxy-PEG maleimide was added and left to react for overnight. For purification the

PEGylated RIT was buffer exchanged into 10mM Tris-HCl, pH 8.0 using a Vivaspin column and

applied to an anion exchange spin column (Pierce). Unreactive methoxy-PEG maleimide was

washed away with four 10 ml washes of the same buffer and the RIT was eluted with 1XPBS.

Samples collected before and after TCEP treatment, and after the final elution were analyzed on

SDS-PAGE gels. For high-performance liquid chromatography (HPLC) analysis, additional




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samples were collected during column washes to make sure methoxy-PEG maleimide was

completely gone.

HPLC Analysis: Reverse phase HPLC was performed to separate PEGylated RIT from the

parental RIT and unreacted PEG. The column was purchased from Water Corporation and

features a non-polar C4 stationary phase with 3.5m particle size and 2.1mm I.D. Mobile phases

A and B contain 5% and 90% acetonitrile, respectively and are supplemented with 0.1%

trifluoroacetic acid. 5l of ~1mg/ml samples were injected into the gradient run, starting from

95% buffer A to 100% buffer B in 10min.

Mesothelin Binding: A 96-well microtiter plate was coated with 50ul of 1g/ml MSLN-hFc, a

fusion protein consisting of human IgG Fc and mesothelin protein and incubated at 4°C

overnight. The plate was blocked with blocking buffer (1x PBS supplemented with 25% DMEM,

25mM HEPES, 0.5% BSA, 0.1% Azide, 5% FBS) and 50l serial dilution of the PEGylated

RITs was added in triplicate and 50l monoclonal anti-PE antibody (IP12) and HRP-labeled goat

anti-mouse IgG were added as a primary and secondary antibodies (4). After washing with

blocking buffer, the plate was developed with TMB substrate kits (Thermos Scientific) and

subsequently read with the plate reader at 640 and 450nm wavelength. Binding curve was

generated using GraphPad and 50% binding (IC50) was calculated. Table 1 summarizes average

IC50 values of two experiments.

ADP-ribosylation: A 20l reaction was set up to contain 4l buffer (20mM Tris-HCl, pH 7.5,

1mM EDTA), 1ul 1M DTT, 1ul 20ng/ul RIT, 5g protein lysate (prepared from KLM1 cell) and




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1l 250uM Biotin-NAD (Trevigen) and incubated at room temperature for 1 hour. Samples were

analyzed in SDS-PAGE gel and western blot was performed. The PVDF membrane was probed

with HRP-streptavidin followed by ECL development to visualize ADP-ribosylated EF2.

Cytotoxicity Assay: Human cell lines KLM1 (provided by Dr. U. Rudloff, National Cancer

Institute; 12), L55 (provided by Dr. T. Yamori, Pharmaceuticals and Medical Device Agency;

13) and A431/H9 (A431 human carcinoma cells transfected with mesothelin cDNA; 14) were

described previously and confirmed by short tandem repeat (STR) testing. MTBM test results

were all negative. Cells were grown in RPMI media supplemented with 10% FBS and 1%

penicillin-streptomycin at 37°C until 70-78% confluency before they were trypsinized. Then

4000 cells were seeded onto each well in a 96-well plate, and serial dilutions of RIT were added

in triplicate rows and incubated for 72 hours. Cytotoxicity activity was evaluated with a WST8

assay according to the manufacturer’s protocol (Dojindo Molecular Technologies). Plates were

read at 640 and 450nm wavelength, and data were plotted using GraphPad to obtain IC50 values.

Half-Life Assay: For each protein two mice (6-8 weeks old, 23-25g) were I.V. injected with

25g RIT. Blood was harvested at 5min, 2, 4, 8 and 24 hours for LMB-203-PEG, LMB-244-

PEG, and LMB-249-PEG and 5min, 1, 4, 24 hours for LMB-163-PEG and LMB-179-PEG

afterward, and serum was isolated. ELISA was used to determine the amount of RIT at each time

point as described in Mesothelin Binding experiment. Half-life and AUC was calculated using

GraphPad, with two-phase decay fitting, except LMB-12 which was fitted with one-phase decay.

The 5min sample was used as the initial amount of RIT in the blood, and the later time points

were normalized against this value.




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Radius of hydration (Rh): The Rh of five PEGylated proteins was measured using Dynamic

Light Scattering (Wyatt Technology). Proteins in the range of 0.5-1mg/ml were spun down at

13,000rpm for 5 min to pellet aggregates and 20l was transferred to a quartz cuvette slowly to

avoid bubbles. Each sample was measured at least two times, and each time constitutes an

average of 30 measurements. Rh values were extracted and plotted against the half-life,

cytotoxicity, and anti-tumor activity using GraphPad. Linear regression analysis was performed

to obtain a line of best-fit and R2 values.

Biodistribution of RIT: 5.8nmol of LMB-249-PEG and 4.0nmol of SS1P were incubated with

23.1nmol and 15.9 nmol of FNIR-Z-759 in PBS buffer, pH 8.5, at room temperature for 1 hour,

respectively. The labeled protein was purified with Sephadex G25 column. 3 mice bearing

~100mm3

A431/H9 tumor were I.V. injected with 41g (580pmol) of labeled LMB-249-PEG or

25g (400pmol) of labeled SS1P, followed by serial dorsal and ventral 800-nm fluorescence

imaging immediately and 15, 30min, 1, 2, 3, 4, 5, 6, 9, 12, 24, 48hours afterward. Fluorescence

accumulation in the kidney, liver, and tumor was monitored. Background fluorescence was also

calculated to obtain the target-to-background ratio. Plots were generated using GraphPad from

average results of 3 mice.

Anti-tumor Experiment: All animal experiments were performed in accordance with NIH

guidelines and approved by the NCI Animal Care and Use Committee. Female (10 per group)

nu/nu mice (6-week-old, 20-25g) were injected s.c. in the flank with 5 x 106 KLM1 cells (12).

When the tumor reached 100mm3, mice were grouped into similar weight and tumor size and




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RIT in PBS supplemented with 0.2% HSA was I.V. injected at 10g/mouse three times per week

for one week. Tumor volume and mice weight were monitored every two to three days. Mice

were euthanized if they lost more than 10% of the body weight. The experiment was repeated for

LMB-244-PEG and LMB-163-PEG (n = 10) and injected at 20g/mouse, two times per week for

two weeks. Mouse weight was monitored every two to three days and were euthanized if they

lost more than 10% of the body weight.

Results

Design of RITs

The RITs used in this study are shown in Figure 1A. SS1P is the first immunotoxin made

to target mesothelin and contains an anti-mesothelin Fv attached to PE38 that contains domains

II and III of PE (15). LMB-12 and LMB-84 both have domain II deleted and contain an 11-

amino acids furin cleavage site that connects the Fv to domain III (PE24). In LMB-12, the Vl

and Vh are connected by a disulfide bond and in LMB-84 they are connected by a flexible 15-

residues (G4S)3 linker. LMB-179, LMB-244, LMB-203, LMB-249, and LMB-163 are all

derived from LMB-84, but have cysteines used for PEGylation. They also contain a 17-residue

GS linker placed either between the Vl and the furin cleavage site (LMB-179, LMB-244, and

LMB-203) or at the N-terminus of the Vh (LMB-249). LMB-163 is similar to LMB-84 but

contains mutations that humanize the Fv portion. Figure 1B shows a cartoon representation of

the RITs. The 20kD PEG is shown as a red wiggly line.

We use site directed mutagenesis to insert cysteine residues at various locations in the

RIT. To choose locations that are less likely to interfere with immunotoxin activity, we modeled

the RIT using the existing structures of its various components. The model of immunotoxin is




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shown in Figure 2A. The structural complex of the immunotoxin bound to mesothelin and EF2 is

shown in Figure 2B, and it was generated by superposing the templates of mesothelin-mAb

(PDB 4F3F (16)), scFV (PDB 3GKZ (17)), PE-EF2 (PDB 1ZM4 (18)) and PE-NAD-AMP (PDB

1DMA (19)) in UCSF Chimera (20). Figure 2C shows a model of 20kD PEG, composed of 454

units of ethylene glycol, conjugated to immunotoxin at D406 of domain III. Five different sites

were mutated to cysteine for PEG conjugation as indicated by red balls. For all five locations, we

tried to diminish interference with mesothelin binding, processing by furin protease and ADP-

ribosylation of EF2.

LMB-179 and LMB-244 have cysteine residues located in domain III at E522 and D406,

respectively (Figure 1). The cysteine residues are located on different sides of PE24 and are

distant from the EF2 binding site (Figure 2A). LMB-203 has the cysteine placed in the middle of

a 17 amino acid linker connecting the Fv to the furin cleavage peptide (Figure 1). It is 8 residues

away from the furin cleavage site in order to diminish effects on cleavage or mesothelin binding

to the Fv or EF2 binding to domain III. In LMB-249 the cysteine residue is located on the amino

terminus of a 17-residue GS linker attached to the first residue of the heavy chain of the Fv

(Figure 1 and 2); this location is unlikely to interfere with the binding of mesothelin to the CDRs

of the Fv. LMB-163 does not contain an extra 17-residues GS linker like the other four RITs.

The cysteine is placed at S63 in the light chain (Figure 1 and 2). We chose S63 to mutate to

cysteine because the residue is surface-exposed and distant from the CDRs and the furin

cleavage peptide so the mutation is less likely to interfere with binding to mesothelin or cleavage

of the RIT by furin protease. All of RITs were expressed in E. coli in the form of inclusion

bodies, which were denatured, refolded and purified through Q Sepharose and Mono Q columns.




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PEGylation of RITs

Because a free thiol can lead to dimer formation, we stopped the purification after the

Mono Q column, where we usually observed a peak of monomer and another of dimer. Figure

3A shows Mono Q fractions of LMB-179, LMB-244, LMB-203 and LMB-249 that are mostly

monomer (lanes 2, 5, 6, and 8) or dimer (lanes 3, 4, 7, and 9, respectively). LMB-163 only has

monomer fractions (lanes 10 and 11). Monomer runs at ~50kD, and dimer runs slightly above

100kD, consistent with the predicted molecular weight of each species. Since both monomer and

dimer can be reduced by TCEP and used for PEGylation, we did not carry out final purification

on size exclusion chromatography as used for other RITs.

The PEGylation reaction was based on modifying a free thiol group of a surface-exposed

cysteine to form a thioester bond with the maleimide functional group of a 20kD linear PEG

(Figure 3B). To determine the best conditions for conjugation, we examined different conditions

and found that the reaction was most efficient in PBS at pH 8.0. TCEP has been shown to

enhance PEGylation efficiency compared to the standard reducing agents such as dithiothreitol

and β-mercaptoethanol, used in our previous studies (9). Since TCEP does not contain a free

thiol, there is no need to remove it prior to PEGylation, and the yields of reduced protein are

much higher than using traditional reducing reagents. A 10-fold molar excess of methoxy PEG-

maleimide was added to the TCEP-treated protein for PEGylation overnight. We performed

HPLC analysis of the reaction mixture to determine the PEGylation efficiency; a typical HPLC

run is shown for LMB-203 and its PEGylated counterpart LMB-203-PEG in Figure 3C. We

quantified the peaks and found that the PEGylation was ~90 percent complete. The unreacted

methoxy-PEG-maleimide was removed on an anion exchange column and the PEGylated protein

was eluted with PBS.




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Eluted proteins were analyzed on the SDS-PAGE gel along with pre and post-TCEP

treated samples (Figure 3D). For LMB-179 and LMB-244, the starting materials were mostly

dimer (lanes 2 and 5). For LMB-203 and LMB-249, there is a mixture of monomer and dimer

(lanes 8 and 11). LMB-163 was all monomer (lane 14) perhaps because the SH group in is not

readily available for dimer formation. After reduction with TCEP, all became monomers (lanes

3, 6, 9, 12 and 15). After addition of PEG, 90% or more of the TCEP-treated monomer was

PEGylated (lanes 4, 7, 10, 13 and 16). The calculated molecular weight of a PEGylated RIT is

about 72kD; however, they all run at ~100kD indicating the molecules are asymmetrical. The

PEGylated proteins were quantified using Image J. The PEGylation efficiencies were ~90%

overall, with LMB-203/LMB-203-PEG being 93-95%, which is consistent with the HPLC

analysis. The PEG-modified proteins were frozen at -70ºC in small aliquots and thawed as

needed for assays.

About 10% of each RIT could not be modified even when we added more methoxy-PEG-

maleimide and incubated for an extended period of time. We also tried removing TCEP before

the addition of methoxy-PEG-maleimide, but this did not improve the yield. We assume the

cysteine is modified during protein purification so that it cannot be reduced to generate a free

thiol by TCEP.

Cytotoxicity Assays

To determine whether PEGylation hampers the activity of the RITs, we performed

cytotoxicity assays on three mesothelin-expressing cancer cell lines. LMB-12 and LMB-84 have

nearly identical cytotoxic activity (Figure S1A); we picked LMB-12 as un-PEGylated control

since it has been widely studied. PEGylated and control RITs were incubated with cancer cells




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for three days, and WST assays were performed. The results are shown in Figure 4A. The cell-

killing data is best fit to a sigmoidal curve. In general, the range of linearity in which the cell-

killing activity is dependent on the concentration of RIT and occurs between 0.1-1ng/ml. Using

KLM1 cells, all of the cysteine containing proteins are as active as the LMB-12 except for LMB-

244 which is somewhat more active (Table 1). For KLM1 (pancreatic) cell line, parental RITs

LMB-249, LMB-203, LMB-163 and LMB-179 have IC50 of 0.22 ± 0.06, 0.29 ± 0.02, 0.22 ±

0.03, and 0.21 ± 0.01ng/ml, respectively. LMB-244 has an IC50 of 0.16 ± 0.03 ng/ml. The PEG

modified immunotoxins were 4-5-fold less active except for LMB-244-PEG, which lost only

35% activity. After PEGylation the IC50s for LMB-249-PEG, LMB-203-PEG, LMB-163-PEG

and LMB-179-PEG were 1.18 ± 0.03, 1.10 ± 0.19, 1.13 ± 0.19, and 1.09 ± 0.13 ng/ml, and

LMB-244-PEG was 0.25 ± 0.05 ng/ml. We also tested activities of LMB-244-PEG and LMB-

163-PEG on A431/H9 epidermoid carcinoma cells and MKN28 stomach cancer cells. Consistent

with KLM1 cell line, LMB-244 was the most active and lost less than 50% of its activity. To

show that PEG doesn’t affects mesothelin-specific cell killing, we tested a few of PEG-modified

RITs on the A431 epidermoid carcinoma cell line that was not transfected with mesothelin

cDNA. The result in Figure S1B shows no specific killing.

Figure 4 B-D shows bar graphs of the activity of PEG-modified RITs relative to the

parental control, which is set to 100%. It is evident that LMB-244-PEG is the most active. In

three tumor cell lines, KLM1 (Figure 4B), A431/H9 (Figure 4C), and MKN28 (Figure 4D),

LMB-244-PEG retains 65, 62 and 70% of the parental activity.

Mesothelin Binding and ADP Ribosylation are not affected by PEG




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The first step in the action of RIT is binding of the mesothelin on the cell surface of the

cancer cell by the Fv. To test if binding is affected by the PEG, we used serial dilutions of each

PEGylated RIT and tested the binding to mesothelin using ELISA. The results are shown in

Figure S2 and 50% maximum binding is indicated. The binding of the PEGylated RITs is close

to that of un-PEGylated LMB-12. The values are: LMB-12 (0.022 ± 0.001g/ml), LMB-249-

PEG (0.027 ± 0.001g/ml), LMB-203-PEG (0.024 ± 0.004g/ml), LMB-163-PEG (0.029 ±

0.006g/ml), LMB-179-PEG (0.023 ± 0.002g/ml) and LMB-244-PEG (0.013 ± 0.001g/ml).

Another crucial step in RIT action is the ADP-ribosylation and inactivation of EF2. This

step can be measured by incubating EF2-containing cell lysate with the ADP precursor NAD-

biotin and PEG-modified RIT and measuring the amount of biotin incorporated into EF2 using

western blots and Strep-HRP antibody. Figure S3 shows an ADP ribosylation assay, in which the

PEGylated RIT were incubated with the EF2-containing cell lysate. The resulting blots show a

RIT-dependent modification of EF2, with only one major band that is consistent with the

molecular weight of EF2 (indicated by arrow). The band is not detected in the negative control

where RIT was omitted (lane 16). We quantified the EF2-ADP bands and normalized each to

LMB-12 positive control. For accuracy, we loaded 5, 10 and 15 l of each reaction. Interestingly,

all RITs retain ~100% of the original activity. This indicates the loss of cytotoxic activity is not

due to the inability of the immunotoxin to inactivate EF2.

Half-life Measurements

To determine the half-lives of the PEGylated RITs in the circulation, we injected 25 g

of each protein IV into nu/nu mice and collected serum at 5-min and 2, 4, 8 and 24 hours for

LMB-203-PEG, LMB-244-PEG, and LMB-249-PEG, and at 5-min and 1, 4 and 24 hours for




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LMB-163-PEG and LMB-179-PEG. ELISA was used to determine the amount of remaining

RIT. To normalize the data, it is plotted relative to the 5-min time point, which is set as 100%

(Figure 5A). LMB-12, which has no cysteine added and is not PEGylated was used as an un-

PEGylated control. The decay for LMB-12 is rapid and mono-exponential. The curves for the

PEG-modified proteins best fit a two-phase decay function; therefore, we calculated two half-life

values for each protein, a fast (⍺) and a slow (β) decay (Table 1). We also calculated Area Under

Curve (AUC) to represent the amount in the blood present over time so that we have a single

value to compare for each protein. Figure 5A shows that all PEGylated proteins have much

longer half-lives than LMB-12. The AUC for LMB-12 is 0.2, while the PEGylated proteins have

AUCs that are at least 10-fold higher and range from 2.2 for LMB-244-PEG to 5.9 for LMB-

249-PEG. LMB-203-PEG has the second largest AUC of 5.1. LMB-163-PEG and LMB-179-

PEG have AUCs of 4.3 and 3.9. Based on the AUC values, the order from the longest to the

shortest half-life is LMB-249-PEG > LMB-203-PEG > LMB-163-PEG > LMB-179-PEG >

LMB-244-PEG. The difference between LMB-244-PEG and LMB-249-PEG is about 3-fold

even though the mass of each protein is identical.

The PEGylated proteins have much longer β than ⍺ decay. LMB-249-PEG has the

longest half-life with an ⍺ of 144-min and a β of 74400-min. LMB-163-PEG has an ⍺ of 29-min

and a β of 62200-min. LMB-244-PEG has the shortest half-life, with an ⍺ of 25-min and a β of

256-min. In summary, PEGylation extends the half-life of RITs significantly, particularly the β

phase. Because protein removal from the blood is controlled by glomerular filtration with

subsequent degradation in the kidney, we postulated that the proteins could have different

hydrodynamic radii that affected their rate of filtration by the glomerulus in the kidney.




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Hydrodynamic Radius

To determine if the addition of PEG at different locations changed the hydrodynamic

radius, we employed the Dynamic Light Scattering. Each sample was measured at least two

times and representative measurements are shown in Figure S4 and summarized in Table 1. The

%Mass is plotted against the radius. Overall, the results show a monomodal size distribution for

LMB-163-PEG, LMB-244-PEG, LMB-12 and LMB-203. For LMB-249-PEG, LMB-203-PEG,

and LMB-179-PEG, we observed polymodal size distributions, which is due to the presence of

trace amount of aggregate that also diffract laser light. LMB-12 and LMB-203 were included as

un-PEGylated controls. LMB-12 has the smallest Rh of 3.4nm. LMB-203 is slightly larger, with

Rh of 3.7nm (Figure S4). With addition of 20kD PEG, the Rhs increase substantially to 7.1, 6.9,

5.9, 5.6, and 5.2nm for LMB-249-PEG, LMB203-PEG, LMB-179-PEG, LMB-163-PEG, and

LMB-244-PEG, respectively. Comparing LMB-203 and LMB-203-PEG, the increase in Rh is

almost 2-fold.

Biodistribution Assay

It is widely-stated that PEG can extend the serum half-life by increasing the

hydrodynamic volume of biologics and reducing their renal filtration (5). To determine how

quickly the PEGylated RITs were removed by the kidney and liver, we performed biodistribution

studies with LMB-249-PEG labelled with FNIR-Z-759, a near-infrared fluorophore that allows

external imaging of living mice (21). Labeled LMB-249-PEG (41g, 580pmol) was injected I.V

into tumor-bearing mice and accumulation in the kidney, liver, and tumor was imaged over 12

hours. The images in the upper panel of Figure S5A are from a dorsal view, showing uptake by

the kidneys and by tumor that are close to the dorsal side of the mouse. The images in the lower




18

panel of Figure S5A show uptake by liver which is close to the ventral side. To compare the

biodistribution with an immunotoxin without PEG, we performed the same experiment with

SS1P, which has a MW of 63kD, close to 72kD for the PEG modified RITs. The upper panel of

Figure S5B shows accumulation of SS1P in kidney and tumor and the lower panel shows liver

uptake.

Quantification of Uptake

To determine the amount of LMB-249-PEG and SS1P taken up by kidney, liver, and

tumor, the images were scanned and quantified. Accumulation as a function of time for kidney is

plotted in Figure 5 C, for liver in Figure 5D, and for tumor in Figure 5E. The graphs show that

uptake of LMB-249-PEG in kidney peaks at 6 hours whereas uptake of SS1P peaks much earlier

at 2 hours. Liver uptake of LMB-249-PEG peaks late at 9 hours, while SS1P uptake much earlier

at 2 hours. The results for tumor follow a different pattern. Accumulation of LMB-249-PEG by

tumor increases steadily over many hours and peaks at 9-12 hours, whereas uptake of SS1P is

much smaller and peaks at 2-3 hours.

Anti-tumor experiments

To do an initial assessment of the anti-tumor activity of the various PEGylated RITs, we

treated tumor-bearing mice IV with 10g of PEG-modified RIT given every other day x3, when

the tumors reached about ~100 mm3 in size. In the PBS treatment group, the tumors grew

rapidly (Figure 6A). All treatment groups responded to PEGylated RITs. LMB-244-PEG and

LMB-163-PEG were the most active and the tumors decreased in size to 69 mm3 and 79 mm

3 on

day 14 and the p-values are both 0.001, respectively. LMB-179-PEG, LMB-203-PEG and LMB-




19

249-PEG slowed tumor growth; on day 14, the tumors reached 259 mm3 in the control group and

only 152, 117 and 143 mm3 in LMB-179-PEG, LMB-203-PEG and LMB-249-PEG,

respectively. The weights of the treated mice decreased by less than 10% (Figure S6A), but two

mice in the LMB-179-PEG group and one in the LMB-249-PEG died.

Since LMB-244-PEG and LMB-163-PEG are the most active, we performed additional

studies with them using a dose and schedule that did not cause weight loss. We compared their

activity with that of LMB-12, which is very similar to LMB-84 in structure and activity and is

not PEG modified. Figure 6B shows that tumors in mice receiving five 20g doses given over 2

weeks of PEG modified RIT regressed to 46mm3 and 48mm

3, whereas tumors in mice treated

with LMB-12 increased in size but grew more slowly than tumors treated with PBS. The p-

values for LMB-244-PEG and LMB-163-PEG compared to LMB-12 are 0.0049 and 0.0048,

respectively. At this schedule the weight of the mice decreased by less than 10% and no mice

died (Figure S6B). This data establishes that increasing half-life greatly increases anti-tumor

activity.

Discussion

We have developed a method to perform site specific PEGylation at cysteine residues

present at different locations in an anti-mesothelin RIT and identified 2 locations that produced

PEG modified RITs with high cytotoxic and anti-tumor activity and very prolonged half-lives in

the circulation. Our strategy utilizes structural information to identify PEGylation sites that are

least likely to interfere with binding to mesothelin or to EF2 or to affect furin cleavage. Since

there are many lysine residues in the RIT, we could not achieve specificity using lysine-specific

PEGylation and chose the highly efficient site-specific PEGylation of cysteine. Although there




20

are two cysteine residues in Fv, they are buried and not available for reduction. Therefore, we

used site-directed mutagenesis to introduce cysteines at locations exposed and distant from

known functional sites in the RIT. We were able to PEGylate these mutant RITs at high

efficiency. The key to this success is the use of TCEP to reduce the protein followed by treating

with a 20 kDa maleimide derivative of PEG at pH 8.0. This that had not been achievable with β-

mercaptoethanol in our previous study (8).

The most active of the PEG modified RITs is LMB-244-PEG, which has a PEG attached

to residue 406C in domain III of the toxin (Figure 1A). We tested LMB-244-PEG on 3 cell lines

and found it retained more than 50% of its cytotoxic activity on all lines. When tested in mice, it

had an 11-fold increase in residence time (AUC) in the circulation compared with an un-

PEGylated protein LMB-12 and this change resulted in a very large increase in anti-tumor

activity (Figure 6). To account for differences in activity, we measured its radius of hydration

and found that LMB-244-PEG had the smallest Rh. This finding suggests that one or more steps

in immunotoxin action are negatively affected by larger Rh of the protein and a smaller Rh is

better. We also observed that LMB-244-PEG has a shorter half-life and AUC than the other

PEGylated RITs indicating it is more efficiently filtered by the kidney glomerulus than RITs

with higher Rh values.

Because we used the same 20 kDa PEG to modify all the RITs, we were surprised to

observe that the Rh values varied widely from 5.2 to 7.1 indicating that the derivatized proteins

adopted different conformations. Since the Rh values correlate with the half-life in the

circulation (Figure 5B), and since kidney is the major organ responsible for removal of RITs, we

assume that the glomerulus in the kidney is sensitive to the size and shape of the proteins and

filters them at different rates. In addition, the PEG-modified proteins with the smallest Rh




21

(LMB-244-PEG) had excellent anti-tumor activity, suggesting that entry into tumors is also

affected by Rh. We plan to use external tumor imaging to analyze the entry into tumors of all 5

PEGylated RITs in a future study.

The mechanism by which RITs kill cells is complex, beginning with binding to

mesothelin on the cell surface and ending with inactivation of EF2 in the cytosol. We are unable

to assess each step in the pathway but did assess two crucial steps and found that binding to

mesothelin was similar for all PEG modified RITs, although there was a small loss of binding to

mesothelin by the two that had PEG added to the Fv (Figure S2). We also found no change in the

ability of the modified proteins to ADP-ribosylate EF2 (Figure S3). We are currently unable to

measure how efficiently RITs are processed by other steps in the pathway.

The half-life data of PEGylated RITs fits well to biexponential decay curves (Figure 5A).

We think it is likely that the ⍺ phase is due to rapid equilibration with the extracellular

compartment, and the β phase is due to the metabolism by the kidney and liver (22). We used

external imaging to determine which organs in mice take up SS1P, a small un-PEGylated RIT

with a half-life of 19 min (AUC 0.2) and LMB-249-PEG with a long half-life and an AUC of

5.9. The data in Figure 5C and 5E show that SS1P is rapidly taken up by kidney and by liver,

whereas the uptake of LMB-249-PEG is delayed. This delay leads to the greatly increased half-

life and AUC. The initial uptake by tumors is the same for both RITs, but the uptake of SS1P is

arrested as blood levels fall (Figure 5D), whereas the uptake of LMB-249-PEG increases for

many hours and must be responsible for the enhanced anti-tumor activity of PEGylated RITs.

It is widely-stated that PEG increases the half-life by increasing the hydrodynamic

volume, and it has been shown increasing the size of PEG attached to mmTrail increases serum

half-life (23). Because a change in PEG size does not account for the different biological




22

properties of our proteins, we employed dynamic light scattering to assess the hydrodynamic

volume of the proteins and found for the first time in our knowledge that the hydrodynamic size

of protein can be fine-tuned depending on the position of the PEG. In Figure 5B, the Rh values

are plotted against AUC. The result shows a good correlation between AUC and Rh with the R2

value of 0.8. In general, larger Rh corresponds to longer half-life. The un-PEGylated LMB-12 is

smallest protein and has a Rh of ~3.4. LMB-203 is slightly larger (RH 3.7) having a 15 amino

acid linker connecting the light and heavy chains and 17 amino acid linker connecting the Fv to

the furin cleavage site (Figure S4). The PEGylated proteins are much larger, with average Rhs of

7.1, 6.9, 5.6, 5.9, 5.2 nm for LMB-249-PEG, LMB-203-PEG, LMB-163-PEG, LMB-179-PEG,

and LMB-244-PEG, respectively. Therefore, our results indicate that the PEGylated RITs adopt

distinct conformations governed by the position of the PEG, and such conformation influences

the half-life. Since Rh is proportional to the hydrodynamic volume, we also concluded that PEG

increases the hydrodynamic volume, which leads to slower renal filtration and longer half-life,

and this is consistent with the reported properties of PEG (5).

The anti-tumor activity of the PEGylated-RITs varied widely. Two of them, LMB-244-

PEG and LMB-163-PEG, produced substantial tumor regressions, but the others were less active

(Figure 6A). The high anti-tumor activity of LMB-244-PEG reflects several factors. The starting

protein, LMB-244, is almost 2-fold more active than the other proteins and PEG addition caused

less than a 2-fold loss of activity. Also, LMB-164-PEG has the smallest Rh indicating it is more

compact and probably enters tumors better than the other PEG modified proteins. However, the

small Rh probably contributes to its low AUC of 2.2. LMB-163-PEG also has good tumor

activity despite being 3-fold less cytotoxic than LMB-264-PEG. The basis of the variable anti-

tumor activity is not understood and will be the subject of further studies.




23

Conclusion

We have developed a new approach to carry out site specific modification of RITs with

PEG and identified two RITs that have a long half-life and high anti-tumor activity. We have

shown that the location of the PEG affects the hydrodynamic size of the protein and contributes

to anti-tumor activity. PEG modified RITs merit further preclinical development for cancer

therapy.

Acknowledgements

Molecular graphics images were produced using the UCSF Chimera package from the Resource

for Biocomputing, Visualization, and Informatics at the University of California, San Francisco

(supported by NIH P41 RR-01081). We thank the Biophysics Resource in the Structural

Biophysics Laboratory, Center for Cancer Research, NCI at Frederick for assistance with light-

scattering studies. This research was supported by the Intramural Research Program of the NIH,

the National Cancer Institute, and the Center for Cancer Research (I.P.).




24

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27

Table 1. Summary of properties of RITs

RIT names

IC50 (ng/ml) in

KLM 1

Half Max

Mesothelin

Binding Half Life (min) AUC Rh

without PEG

with PEG (%

Activity) (g/ml) Fast () Slow ()

(arbitrary unit,

x104)

(nm)

SS1P 0.19 n/a nt 19 n/a nt nt

LMB-12 0.22 n/a 0.022 0.001 11 n/a 0.2 3.4

LMB-84 0.19 n/a nt nt n/a nt nt

LMB-249 0.22 0.06

1.18

0.03(18%) 0.027 0.001 144 74400 5.9 7.1

LMB-203 0.29 0.02

1.10 0.19

(26%) 0.024 0.004 59 51600 5.1 6.9

LMB-163 0.22 0.03

1.13 0.19

(19%) 0.029 0.006 29 62284 4.3 5.6

LMB-179 0.21 0.01

1.09 0.13

(19%) 0.023 0.002 38 26600 3.9 5.9

LMB-244 0.16 0.03

0.25 0.05

(64%) 0.013 0.001 25 256 2.2 5.2

nt: not tested; n/a: not applicable

Bolded numbers refer to PEGylated RITs




28

Figure Legends

Figure 1. Schematics of anti-mesothelin RITs. A. SS1P contains an anti-mesothelin disulfide

linked Fv connected to PE38 (domain II and III of PE). LMB-12 is derived from SS1P with

domain II deleted and replaced with an 11 amino acid furin cleavge peptide connecting the Fv to

domain III. LMB-84 contains a single-chain Fv instead of a dsFv and is the parental RIT from

which the cysteine containing PEG-modified proteins were prepared. LMB-179, LMB-244, and

LMB-203 have an additional 17-GS linker that separates Vl from the furin cleavage site. LMB-

249 has GS linker placed at the N-terminus of Vh. LMB-163 contains mutations that humanize

the Fv portion. For each construct, the engineered cysteine for site-specific PEGylation is

indicated by a bolded red C. B. Cartoon representations of RITs shown in A. PEGs on cysteines

are represented by red lines. Domains are not drawn to scale.

Figure 2. Ribbon diagrams of anti-mesothelin RITs. A. The ribbon diagram was generated

with UCSF Chimera. Various domains are colored as: N terminal linker = grey, Vh = blue, Vl =

cyan, CDR = orange, furin site = green, and PE24 = yellow. Sites that were mutated to cysteine

are shown as red spheres, and they were chosen to avoid interfering with functional binding to

mesothelin, furin protease, and EF2. B. A structural model of the complex of the RIT in A bound

to mesothelin (olive green) and EF2 (dark gray). C. A hypothetical 20kD linear PEG composed

of 454 repeating units of ethylene glycol is connected to the cysteine at D406 of PE24 (LMB-

244). The structure of PEG is random, and the overall size of the molecule is modeled to

approximate the average radius of hydration measured for all five PEGylated RITs.




29

Figure 3. PEGylation of RITs. A. RITs eluted from Mono Q anion exchange column. Fractions

A, B, and C correspond to separate peaks in the Mono Q chromatogram and were pooled

separately. B. Schematic showing a hypothetical site-specific PEGylation reaction, which occurs

by the formation of a thioester bond between a free thiol of a surface-exposed cysteine and a

maleimide functional group of a PEG. C. Representative HPLC analysis of LMB-203/LMB-203-

PEG. D. Non-reducing SDS-PAGE gel analysis of parental, TCEP-reduced, and PEGylated

RITs. Gels were visualized by Coomassie Blue staining. The efficiencies of PEGylation were

quantified by counting the number of pixels using Image J. PEGylated RIT is indicated by the

blue square. White lines indicate the point of merge between two separate gels.

Figure 4. WST assays of parental and PEGylated RITs. A. Representative cytotoxicity assays

after 3 days incubation of mesothelin-positive cancer cell lines with different RITs. KLM1,

pancreatic cancer cell line; A431/H9, epidermoid carcinoma cell line transfected with mesothelin

cDNA; MKN28, gastric cancer cell line. B-D. The IC50 values of PEGylated RITs were

normalized against the un-PEGylated controls which were set to 100%. The percent remaining

activity is indicated.

Figure 5. Biological properties of RITs. A. Pharmacokinetics of PEGylated RITs. 25g of

LMB-203-PEG, LMB-244-PEG, LMB-163-PEG, LMB-179-PEG, and LMB-249-PEG were

injected into nu/nu mice and blood was collected at 5 min, 1 (LMB-163-PEG and LMB-179-

PEG), 2, 4, 8 (LMB-203-PEG, LMB-244-PEG, and LMB-249-PEG), and 24 hour time points.

ELISA was performed to determine the level of RIT and plotted in two-phase decay using

GraphPad. Amount of RIT at 5 min was set to 100% and used to normalize the remaining time




30

points. LMB-12 was data from our previous study and plotted as one-phase decay. The AUC

values are summarized. B. Rh of PEGylated protein was measured using Dynamic Light

Scattering. 20l aliquots of proteins (0.5-1mg/ml) were measured at least two times and Rh was

plotted against half-life using GraphPad. The same color scheme was used as in A. The line of

best fit was generated with R2 value indicated. C-E. Biodistribution of LMB-249-PEG and SS1P

in mice. Fluorescently labeled LMB-249-PEG and SS1P was I.V. injected into A431/H9 tumor-

bearing mice (n=3, tumor ~100mm3), respectively. Serial dorsal and ventral 800nm fluorescence

images were obtained with focus placed on the tumor, kidney, and liver. The fluorescence

intensity at each time point was quantified and plotted to show the accumulation in the

kidney(C), tumor(D) and liver(E). Plots were generated from the average results of 3 imaged

mice.

Figure 6. Anti-tumor activity of parental and PEGylated RITs. A. Antitumor activity of

PEGylated RITs in nude mice (n = 10). Mice were implanted with mesothelin-expressing KLM1

tumor cells. When tumors reached ~100mm3, mice were I.V. injected with 10g/mouse

PEGylated RITs qod x3 as indicated by arrows. Tumor burden was monitored over 2 weeks. B.

Comparison of anti-tumor activities of LMB-12, LMB-163-PEG and LMB-244-PEG. Groups of

10 mice were treated with 20 g of each agent as shown by arrows and tumor burden was

monitored over 2 weeks.




Published OnlineFirst December 23, 2019.Mol Cancer Ther Zeliang Zheng, Ryuhei Okada, Hisataka Kobayashi, et al. immunotoxins increases half-life and anti-tumor activitySite-specific PEGylation of anti-mesothelin recombinant

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