Saporin Conjugated Monoclonal Antibody to the Transcobalamin Receptor TCblR/CD320 Is Effective in Targeting and Destroying Cancer Cells

doi:10.4236/jct.2013.46122

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Journal of Cancer Therapy, 2013, 4, 1074-1081

http://dx.doi.org/10.4236/jct.2013.46122 Published Online August 2013 (http://www.scirp.org/journal/jct)

Saporin Conjugated Monoclonal Antibody to the

Transcobalamin Receptor TCblR/CD320 Is Effective in

Targeting and Destroying Cancer Cells

Edward V. Quadros1,2, Yasumi Nakayama1, Jeffrey M. Sequeira1

1Department of Medicine, SUNY-Downstate Medical Center, Brooklyn, USA; 2Department Cell Biology, SUNY-Downstate Medical

Center, Brooklyn, USA.

Email: edward.qua[email protected]

Received May 29th, 2013; revised June 30th, 2013; accepted July 8th, 2013

cense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ABSTRACT

Cobalamin uptake into cells is mediated by the CD320 receptor for transcobalamin-bound cobalamin. Optimum recep-

tor expression is associated with proliferating cells and therefore, in many cancers this receptor expression is up regu-

lated. Delivering drugs or toxins via this receptor provides increased targeting to cancer cells while minimizing toxicity

to the normal tissues. Saporin conjugated monoclonal antibodies to the extracellular domain of TCblR were effectively

internalized to deliver a toxic dose of Saporin to some cancer cell lines propagating in culture. Antibody concentration

of 2.5 nM was effective in producing optimum inhibition of cell proliferation. The cytotoxic effect of mAb-Saporin

appears to be dictated primarily by the level of receptor expression and therefore normal primary cells expressing low

levels of CD320 were spared while tumor cell lines with higher CD320 expression were destroyed. Targeting the path-

way for cellular uptake of vitamin B12 via the CD320 receptor with toxin-antibody conjugates appears to be a viable

treatment strategy for certain cancers that over expresses this receptor.

Keywords: Transcobalamin Receptor; CD320 Gene; Cobalamin; Cancer; Toxin

1. Introduction

The transcobalamin receptor TCblR/CD320 [1] is ubiq-

uitously expressed in most cell types and mediates up-

take of transcobalamin (TC), a plasma protein saturated

with cobalamin (Cbl, B12). Cbl in blood is bound to two

plasma proteins; haptocorrin (HC) which carries ~70% of

the vitamin and TC which is ~30% saturated with Cbl.

The latter, a nonglycosylated protein secreted by vascular

endothelial cells [2] has a relatively short half life and

plays the all important role of transporting the newly

absorbed Cbl in the distal ileum to all tissues where it

binds to the receptor on the cell surface with high affinity

[3]. TC-Cbl is internalized by receptor-mediated endo-

cytosis of the ligand [4,5]. It is generally accepted that

the TC is degraded in the lysosome and the Cbl is re-

leased [6]. Even though published data supports recy-

cling of the receptor [7,8], unequivocal evidence in sup-

port of this is lacking. TCblR expression appears to be

cell cycle associated with highest expression in actively

proliferating cells and is substantially down regulated in

quiescent cells [7-9]. This differential expression of the

receptor serves to provide optimum delivery of the vita-

min to cells during the early phase of DNA synthesis.

This process ensures adequate functioning of Cbl de-

pendent enzymes, especially the methionine synthase that

is essential for recycling of methyl folate to generate

folates needed for purine and pyrimidine biosynthesis.

The more proliferative a cell, higher the need for folates

and Cbl and this need for Cbl is met by the increased

expression of TCblR in cancer cells that may have in-

herently lost the ability to stop dividing and differentiate.

Selective targeting of cancer cells for destruction by de-

livering drugs and toxins preferentially to these cells has

been the ultimate objective of cancer therapy. The search

for tumor specific markers and the strategies to utilize

these in cancer therapy have been pursued for decades

with mixed results. Success in this approach has been

limiting and can be attributed to multiple factors that

include the lack of specificity of the target antigen, cel-

lular events that can alter this targeting and to the com-

plex and diverse nature of cancer itself. TCblR is struc-

turally related to the LDL receptor family with two

Saporin Conjugated Monoclonal Antibody to the Transcobalamin Receptor

TCblR/CD320 Is Effective in Targeting and Destroying Cancer Cells

1075

LDLR type A domains separated by a CUB like domain

and contains a single transmembrane region followed by

a short cytoplasmic tail [1]. We have generated mono-

clonal antibodies to the recombinant extracellular 200aa

domain of TCblR and have defined the epitope specific-

ity of these antibodies [10]. We have previously demon-

strated the utility of anti TCblR antibody to deliver sec-

ondary antibody conjugated to Saporin [11], an inhibitor

of ribosomal assembly [12] to cancer cells propagating in

culture [13,14]. The present study extends this prelimi-

nary observation by demonstrating enhanced targeting

and destruction of cancer cells by Saporin conjugated di-

rectly to monoclonal antibodies to the extracellular do-

main of TCblR/CD320.

2. Methods

All tumor cell lines obtained from ATCC were cultured

to passage 2 to 4 and frozen for use in this study. MCH

064, MCH 065 and RF peripheral skin fibroblast cultures

in passage 9 - 12 were from The Repository for Mutant

Human Cell Strains, Montreal Children’s Hospital, Can-

ada. Fresh human bone marrow mononuclear cells were

obtained from Lonza, Walkersville, MD. Human chori-

onic trophoblasts, ED cells were obtained as previously

described [15]. Cells were cultured in Dulbecco’s modi-

fied Eagle’s minimal essential medium (DMEM) with

10% fetal bovine serum (FBS) and antibiotics. Mono-

clonal antibodies generated to the extracellular domain of

TCblR [10] were purified by affinity chromatography on

a protein G agarose column and used for covalent conju-

gation of Saporin. The coupling of Saporin and purifica-

tion of monoconjugate of the antibody was done at Ad-

vanced Targeting Systems, San Diego, CA. The anti-

body-Saporin conjugates were stored in aliquots at −20˚C

and used in the present study. All data reported represent

mean results of duplicate analyses.

Determination of optimum concentration of mAb-

Saporin conjugate:

HEK293 cells (2 × 103 in 100 ul complete DMEM)

were seeded in 96 well culture plates with 0.156 - 5 nM

mAb-Saporin for 72 hours and viable cells were quanti-

fied using the CellTiter96 Aqueous One solution cell

proliferation assay (Promega). Optical density at 495nm

was read in a microplate reader (Bio-Rad). Statistical

analysis was done using GraphPad software Prism 3.03.

Effect of Cell seeding density on efficacy of mAb-

Saporin:

Initial seeding density defines duration of the prolif-

erative phase in the culture and therefore cells seeded at

lower density would continue to divide for a longer pe-

riod compared to cells seeded at a higher density until the

cell population reaches confluency. Since TCblR expres-

sion is highest in actively dividing cells and down-regu-

lated in resting cells, we tested cell lines at seeding den-

sities ranging from 1000 - 10,000 cells/well with 2.5 nM

mAb-Saporin concentration.

Measuring functional receptor on cells:

Functional receptor activity in whole cells was deter-

mined by incubating cells with 57CoB12 labeled TC. For

suspension cultures, cells are collected by centrifugation,

resuspended in DMEM and 0.5 to 1 × 106 cells are sus-

pended in 1 ml DMEM containing 10,000 CPM of radio-

labeled TC at 4˚C, or 37˚C. An identical tube containing

10 mM EGTA is included to show blocking of the uptake.

After 1 hour incubation, cells are pelleted by centrifuga-

tion at 2000 g for 5 min. washed once with buffer and

counted for radioactivity. Adherent cells are seeded in 6

well culture plates, washed with DMEM and incubated

with 57CoB12-TC in 1 ml DMEM for 1 hour. The cell

layer is washed with HBS, incubated with 0.5 ml trypsin/

EDTA for 5min at 37˚C, cells collected with solution and

counted for radioactivity.

Determination for mAb-Saporin concentration for in-

hibiting cell growth by 50% (IC50):

Since the toxic effect of mAb-Saporin was more pro-

nounced in cell cultures seeded at lower density, the

IC50 determinations were done with cells seeded at 2000

cells/well in 96 well plates with a mAb-Saporin concen-

tration of 0.156 to 5 nM for 96 hours. The number of

viable cells was determined by the MTS assay.

Specificity of TCblR pathway for delivering the mAb-

Saporin toxin:

The specificity of the TCblR-mediated pathway for

uptake and internalization of the mAb-Saporin complex

was determined by adding recombinant soluble extracel-

lular fragment of receptor to the culture medium. The

soluble receptor would compete with the cell surface

receptor for the antibody and this would reduce the Ab-

toxin available for cellular uptake resulting in a decrease

in percent inhibition. For these studies, K562 cells and

both normal and stably transfected HEK293 cells to over

express the receptor were used. Cells were seeded in 96

well plates at 2000 cells/well and the amount of mAb-

Saporin-Ab used was equivalent to the IC50 concentra-

tion.

Specificity of mAb-Saporin for the TCblR receptor A

100 fold excess primary mAb or normal mouse IgG was

added to the incubation medium containing a mAb-

Saporin concentration of 2.5 nM. A decrease in the mAb-

Saporin induced inhibition of cell growth should be ob-

served when excess primary Ab without Saporin is pre-

sent since the ratio of mAb-Saporin to unlabelled mAb

should be lower and this increases the probability of

unlabelled mAb binding to TCblR. The addition of nor-

mal mouse IgG should not result in a decrease in cell-kill

since this cannot bind to TCblR and therefore would not

Saporin Conjugated Monoclonal Antibody to the Transcobalamin Receptor

TCblR/CD320 Is Effective in Targeting and Destroying Cancer Cells

1076

compete with mAb-Saporin for binding to TCblR.

Effect of mAb-Saporin on normal and cancer cells in

culture:

In order to determine any differential effect of the

mAb-Saporin in targeting cancer cells in culture, various

normal and cancer cell lines seeded at 1000, 2000 and

4000 cells/well were exposed to mAb-Saporin at an an-

tibody concentration of 2.5 nM.

Immunohistochemical staining of TCblR on cells:

Cell lines cultured on gelatin-coated cover slips were

fixed in 70% ethanol for 2 hrs at 4˚C and washed in PBS.

Human placental tissue was fixed in paraformaldehyde,

embedded in Paraplast and 5 um sections were used for

immunostaining. Rabbit polyclonal Ab to human TCblR

(RPI) at 1:500 dilution was added for 1 hr, washed and

incubated with biotin conjugated secondary Ab for 1 hr

followed by ABC reagent (Vector Labs) for 30 min and

color development with DAB (Vector Labs).

3. Results

A number of adherent and suspension cell cultures were

propagated in medium containing 0.156 to 5 nM mAb-

Saporin. The effective concentration determined by per-

cent decrease in viable cell number showed maximum

effect at an antibody concentration of 2.5 to 5 nM. An

example of this is shown in Figure 1(a) whereby normal

HEK293 cells (293N) and those stably transfected to

over express TCblR (293OE) showed a dose dependent

effect with maximum inhibition of cell growth between

2.5 and 5 nM mAb-Saporin concentration. In addition, in

293N cells, inhibition of proliferation was influenced by

initial seeding density which influences the rate and du-

ration of proliferation and this effect was abrogated in

293OE cells which constitutively over express the re-

ceptor, independent of the cell cycle. In addition, 293OE

cells were more severely affected compared to 293N

cells due to sustained over expression of TCblR (Figure

1(b)). A mAb concentration of 2.5 nM was chosen for

subsequent studies to ensure adequate extra cellular con-

centration of mAb-Saporin for all cell lines tested. Un-

conjugated Saporin at 10 to 100 fold molar excess had no

effect on cell proliferation (data not shown).

Since initial seeding cell density is likely to affect the

rate of proliferation and time to confluence, we tested a

number of suspension and adherent cancer cell lines at

initial seeding densities of 2000 - 10,000 cells per well in

96 well plates. The effect of mAb-Saporin was least in-

fluenced by the initial seeding density in both suspension

and adherent tumor cells in culture, but the effect on cell

proliferation was more pronounced in suspension cul-

tures (Figure 2). A skin fibroblast cell line (RFP3) and a

trophoblast line (ED) derived from human placenta (15)

served as non-cancerous cell lines and were not affected

or minimally affected (Figure 2).

(a)

(b)

Figure 1. Effect of antibody concentration (a) and seeding

cell density (b) on cell proliferation. Normal HEK 293 cells

(N) and HEK 293 cells transfected to stably over express

TCblR (OE) were tested for inhibition of cell proliferation

during a 72hour exposure to anti TCblRmAb-Saporin con-

jugate in culture. Results shown are mean of duplicate

samples that varied <5%.

Since in normal cells, the receptor expression is cell

cycle assocated and lower seeding density and higher

receptor expression appear to have a greater inhibitory

effect on cell proliferation, the lack of this effect in can-

cer cells was puzzling and therefore, we evaluated the

expression of functional TCblR in a number of tumor and

normal cell lines. Since TCblR expression appears to be

cell cycle dependent, we determined cell surface receptor

expression by TC-Cbl binding at various time points

during a 96 hour culture period. As seen in Table 1,

TCblR expression varied considerably among the various

tumor lines. In some cell lines TCblR expression peaked

between 8 - 48 h and was followed by a substantial de-

crease in TC-Cbl binding by 96h, a profile observed for

Saporin Conjugated Monoclonal Antibody to the Transcobalamin Receptor

TCblR/CD320 Is Effective in Targeting and Destroying Cancer Cells

1077

Figure 2. Effect of initial seeding density on proliferation of

cells in the presence of antibody-Saporin conjugate. Normal

primary cell lines and neoplastic cell lines were seeded at a

density of 1000 - 10,000 cells per well in 96 well plates with

2.5 nM mAb-Saporin conjugate. Results shown are mean of

duplicate samples that varied <5%.

most normal cell lines. In some tumor lines, TC-Cbl was

substantially up regulated initially and did not decrease

to the level seen in normal cells. On the other hand, some

tumor lines expressed moderate level of TCblR through-

out the culture period without the peak and decrease seen

in normal cells. Immunohistochemical visualization of

the receptor showed specific membrane staining of

TCblR (Figure 3). While the level of TCblR expression

is too low to detect in most normal cell lines such as skin

fibroblasts, the expression is adequate to visualize in

human placenta and most tumor cell lines.

Because of the wide range of doubling time and the 8 -

48 h period for peak receptor expression in various tumor

lines, the cytotoxic effect of the mAb-saporin was deter-

mined at three different initial cell densities of 1000,

2000 and 4000 cells and we evaluated three monoclonals

with specificity for different regions of the protein (10).

As shown in Figure 4, typically, lower seeding density

showed higher inhibition of cell growth and the effect

varied with different cell lines. Table 1 provides the

IC50 inhibitory dose for the three mAb-Saporin conju-

Figure 3. Immunohistochemical identification of TCblR

expression in various human tissues. (A) KB epidermoid

carcinoma; (B) HL60 acute promyelocytic leukemia; (C)

U266 myeloma; (D) human placenta, E: U373 glioblas-

toma-as- trocytoma; (F) HEK293 embryonic kidney; (G)

PC3 prostate adenocarcinoma.

Figure 4. Inihibition of cell proliferation in the presence of

mAb-Saporin conjugates. Data shows various tumor cell

lines with differences in susceptibility to the toxin. Results

shown are mean of duplicate samples that varied <5%.

Saporin Conjugated Monoclonal Antibody to the Transcobalamin Receptor

TCblR/CD320 Is Effective in Targeting and Destroying Cancer Cells

1078

Table 1. TCblR expression profile and c oncentration of mAb-Saporin required for inhibiting cell proliferation.

Cbl-TC binding IC50/2000 cells (nM)

Cell Line Source Cell line discription Doublingpg/106 cells 1 - 10

saporin

1 - 19

saporin

1 - 25

saporin

Time hr8 - 18 hr24 hr48 hr72 hr 96 hr

K-562* ATCC CCL-243 chronic myelogenous

leukemia (CML) 24 - 4010 1910 7 3 <0.05 <0.05<0.05

HL-60 ATCC CCL-240 acute promyelocytic leukemia 24 4 6 5 4 3 11 10 10

Jurkat ATCC TIB-152 acute T-cell leukemia 25 - 3520 2115 6 4 2.4 2.5 2.08

U266B1

[U266]* ATCC TIB-196 myeloma plasmacytoma 55 10 107 7 8 0.8 <0.05<0.05

RPMI8226* ATCC CCL-155 plasmacytoma; myeloma,

B lymphocyte 60 - 706 1515 8 7 1.92 1.621.54

NCI-H929

[H929]* ATCC CRL-9068 Plasmacytoma, Myeloma 70 20 2424 3315 1.89 1.421.49

U4937

(U937)*

ATCC

CRL-1593.2 monocytic cell, histocytic lymphoma20 - 489 9 10 3 3 1.87 1.581.64

SW48

[SW-48]* ATCC CCL-231 colorectal adenocarcinoma, epithelial35 20 3718 107 <0.05 1.7 <0.05

SW480

[SW-480] ATCC CCL-228 colorectal adenocarcinoma, epithelial25 - 4819 1512 7 10 3.29 3.382.45

RKO* ATCC CRL-2577 carcinoma colon,epithelial 14 26 2711 8 8 1.71 1.811.52

LoVo ATCC CCL-229 colorectal adenocarcinoma, epithelial36 - 489 7 10 1214 7.81 41.76.94

Caco-2 ATCC HTB-37 colorectal adenocarcinoma 24 - 626 8 7 5 4 3.21 4.812.98

Hep G2 ATCC HB - 8065 hepatocellular carcinoma (epithelial)50 - 6017 149 3 3 10.4 10.44.81

Hep 3B ATCC HB - 8064 hepatocellular carcinoma (epithelial)40 - 5011 1110 9 6 5.95 6.254.03

KB ATCC CCL-17 HeLa Derivatives 30 - 4038 1615 115 4.03 4.812.84

MDA-MB-231 ATCC HTB-26 adenocarcinoma,

mammary gland; breast 50 - 6033 4510 6 8 11.3 17.911.3

MCF7 ATCC HTB-22 adenocarcinoma,

mammary gland; breast 50 12 2821 107 6.25 8.935

HeLa ATCC CCL-2 cervix,adenocarcinoma (epithelial)48 13 1213 9 9 2.11 2.551.84

A431NS ATCC CRL-1555 epidermal carcinoma: skin 80 - 1003 3 4 4 3 6.58 9.624.63

MIA PaCa-2 ATCC CRL-1420 Pancreatic carcinoma 40 22 1111 7 7 5 10.45.2

PC-3* ATCC CRL-1435 prostate adenocarcinoma 50 16 1618 1010 2.19 2.081.81

U-373 MG ATCC HTB-17 identities in question 24 - 4819 8 8 6 7 3.1 3.792.91

HEK-293

Normal ATCC CRL-1573 human embryonic kidney cells 24 - 309 9 9 5 6 4.3 4.1 3.7

EC304

(ECV 304) ATCC CRL-1998 T24 (human bladder cell) derivative48 13 166 5 5 2.91 3.292.5

ED Quadros lab embryonic cells from human placenta48 8 132 2 2 12 14 12

RFP3 Quadros lab human skin fibroblast 18 - 243 132 2 2 30 24 26

2M-125C Lonza 2M-125C human bone marrow MNC Not tested 5 4.8 4.8

Cells for TCblR expression were seeded in 6 well plates at a density of 0.2 × 106 cells/well and Cbl-TC binding was determined by incubating with [57Co]

Cbl-TC (10,000 cpm) for 1hr at 37˚C in 1ml medium. The effect of mAb-Saporin on cells was determined at 72 hr following seeding of 2000 cells/well in a 96

well plate with 0.156 - 5 nM mAb-Saporin and IC50 (concentration for inhibition cell proliferation by 50%) was calculated from the slope of the growth curve.

Asterisk indicates cells most susceptible to mAb-Saporin.

Saporin Conjugated Monoclonal Antibody to the Transcobalamin Receptor

TCblR/CD320 Is Effective in Targeting and Destroying Cancer Cells

1079

gates on cell proliferation. All three antibodies were ef-

fective as carriers of Saporin and the average inhibition

observed for a specific cell line was similar for all three

antibodies. However, there were major differences in

susceptibility of different cell lines to the mAb-Saporin

conjugate. On average, most of the suspension cell lines

showed higher inhibition of cell proliferation with K562

and RPMI18266 showing the most inhibition. Among the

adherent cell lines also, the effect varied with RKO,

SW48, HeLa and PC3 showing the most inhibition and

LoVo. Hep G2, Hep 3B, MCF7, MDA-MB-231 and

MIA PaCa-2 cell lines, showing least inhibition. Inhibi-

tion of cell proliferation was more pronounced in sus-

pension cultures than in adherent cultures at all concen-

trations of the Saporin-antibody and cell density tested.

In cell lines with 50% or less inhibition of growth, in-

creasing the antibody concentration did not increase the

inhibition. Based on the above data, the IC 50 for most

tumor cell lines appears to be below 100 pM (Table 1).

The specificity of Saporin conjugated antibodies was

evident from a decrease in inhibition with the addition of

excess unconjugated specific antibody and lack of any

effect with excess normal mouse IgG (Figure 5). The

specificity of receptor targeting by the antibodies was

further confirmed by adding soluble recombinant TCblR

to the incubation, which reduced the effect of the Saporin

conjugated antibodies (Figure 6).

4. Discussion

Our previous study described the utility of monoclonal

antibodies to the extracellular domain of TCblR as effec-

tive carriers of Saporin conjugated secondary antibody

into cells [11]. Encouraged by this preliminary observa-

tion, we have extended the approach to evaluate the effi-

cacy of Saporin directly conjugated to primary antibodies.

Two of the three representative antibodies (TCblRKB1-

10 and TCblRKB 1-19) selected were binding antibodies,

in that binding of these antibodies to their epitopes on

TCblR did not prevent the binding and internalization of

the physiologic ligand, TC-Cbl. The third antibody

(TCblRKB1-25) was a blocking antibody since binding

of this antibody to TCblR, prevented the binding and

cellular uptake of TC-Cbl. The epitope specificity of

these antibodies and properties are described elsewhere

[10]. All three mAb-Saporin conjugates are fully func-

Figure 5. The specificity of mAb-Saporin conjugate for

TCblR. Figure shows excess unconjugated specific mAb

decreases the inhibitory effect of the mAb-Saporin conju-

gate while normal mouse IgG does not (A) & (C). This ef-

fect is less pronounced in suspension cultures (B) and in

cells over expressing TCblR (D). Results shown are mean of

duplicate samples that varied <5%.

Figure 6. The specificity of targeting TCblR by Mab-Saporin conjugate. The recombinant extracellular domain of TCblR

added to culture decreases the inhibitory effect of the mAb-Saporin conjugate in culture. Results shown are mean of dupli-

cate samples that varied <5%.

Saporin Conjugated Monoclonal Antibody to the Transcobalamin Receptor

TCblR/CD320 Is Effective in Targeting and Destroying Cancer Cells

1080

tional in recognizing the target antigen with high affinity,

an important requirement for mAbs to be used as carriers

of drugs and toxins. Even though a marginal increase in

internalization of mAb 1 - 10 and 1 - 19 is observed with

TC-Cbl in the culture medium, the binding of the native

ligand is not a pre requisite for antibody binding and in-

ternalization. Since in the absence of TC-Cbl, the apo re-

ceptor remains on the plasma membrane, TC-Cbl binding

appears to trigger the necessary response for internaliza-

tion. The effective binding and internalization of all an-

tibodies, including the ligand blocking antibody, mAb 1 -

25 is strong evidence that antibody binding triggers a

response similar to that of ligand TC-Cbl binding. Highly

potent toxins such as ricin, cholera toxin, gelonin and

saporin, drugs or radionuclides are very effective in de-

stroying cancer cells if a toxic dose can be delivered spe-

cifically to these cells [16,17]. This strategy requires a

tumor specific carrier to transport the toxin across the

plasma membrane into cells since these molecules cannot

cross the cell membrane by either specific or nonspecific

transport mechanisms. An ideal target protein would be a

receptor or cell surface protein that is expressed and in-

ternalized only in cancer cells. However, such proteins

are scarce and not easy to identify. Many proteins and

receptors are expressed in all cell types and some of

these are cell cycle associated or expressed only in ac-

tively dividing cells. Such proteins can be carriers for

drugs and toxins and may provide some degree of en-

hanced targeting to cancer cells. However selective tar-

geting to cancer cells and lack of toxicity to the normal

cell population will depend not only on the differential

expression but also on the density of the target protein in

the two cell types. For example, a protein with relatively

high expression such as the transferrin receptor [18],

even though differentially expressed in cancer and nor-

mal cells, would not be suitable for delivering a toxin

because the normal cells would internalize sufficient

toxin to kill the cell. The ideal target protein is one with

fairly low expression in normal cells and cannot inter-

nalize toxic amounts of drugs but is adequately over ex-

pressed in cancer cells to internalize cytotoxic amounts

of the drug. The TCblR is one such protein whose ex-

pression is sufficiently low to render any toxin internal-

ized in normal cells to be ineffective and is adequately

over expressed in some cancers to internalize sufficient

toxin to kill the cell. In addition, the cell cycle associated

expression of this protein makes highly proliferative

cancer cells with sustained expression, an ideal target for

this approach. Blocking Cbl uptake into cells with mono-

clonal antibody to TC can deplete cells of Cbl and ulti-

mately inhibit DNA synthesis leading to inhibition of cell

replication [19,20]. A specific antibody to TCblR that

blocks the binding of TC-Cbl could also have the same

effect [10]. This approach, even though less toxic, is

likely to be slow and many cancers require a faster effect

to destroy the malignant tissue before it metastasizes.

Inhibiting CD320 expression with siRNA also affects

proliferation by depleting intracellular Cbl [21]. The use

of potent drugs or toxins conjugated to antibody that can

deliver the payload to its target antigen is a highly effec-

tive strategy that can provide the specificity and speed of

action demanded in cancer therapy. The present data on

the use of mAb-Saporin conjugate to target TCblR ap-

pears to be specific for certain cancers and provides

proof of concept for utilizing this receptor for targeted

delivery of drugs and toxins and awaits confirmation of

in vivo targeting efficacy of this pathway.

5. Acknowledgements

This work was supported by NIH grant DK064732 to

EVQ and by KYTO Biopharma, Toronto, CA. EVQ and

JMS are inventors on patent applications US2011/

052154 and WO/2013/015821 by The Research Founda-

tion of SUNY. YN declared no potential conflicts.

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