ORIGINAL ARTICLE

Farletuzumab, an anti-folate receptor a antibody, does not blockbinding of folate or anti-folates to receptor nor does it alterthe potency of anti-folates in vitro

B. A. Kamen • A. K. Smith

Received: 2 February 2012 / Accepted: 9 May 2012 / Published online: 27 May 2012

� Springer-Verlag 2012

Abstract

Purpose Folate is a cofactor in the synthesis of purines and

pyrimidines; folate analogs are potent cytotoxic drugs.

Folate receptor alpha (FRa), a protein-mediating cellular

accumulation of folate (and anti-folates), has limited

expression in normal tissues and is overexpressed by

numerous carcinomas. Limited distribution and high affinity

for folic acid have resulted in the development of antibodies

or the use of folic acid coupled to toxins or radionuclides as

therapeutic and imaging agents. Farletuzumab is an anti-

FRa antibody in clinical trials for ovarian and non-small cell

lung cancers. Our goal was to evaluate the effect of far-

letuzumab on binding and uptake of folates and anti-folates

and the potency of anti-folates in vitro.

Methods Direct binding and uptake of radiolabeled folates

and anti-folates and the assessments of drug concentration of

drug that inhibited cell growth 50 % (IC50) in vitro in the

presence or absence of antibody.

Results Farletuzumab did not block membrane binding of

radiolabeled folic acid, 5-methyltetrahydrofolate, pemetr-

exed, and other anti-folates; folic acid blocked [95 %.

Farletuzumab had a minimal effect on the cytoplasmic

accumulation of 5-methyltetrahydrofolate or pemetrexed;

folic acid had a considerable but variable effect on the

different cell lines. As a single agent, farletuzumab did not

affect cell viability or the IC50 of pemetrexed and other

anti-folates in vitro.

Conclusions Farletuzumab does not block FRa binding of

folates and anti-folates, minimally retards folate delivery

via FRa-mediated transport, and minimally retards the

growth of cells in vitro. Concomitant use of farletuzumab

and pemetrexed is not contraindicated.

Keywords Anti-folate � Folate receptor � Farletuzumab �Pemetrexed

Introduction

Folate is a water-soluble vitamin, a cofactor in the synthesis of

purines and pyrimidines [1]. The critical nature of these

pathways has made them targets for anti-folates (folate ana-

logs), drugs used as antibiotics, immunosuppressives, and

anticancer agents. For example, methotrexate (MTX) has

been a mainstay for patients with acute lymphoblastic leuke-

mia for about 60 years and also remains one of the most

commonly used disease-modifying antirheumatic drugs

(DMARD). Concomitantly, with the empiric clinical suc-

cesses, the study of folate and anti-folate transport and intra-

cellular metabolism has led to a greater understanding of the

mechanisms of resistance to anti-folates and subsequently, the

development of newer agents which are either transported

more robustly or are better substrates for folylpolyglutamate

synthase [2]. One such drug is pemetrexed (Alimta�, Eli Lilly

& Company), initially referred to as the multitargeted anti-

folate because it inhibited three enzymes: thymidylate syn-

thase, dihydrofolate reductase, and glycinamide ribonucleo-

tide formyl transferase. It is approved for patients with

mesothelioma and non-small cell lung cancer (NSCLC).

A greater understanding of folate accumulation has also

led to the development of agents that target a specific

molecule on the membrane. Folate receptor a (FRa), one

of the four members of the folate receptor family, has a

high affinity for the natural, major serum folate,

B. A. Kamen (&) � A. K. Smith

Cancer Institute of New Jersey, Robert Wood Johnson

Medical School, 195 Little Albany Street,

New Brunswick, NJ 80901, USA

e-mail: kamenbart@gmail.com

123

Cancer Chemother Pharmacol (2012) 70:113–120

DOI 10.1007/s00280-012-1890-2

5-methyltetrahydrofolate (Kd * 10-9 M), and an even

greater affinity for folic acid (Kd * 10-12 M). It also binds

pemetrexed and, to a much lesser extent, MTX [3]. FRa has

a limited normal tissue distribution and is overexpressed by

a number of carcinomas [4]. This specificity, affinity, and

limited tissue distribution have led to using folic acid

coupled to toxins or radionuclides and the development of

antibodies as therapeutic or imaging agents [5].

Farletuzumab, an anti-FRa antibody (Ab), is currently in

multiple clinical trials for women with ovarian cancer and,

more recently, in NSCLC [6, 7]. Pemetrexed is indicated for

the treatment for NSCLC and mesothelioma and has shown

some activity in patients with ovarian cancer [8, 9]. If far-

letuzumab and anti-folates are to be used concomitantly, it is

important to know the effect of the antibody on folate and

anti-folate binding and accumulation as well as potency of

the latter in FRa? tumors. Previously, it has been shown

that farletuzumab mediates antibody-dependent cellular

cytotoxicity (ADCC) and complement-dependent cytotoxic-

ity (CDC) and has some effects on the growth of a Chinese

Hamster Ovary (CHO) cell line transfected with FRa (CHO-

FR) when grown in low folate, but its effects on folate uptake

and toxicity in human cell lines naturally expressing FRa have

not been reported [10]. Here, we show that, in several cell

lines, naturally expressing FRa farletuzumab did not block

binding of folic acid, 5-methyltetrahydrofolate, pemetrexed,

and other anti-folates and only minimally altered FRa-medi-

ated folate uptake and cell growth in vitro. Moreover, the

concentration of drug that inhibited cell growth 50 % (IC50) of

MTX, aminopterin, and pemetrexed (PMX) in vitro were not

significantly affected by the same Ab concentration that

mediated ADCC and CDC [10]. As important controls, we

found that (a) exogenous folate in the medium did decrease

PMX potency, and (b) functionally disabling FRa by satu-

rating with low-dose folic acid decreased PMX accumulation

but not cytotoxicity. This latter point confirms that FRa is not

the major transport system for pemetrexed or folates. This is in

agreement with the work of others [11, 12].

Materials and methods

Materials

Radiolabeled compounds (5-methyltetrahydrofolic acid

[3H-MTHF], folic acid [3H-FA], and pemetrexed [3H-PMX])

were obtained from Moravek Biochemicals. The specific

activity of these compounds varied from batch to batch and

ranged from 10,000 to 40,000 DPM/pmole. They were puri-

fied by high-performance liquid chromatography (HPLC)

when the non-specific background was[5 %.

Roswell Park Memorial Institute (RPMI) folic acid-free

medium was from Invitrogen #27016-021 and fetal bovine

serum (FBS) was purchased from Gibco�. All other com-

pounds for buffers (e.g., Tris, saline) were purchased from

commercial sources such as Sigma. Pemetrexed, commer-

cially available, was supplied by our Pharmacy and ami-

nopterin and MTX purchased from Sigma. Farletuzumab

was provided by Morphotek, Inc.

Cell lines

IGROV-1, MA104, JAR, BeWo, HK2, and CHO cells

(both transfected with FRa and parent) were maintained in

RPMI folic acid-free medium containing 10 % FBS, with

20 pmol/ml fresh 5-methyltetrahydrofolate added to stock

flasks only at time of split (approximately weekly), so that

the final concentration of reduced folate in the medium of

experimental cells was 5 nM, with the exception of CHO

parent, which required additional folate in experimental

plates as well for adequate growth [13–16].

Folate and anti-folate uptake

Cells were grown in RPMI without folic acid with 10 %

FBS and Penicillin and Streptomycin until the desired level

of confluence was reached. Typically, T25 flasks were

seeded with 0.2 million cells each in 3–5 ml media. For

multi-well plates, approximately 1.5 million cells from

stock flask were diluted with 50 ml growth medium

(30,000 cells/ml) and then plated at: 2 ml/well for 6-well

plates, 1 ml/well for 12-well plates, 0.5 ml/well for 24-well

plates, 0.25 ml/well for 48-well plates, or 0.1 ml/well for

96-well plates (yielding final plating density of approxi-

mately 7,000–9,000 cells/cm2). On the day of an experi-

ment, the media were aspirated and cells rinsed once with

Hanks Balanced Salt Solution (HBSS).

For assessing vitamin or drug accumulation, the growth

media were replaced with incubation media (either HBSS

or RPMI without folate or FBS) and the desired 3H-labeled

compound as indicated in each experiment. To control for

non-specific uptake, a flask or well of each condition/

concentration was incubated with a 200–5009 excess of

non-labeled compound. To harvest, cells were chilled on

ice, the media aspirated, and plates were washed 39 with

large volumes of ice-cold phosphate-buffered saline (PBS).

Freeze–thaw (FT) buffer (10 mM Tris, 1 % beta mercap-

toethanol) and 0.5 lM MTHF were added in the same

volume as initial incubation, and the flasks or plates were

frozen flat at -80 �C for at least 15 min to lyse the cells

(plates are wrapped in plastic wrap first to deter evapo-

ration; flasks caps are tightened). The cells were then

thawed on ice, scraped with a plastic cell scraper, and

collected into microcentrifuge tubes along with a FT

buffer rinse. Tubes were then centrifuged at 1009g for

20 min at 4 �C, and the supernatant carefully removed.

114 Cancer Chemother Pharmacol (2012) 70:113–120

123

The entire supernatant (intracellular uptake) was aspirated

and counted for radioactivity. The pellet, representing

membrane-bound compound, was also subjected to analy-

sis by liquid scintillation counting after solubilizing in

0.1 N NaOH. These techniques are detailed in our previous

reports [14–16].

Folic acid binding to folate receptor (AL and AR)

Direct binding

3H-folic acid was added to the incubation medium. At the

end of incubation, the cells were chilled on a bed of ice, the

media aspirated, and cells washed 39 with large volumes

of ice-cold PBS. Acid saline (pH 3.5 with acetic acid) was

added (same volume as 3H incubation), and the flask or

plate rocked for 30 s. The acid wash was collected into

scintillation vials, and the cells rinsed with PBS (about

5–10 s) and that wash added to the first. This represents the

surface-bound ligand referred to as the acid-labile (AL)

fraction. The radioactivity remaining with the acid-resis-

tant (AR) cells was determined either by trypsinizing the

cells and collecting them with a rinse or by solubilizing the

cells with 0.1 N NaOH. At least one of the non-specific

wells was collected by trypsinization, so that a cell count

could be done (if growth conditions or experimental

treatment was likely to have changed the final cell count,

then one well from each condition was counted). These

techniques are detailed in our previous efforts [14–16].

Cytotoxicity

Cells were plated in 96-well plates in 100 ll growth media

(RPMI without folic acid, with 10 % FBS and Pen-Strep) at

3,000 cells/well. Cells were usually allowed to settle and

attach before additives or treatments were begun (generally

2–4 h). Antibody and/or folate was added in a 50 ll volume

at 49 concentration to appropriate wells. Drug or further

treatments were added in additional 50 ll (also 49), and

cells were allowed to grow as indicated in experiment.

Phenazine methosulfate (PMS)-activated MTS (Promega)

was added to wells (usually 25–30 ll/well) and incubated for

2–4 h. Absorbance at 490 nm was read on a plate reader and

the IC50 calculated from the maximum and minimum read-

ings for each set of conditions. A media/MTS control was

subtracted as background. All cell lines had been adapted to

grow in folic acid-free medium with 10 % FBS, with no

additional folate added except as indicated.

Analysis

All experimental points shown here were done at least in

triplicate and repeated at least twice on separate days. In

order to account for non-specific binding and uptake, folic

acid or 5MTHF at 200–500 times the radiolabeled com-

pound was also included in each experiment. This back-

ground was generally \5 % of total counts or the

radiolabeled compound was purified before using. The data

are plotted showing the 95 % confidence limits using Prism

IV (Graph Pad, San Diego California).

Results

Farletuzumab does not block binding of folic acid

to FRa

FRa, a glycosyl phosphatidyl inositol (GPI) linked pro-

tein, is clustered in lipid rafts on the membrane and

cycles between a surface (acid labile) and internalized,

but still membrane-bound, compartment (acid-resistant)

[13]. To begin the assessment of potential effects of far-

letuzumab on FRa function/cellular location, we first

CHO-FR cells

0 1 10 1000

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Farletuzumab (ug/ml)

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/105 c

ells

control 1 10 1000

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Farletuzumab (ug/ml)

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ells

Fig. 1 Farletuzumab has little effect on the steady-state distribution

of FRa bound folic acid: CHO-FR and Ma104 cells were incubated

for 2 h with 10 nM 3H-folic acid at 37�, and then the cells were

processed to determine the surface binding (acid labile, AL) and

internal pool (acid-resistant, AR) using an acid buffer wash as

detailed in ‘‘Materials and methods.’’ The open bars are the AL and

the closed bars the AR pool of bound 3H-folic acid. Farletuzumab

was added 2 h before the 3H-folic acid and remained in the medium

for the entire incubation

Cancer Chemother Pharmacol (2012) 70:113–120 115

123

compared the binding of 10 nM 3H-folic acid by MA104

cells (monkey kidney proximal tubule cell) and CHO cell

transfected with FRa (CHO-FR). The former was the

initial model for FRa-mediated folate transport and the

latter the cell line used to initially characterize far-

letuzumab effects in vitro [10, 13–15]. Using up to a 109

greater concentration than what was used in cytotoxicity

studies previously reported [10], farletuzumab had little

effect on folic acid binding (Fig. 1). Confirming an earlier

report [13], the CHO-FR cells bind 6–7 times more folic

acid than MA104 cells, and the ratio of the AL/AR pool

is approximately 4:1, that is, the majority of the receptor

was in the external pool, whereas in the MA104 cell the

ratio was approximately 1.5:1. To more thoroughly study

the effects of the Ab, we studied the binding of folic acid

by the naturally overexpressing IGROV-1 cells. Binding

was essentially complete by 2 h, and the ratio of AL/AR

was about 4:1 (Fig. 2a). Importantly, even at 6 h, there is

no significant cytoplasmic accumulation of folic acid

(solid circle at 6 h time point). Folic acid has such a great

affinity for FRa that at this low, but saturating, concen-

tration, it is not released into the cytoplasm of the cell;

therefore, it is an excellent marker of membrane binding

[13]. Cells in Fig. 2b (IGROV-1), c (HK2), and d (JAR)

show that farletuzumab does not block binding of 3H-folic

acid, as total membrane radioactivity (AL ? AR) was

similar in controls and Ab-treated cells. The JAR cells

have an AL/AR ratio similar to the IGROV-1 cells

(*4:1) and other malignant cell lines, such as KB, HCT8,

and SKOV, which we have studied (data not shown)

while the HK2 is similar to the MA104 cells (1.5:1). Why

cancer cells and cells transfected with FRa have the

majority of the receptor on the external surface while the

nonmalignant cells have a more equal distribution is not

known. Interestingly, treating MA104 cells with phorbol

myristic acid (PMA) externalizes the majority of the

receptor, and PMA has no effect on malignant cells [21].

There does appear to be a small decrease in binding of3H-folic acid-resistant (AR) compartment (Fig. 2c, d) and

slight increase in the acid-labile fraction in Fig. 2b. We

speculate that these small differences in the distribution of

FRa on the membrane may be a result of the fact that

compartmentalization of a GPI-anchored protein affected

by the bulk of the protein attached to the lipid anchor [17].

0 1 2 3 4 5 60

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AR

Hours

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ell

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ell

Fig. 2 Time course of 3H-folic acid binding to membrane in naturally

expressing FRa human cell lines in the presence and absence of

farletuzumab: Cells (IGROV-1, ovarian cancer), JAR (choriocarci-

noma) and HK2(human kidney epithelial cells) were incubated with

10 nM 3H-folic acid at 37� for the times indicated, and then surface

membrane binding (i.e., acid labile, AL) and the internalized membrane

fraction (i.e., acid-resistant, AR) were determined. a The time course to

the saturation of the receptor pool and the lack of cytoplasmic folic acid

(solid circle at 6 h) accumulation in IGROV-1 cells. b (IGROV-1);

c (HK2) and d (JAR) Farletuzumab (10 lg/ml) added 2 h before 3H-

folic acid had no significant effect on the binding of 3H-folic acid or the

distribution of the receptor in the AL or AR compartment. Solid line is

AL and dotted lines are AR. Squares = control; Triangles = sample

with Ab

116 Cancer Chemother Pharmacol (2012) 70:113–120

123

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+ Folic Acid

+ Ab (10ug)

+ Ab (50ug)

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Time (hours)

% m

ax u

pta

ke

Fig. 3 Total membrane binding and cytoplasmic accumulation of3H-MTHF and 3H-PMX in the presence or absence of farletuzumab or

folic acid: a (IGROV-1 cells) and b (HK2 cells) incubated with 3H-

MTHF (20 nM) and c (IGROV-1) and d (HK2 cells) with 3H-PMX

(50 nM). e 3 H-MTHF uptake in the presence of either 10 or 50 ug/ml

Ab or 5 nM folic acid normalized to control. In a–d, Farletuzumab

(10 lg/ml) or folic acid (5 nM) when present was added 2 h before

vitamin or drug. Squares = control; Triangles = farletuzumab; Circles =

folic acid. Solid line = cytoplasmic uptake; dashed line = membrane

binding. a–d 95 % Confidence limits

Cancer Chemother Pharmacol (2012) 70:113–120 117

123

The Ab (160,000 kD) would add significant mass to the

approximately 40 kD FRa. This observation and the

potential effect on FRa cycling (and other proteins that

may also be in the same lipid raft) are under further study

and will be discussed below and in greater detail

elsewhere.

Folic acid, but not farletuzumab, blocks binding

of 5-methyltetrahydrofolate and pemetrexed

and also decreases cytoplasmic accumulation

of these ligands more than Ab

As shown in Fig. 2a, FRa has such a great affinity for folic

acid (Kd * 10-12 M) compared with the natural serum

folate, 5-MTHF, that it does not accumulate to any sig-

nificant degree under low physiological concentrations

(e.g., 10–20 nM) because it is not released from the

receptor and is a very poor substrate for the reduced folate

carrier (Kt * 100–200 lM). Therefore, to more com-

pletely assess the effect of farletuzumab on FRa-mediated

vitamin or drug transport, we measured membrane binding

and cytoplasmic accumulation of the natural serum folate,

MTHF, and PMX in the presence of Ab and/or folic acid.

The latter will block binding to FRa; therefore, uptake of

any substrate under these conditions is a measure of non-

FRa-mediated uptake, likely the ubiquitous and well-

described RFC or PCFT [18, 19]. Similar to the lack of the

effect of farletuzumab on binding of 3H-folic acid by FRashown in Figs. 1 and 2, we observed no significant effect

on total membrane binding of either 3H-MTHF or3H-PMX, whereas folic acid pre-incubation blocked

[95 % binding of both ligands (dashed lines in Fig. 3).

Cytoplasmic accumulation of 3H-MTHF and 3H-PMX was

minimally blocked by farletuzumab under the conditions

tested (triangles, solid lines Fig. 3). However, folic acid

blocked 34 and 23 % of MTHF uptake and 93 and 53 % of

PMX in IGROV-1 and HK2 cells, respectively. Similar

results for 3H-MTHF were seen when confluent MA104

and CHO cells were studied, although the inhibition of3H-PMX was more in the 50 % range, not [90 % as seen

for the IGROV-1 cells. The accumulation of MTHF and

PMX when FRa is blocked with folic acid shows that other

transport mechanisms for vitamin and drug uptake are

active to varying degrees in all the lines studied. The

importance, or lack thereof, of FRa in log-phase growth of

MA104 cells has been previously shown by us and further

highlights that FRa function on normal epithelial cells such

as in the kidney and choroid plexus [15, 16] is to help

package and conserve folate under the conditions of folate

deficiency [20] and, in general, is not likely critical for

folate uptake in a proliferative cell.

The slightly decreased FRa-mediated uptake of 3H-MTHF

in the presence of farletuzumab is in agreement with the

data in Fig. 2, which allowed the suggestion that receptor

cycling may be altered since ligand binding is not. This is

more completely shown in Fig. 3e, in which the effects

of two concentrations of farletuzumab or folic acid on3H-MTHF cytoplasmic accumulation were studied over an

extended time in IGROV-1 cells. As seen, by 8 h, the total

accumulation of MTHF in the presence of farletuzumab

was equivalent to the control cells and only about 80 %

maximum in the presence of folic acid. To see whether this

attenuated rate of delivery was critical to the cell (since it

was not the major pathway for uptake of MTHF), we more

completely evaluated the effect of Ab on cell proliferation

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C

5-methyltetrahydrofolate (nM)

MT

S (

abs

@ 4

90 n

m)

Fig. 4 Three-day growth of cells in the presence of farletuzumab and

increasing concentrations of 5-methyltetrahydrofolate. a Ma104 cells,

b CHO-FR cells, and c IGROV-1 cells. All cells were plated in

96-well plate in RPMI (folic acid-free ?5 % FBS, day 0). On day 1, 1 or

10 lg/ml farletuzumab was added in addition to increasing concentra-

tions of 5-MTHF. After 3 days, MTS was added (30 ll/0.5 ml) and

incubated *1 h and then absorbance was read at 490 nm

118 Cancer Chemother Pharmacol (2012) 70:113–120

123

in vitro as earlier work did show some growth inhibition of

CHO cells transfected with FRa when grown in low folate

for 96 h [10]. The growth of CHO-FR, MA104, and

IGROV-1 cells grown in the presence of Ab and increasing

MTHF for 3 days is shown in Fig. 4. Figure 4a, b shows

that farletuzumab at best may minimally result in some

growth retardation in the MA104 and CHO-FR cells (as

already reported for CHO-FR) that was abrogated as

5-MTHF was increased. IGROV-1 cells were unaffected

(Fig. 4c). This supports the concept that the decreased/

delayed delivery of 5-MTHF in the presence of far-

letuzumab seen in Fig. 3 does not likely cause much

growth arrest.

Folate concentration, but not farletuzumab, decreases

the potency of pemetrexed in vitro

The potency of PMX and several other anti-folates with a

lower affinity for FRa was assessed in the presence of low

and physiological folate concentrations in vitro. In the

presence of increasing amount of folate, the IC50 for PMX

increased but the presence of farletuzumab did not make

any difference (Fig. 5). Specifically, as the folate content

was increased from 5 to 50 nM, the potency of PMX

decreased by as much as 15-fold for IGROV-1 cells

(Fig. 5a) and *twofold for MA104 cells (Fig. 5b). The

potency of PMX against CHO-FR cells was also not altered

by farletuzumab (IC50 2 nM), but overall growth rate was

stimulated by increasing 5-MTHF (data not shown). In this

regard, to confirm the absence of any ‘‘off-site effects,’’ we

also found that farletuzumab had no effect on the potency

of PMX in a receptor-negative cell line (CHO parent), and

the IC50 of PMX for CHO cells transfected with FRa and

the parent line was the same. This is more evidence that

FRa is not important in PMX potency. Finally, as a further

indication that FRa was not overly important with regard

to anti-folate cytotoxicity, we also tested MTX and

aminopterin, two very potent anti-folates with a very low

affinity for FRa, compared to MTHF and PMX. We

found that the IC50 was not altered by farletuzumab (IC50

values of *5 and 0.1 nM for MTX and aminopterin,

respectively).

Discussion

Since PMX is currently approved for patients with can-

cers that may express FRa, it was important to know if

farletuzumab interfered with its potency. Overall, the

results here provide more evidence that (a) FRa is not

the major transport route for intracellular accumulation of

physiological folates or PMX, and (b) that farletuzumab,

an anti-FRa antibody has no significant effect on folate or

anti-folate accumulation or potency (in the case of the

drug). These results support previous studies that PMX

potency is not affected by the presence or absence of the

receptor [11, 12]. Moreover, since folic acid is routinely

prescribed when patients are receiving PMX, the results

here confirm that the potency of PMX may be decreased,

but this is balanced by the need to maintain adequate

folate stores to minimize toxicity, that is maintain accept-

able efficacy.

Farletuzumab has been shown to be active via ADCC

and CDC [10] by targeting FRa as a unique antigen on the

tumor cell and because it has little effect on anti-folate

accumulation or potency, supporting the concept that anti-

folates and antibody can be used together if both agents

have activity as single agents is supported.

More studies of the Ab effect on FRa cycling and dis-

tribution in lipid rafts as a GPI-anchored protein may yield

interesting insights from a cell biology view of membrane

integrity and function, but for now we conclude that far-

letuzumab, although targeting FRa, is not functioning as an

anti-folate.

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Fig. 5 Folate but not

farletuzumab decreases the

potency of pemetrexed:

IGROV-1 cells (a) and Ma104

Cells (b) were grown in the

presence of 10 lg/ml antibody,

or just increasing folate without

Ab, for 72 h along with

increasing PMX (10-10–10-7

M). The addition of folate

increased the IC50 from *2 to

*30 nM for IGROV-1 cells

and from *7 to *13 nM for

Ma104 cells, whereas the Ab

had no significant effect

Cancer Chemother Pharmacol (2012) 70:113–120 119

123

Acknowledgments The authors acknowledge medical writing

assistance by Robyn Boyle, RPh under the direction of the authors as

well as financial assistance provided by Morphotek.

Conflict of interest Barton Kamen is now a consultant for Mor-

photek, Inc. Angela Smith reports no conflicts of interest. The authors

have full control of all primary data.

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