conventional nontargeted 
 chemotherapeutics , such as antimetabolites , microtubule inhibitors , and dna intercalating / alkylating agents , are effective 
 at killing cancer cells , but due to their indiscriminate penetration 
 into nearly all cells , they can also damage healthy cells , causing 
 such toxicities as myelosuppression , alopecia , mucositis , peripheral 
 neuropathy , and cardiotoxicity . to minimize such collateral 
 damage to healthy tissues , physicians must often either reduce the 
 dosage or decrease the frequency of drug administration , leading to 
 incomplete elimination of diseased tissue .
on the basis of these considerations , 
 recent approaches to cancer therapy have focused on developing methods 
 that specifically target chemotherapeutic agents to cancer cells , 
 allowing for improved tumor suppression with fewer adverse events .
the most common approach to drug targeting has relied on the specificity 
 of a monoclonal antibody for its tumor - specific antigen . through the 
 conjugation of a highly cytotoxic drug to a tumor - specific antibody , 
 tumor - selective drug delivery
examples of such tumor - targeted 
 antibody  drug conjugates ( adcs ) include trastuzumab emtansine and brentuximab vedotin .
although several 
 adcs have shown significant success in preclinical and clinical settings , 
 some questions as to their ability to penetrate solid tumors have 
 been raised .
a related strategy to achieve tumor - selective drug delivery involves 
 the use of low molecular weight targeting ligands that can similarly 
 deliver attached drugs specifically to cancer cells .
drug conjugates ( figure 1a ) also target receptors that are overexpressed on malignant cells , 
 and their much smaller sizes may permit more thorough tumor penetration .
( a ) general representation of 
 ligand conjugated to cytotoxic payload via a peptide linker .
the circle 
 represents the cholecystokinin 2 receptor ( cck2r ) binding ligand , 
 whereas the linker is represented by an oval . the cytotoxic drug , 
 or payload ,
the solid black line represents 
 a covalent bond between the ligand and the linker , and the dotted 
 line symbolizes a cleavable self - immolative bond .
( b ) chemical structures 
 of the cck2r ligand crl conjugated to the cytotoxic antimicrotubule 
 agents desacetyl vinblastine hydrazide and tubulysin b hydrazide via 
 a hydrophilic peptide linker . in this paper , z-360 , a low 
 molecular 
 weight ligand of the cholecystokinin 2 receptor ( cck2r ) , is modified 
 to deliver two of the more potent antimicrotubule agents currently 
 available ( desacetyl vinblastine monohydrazide and tubulysin b hydrazide ) . 

cck2r is a transmembrane receptor primarily found in epithelial cells 
 of the gi tract and brain , where it can bind gastrin and other amidated 
 peptides of the gastrin - cholecystokinin family .
cck2r is overexpressed on many cancers of the lung , pancreas , liver , 
 and gi tract ( esophagus , colon , and gastrointestinal stromal tumors ) . because cck2r in the brain is inaccessible to drugs that can not 
 pass the blood brain barrier
, this restricted expression pattern renders 
 cck2r a possible candidate for ligand - targeted drug delivery .
we report 
 here the design , synthesis , and biological evaluation of two highly 
 potent cck2r - targeted chemotherapeutic agents and demonstrate their 
 efficacy and tolerability in treating cck2r - expressing human tumor 
 xenografts in nu / nu mice .
h - cys ( trt)-2-cl - trt 
 resin was obtained from novabiochem ( san diego , ca ) .
diisopropylethylamine ( dipea ) , piperidine , dimethylformamide 
 ( dmf ) , isopropyl alcohol ( i - proh ) , and all other 
 reagents were purchased from sigma - aldrich .
tubulysin b and desacetyl 
 vinblastine hydrazide and their activated derivatives were a kind 
 gift from endocyte inc .
dulbecco s modified 
 eagle medium ( dmem ) , roswell park memorial institute medium ( rpmi ) , 
 fetal bovine serum ( fbs ) , and g418 ( geneticin ) were all purchased 
 from gibco ( grand island , ny ) .
the 
 hek 293 cell line transfected with 
 wild type cck2r was a generous gift from dr .
cells were cultured as monolayers in dmem supplemented with 
 10% fetal bovine serum , g418 disulfate ( 400 g / ml ) , 1% of 2 
 mm glutamine , and 1% of penicillin - streptomycin at 37 c in a 
 5% co2:95% humidified - air atmosphere .
kb cells were cultured 
 from frozen stocks in rpmi and supplemented with the same concentrations 
 of fetal bovine serum , penicillin - streptomycin , and glutamine as the 
 hek 293 cells .
the cck2r ligand ( z-360 ) was synthesized 
 as previously described and abbreviated 
 crl to be consistent with previous 
 publications .
the peptide spacer was 
 prepared using fmoc - protected solid phase peptide synthesis as outlined 
 in scheme 1 ( supporting information ( si ) , figure 1 ) and named l1 .
as shown in scheme 1 ( si , figure 1 ) , crl was coupled to the peptide spacer on the 
 solid phase and cleaved from the resin using a standard cleavage cocktail 
 solution .
crude crl - l1 was purified by preparative rp - hplc [ a = 2 
 mm ammonium acetate buffer ( ph 5.0 ) , b = ch3cn , solvent 
 gradient : 5% b to 80% b in 25 min ] to yield the requisite product . 

lrms - lc / ms ( m / z ) : [ m + h ] calcd for c72h110n12o27s , 1607.79 ; found , 1608 .
a solution of saturated sodium bicarbonate 
 ( 2 ml ) and hplc grade 
 water was bubbled with argon continuously for 10 min .
crl - l1 ( 36 mg , 
 0.0226 mmol ) was dissolved in argon - purged hplc grade water ( 2.0 ml ) , 
 and the ph of the reaction mixture was increased to 7 using 
 argon - purged sodium bicarbonate .
a solution of disulfide activated - davbh 
 ( 11.10 mg , mmol ) in thf ( 2.0 ml ) was then added to the reaction mixture 
 ( si , figure 2 ) .
the progress of the reaction 
 was monitored using analytical lrms - lcms , and after stirring for 20 
 min , the reaction was found to reach completion .
crude crl - l1-davbh 
 was purified by preparative rp - hplc [ a = 20 mm ammonium acetate ( ph 
 7.2 ) , b = ch3cn , solvent gradient : 5% b to 80% b in 30 
 min ] , yielding the desired product .
lrms ( lc / ms ) ( m / z ) : [ m + h ] calcd for c118h168n18o36s2 , 2478 ; found , 
 2478 . crl - l1-tubbh was synthesized from activated tubulysin b hydrazide 
 following the same procedure used for the synthesis of crl - l1-davbh 
 ( si , figure 2 ) . after removing the thf 
 under reduced pressure
, crl - l1-tubbh was purified by preparative rp - hplc 
 [ a = 2 mm ammonium acetate buffer ( ph 7.0 ) , b = ch3cn , 
 solvent gradient : 5% b to 80% b in 25 min ] to yield the requisite 
 product . lrms - lc / ms ( m / z ) : [ m + 
 h ] calcd for c117h177n19o38s3 , 2553.96 ; found , 2554 . l1-davbh and 
 l1-tubbh were synthesized from activated davbh and tubbh , respectively , 
 following the procedure outlined for the synthesis of crl - l1-davbh 
 ( si , figure 1 and 3 ) .
each compound was 
 then purified by reverse phase hplc [ a = 2 mm ammonium acetate buffer 
 ( ph 7.0 ) , b = ch3cn , solvent gradient : 5% b to 80% b in 
 25 min ] to yield the requisite product .
lrms - lc / ms ( m / z ) : [ m + h ] calcd for l1-davbh , c89h134n14o32s2 , 
 1976.22 ; found , 1976 ; and ( m / z ) : 
 [ m + h ] calcd for l1-tubbh c88h143n15o34s3 , 2051.35 ; found , 2051 . 
 all conjugates were found to be stable in saline for at least four 
 weeks as monitored by lc - ms .
cck2r - transfected hek 293 cells ( 100,000 
 cells / well ) were seeded on amine - coated 24-well plates and allowed 
 to form monolayers .
the spent medium in each well was replaced with 
 fresh medium containing increasing concentrations of the test agents . 
 after incubating for 2 h at 37 c
, cells were rinsed 3 
 with fresh medium and then incubated an additional 66 h at 37 c 
 in fresh medium .
spent medium in each well was again replaced with 
 fresh medium ( 0.5 ml ) containing h - thymidine ( 1 ci / ml ) , 
 and the cells were incubated for an additional 4 h. after washing 
 the cells 3 with medium , they were dissolved in 0.5 ml of 0.25 
 m naoh .
thymidine incorporation was then determined by counting cell - associated 
 radioactivity using a scintillation counter ( packard , packard instrument 
 company ) .
the ic50 value was derived from a plot of the 
 percent of h - thymidine incorporation versus log concentration 
 using graph pad prism 4 and tablecurve 2d software .
hek 293 cells expressing cck2r 
 ( 5.0  10 in 50% hc matrigel ) were injected into 
 the shoulders of 56 week old female nu / nu mice .
an age - matched group of animals was similarly implanted with 
 1  10 kb cells in 100 l of cell culture medium . 
 because kb cells do not overexpress cck2r , the kb xenograft model 
 served as a negative control .
tumors were measured in two perpendicular 
 directions 3 per week with vernier calipers , and their volumes 
 were calculated as 0.5 x l x w , where l is the longest 
 axis ( in millimeters ) , and w is the axis perpendicular to l ( in millimeters ) . 

solutions were administered 
 either intraperitonealy ( crl - l1-davbh , l1-davbh ) or intravenously 
 ( crl - l1-tubbh , l1-tubbh , crl - l1 ) .
each mouse received 2 mol / kg 
 of the test or control agent in 100 l of saline per injection . 

injections were given 3 per week for 3 weeks , and the mice were 
 weighed at each dosing as a measure of gross toxicity .
all animal 
 work was performed under the guidance of the purdue laboratory animal 
 program and was reviewed by the purdue animal care and use committee .
tumors were excised , fixed in 
 formalin , embedded in paraffin , sectioned , and stained with hematoxylin 
 and eosin by the purdue histology and phenotyping laboratory .
proper design of a ligand - targeted chemotherapeutic agent requires 
 ( i ) selection of a high affinity ligand with good selectivity for 
 a cancer - enriched receptor , ( ii ) identification of a therapeutic agent 
 with sufficient potency to kill cancer cells when captured by a cancer - specific 
 receptor , and ( iii ) construction of a linker that will enable delivery 
 and release of the attached drug preferentially within the targeted 
 cells . because cholecystokinin receptor ligand ( crl ) has been shown 
 to exhibit high affinity ( 0.47 nm ) and strong selectivity for cck2r 
 ( > 600-fold specificity over cck1r ) , 
 it 
 was selected for exploration as a targeting ligand for drug delivery 
 to cck2r - expressing cancer cells ( figure 1b ) . to avoid nonspecific adsorption to cck2r negative cells , we 
 incorporated 
 a water - soluble peptide spacer , referred 
 to as l1 , between the ligand and its therapeutic payload ( figure 1b ) .
previous results from our lab have shown only 
 a slight loss of affinity with no effect on specificity when crl is 
 conjugated to its payload via hydrophilic linkers . in the present study ,
two highly potent microtubule 
 inhibitors , 
 desacetyl vinblastine hydrazide ( davbh ) and tubulysin b hydrazide 
 ( tubbh ) , were attached to the crl - l1 peptide spacer through a self - immolative 
 disulfide linker .
this linker allows 
 for selective release of the cytotoxic agent upon entry into the reducing 
 environment of cancer cells .
detailed schemes for the synthesis of 
 crl conjugates of davbh and tubbh are described in si figures 2 and 3 and complete chemical structures for all 
 conjugates can be found in si figures 47 . to determine the cytotoxicity and targeting specificity of the 
 crl - l1-davbh conjugate , we incubated crl - l1-davbh , free davbh , and 
 nontargeted l1-davbh with cck2r - transfected hek 293 cells for 2 h 
 followed by incubation of the cells in drug - free medium for 66 h. 
 cell viability was then measured via incorporation of h - thymidine . as shown in figure 2 , the potency 
 of free davbh and crl - l1-davbh was 9 and 29 nm , respectively , whereas 
 the potency of the nontargeted conjugate l1-davbh was markedly reduced 
 by a factor of > 1000 ( ic50 value > 50 m ) .
importantly , 
 crl by itself was found to show no cytotoxicity toward hek 293-cck2r 
 cells ( si , figure 8) , demonstrating that 
 the aforementioned cytotoxicity was due to the targeted therapeutic 
 agent rather than obstruction of cck2r by crl . in vitro cytotoxicity 
 of davbh derivatives .
the cytotoxicity of 
 free davbh ( circles ) , the nontargeted l1-davbh ( triangles ) , and the 
 targeted crl - l1-davbh ( squares ) conjugates in hek 293 cells transfected 
 with cck2r when pulsed for 2 h at 37 c , washed with culture 
 medium three times , and then incubated for an additional 66 h. cells 
 were incubated with h - thymidine for 4 h and washed three 
 times . after the final wash , the cells were dissolved in 0.5 ml of 
 0.25 m naoh .
viability of the cells was assessed by analyzing the 
 incorporation of h - thymidine .
, 
 we injected 2 mol / kg crl - l1-davbh 
 intraperitoneally into mice bearing cck2r - transfected hek 293 xenografts 
 at a frequency of 3x per week for 3 weeks ( n = 5 ) .
this dose was selected based on previous studies showing significant 
 therapeutic benefit when similar folate- and dupa - targeted chemotherapeutics 
 were administered at the same concentration . as shown in figure 3a , crl - l1-davbh
was found to markedly delay tumor growth but not 
 lead to complete tumor eradication . to determine whether the residual 
 lesion was comprised predominately of cancer or stromal cells , h&e 
 staining
was performed on excised tumors ( day 33 ) . unlike the saline 
 control ,
which was comprised of almost entirely of cancer cells ( figure 4a ) , crl - l1-davbh - treated tumors exhibited a greatly 
 decreased ratio of cancer to stromal cells ( figure 4b ) . as expected , the nontargeted l1-davbh exerted little impact 
 on tumor growth , demonstrating the critical role of ligand targeting 
 on crl - l1-davbh efficacy ( figure 3a ) .
importantly , 
 body weights in the crl - l1-davbh - treated group remained essentially 
 constant over the course of the study , suggesting that the targeted 
 therapy was not grossly toxic to the animals ( figure 3b ) . in vivo therapeutic efficacy and gross toxicity of davbh conjugates . 

( a ) volume of tumors and ( b ) percent weight change in nu / nu mice subcutaneously injected with cck2r - transfected hek 293 cells 
 ( 5.0  10 in 50% hc matrigel ) on day 0 .
mice were 
 randomized to different treatment groups ( n = 5 ) 
 and began treatment on day 15 , when tumor volume was approximately 
 100 mm .
saline ( diamonds ) , nontargeted l1-davbh ( triangles ) , 
 and targeted crl - l1-davbh ( squares ) conjugates were administered intraperitoneally 
 following a tiw schedule for 3 weeks .
excised tumors from mice treated with ( a ) saline or ( b ) crl - l1-davbh 
 were sectioned and stained with hematoxylin and eosin .
arrows show 
 the abundance of cancer cells in panel a and a reduced number of cancer 
 cells in panel b. because complete tumor 
 remission was not achieved at a dose of 
 2 g / kg with crl - l1-davbh , we elected to develop a more potent 
 cck2r - targeted conjugate .
for this purpose , tubulysin b hydrazide 
 ( tubbh ) , a microtubule inhibitor with 10 the potency 
 of davbh , was conjugated to crl via the same l1 linker ( figure 1 , si figures 2 and 3 ) . 

as shown in figure 5a , free tubulysin b hydrazide 
 was found to be very potent in vitro , exhibiting an ic50 of 2.7 nm on hek 293-cck2r cells .
similarly , the targeted crl - l1-tubbh 
 conjugate yielded an ic50 of 2 nm , whereas nontargeted 
 l1-tubbh displayed the anticipated significant drop in potency ( ic50 of 310 nm ) due to its membrane impermeability and 
 lack of targeting . to ensure
that the cytotoxicity of crl - l1-tubbh 
 was receptor mediated , cells were incubated with crl - l1-tubbh in the 
 presence of 100-fold excess crl - l1 .
as expected , the ic50 value of the competed crl - l1-tubbh was nearly identical to that 
 of the nontargeted l1-tubbh conjugate ( ic50 of 340 
 and 310 nm , respectively ) . in vitro cytotoxicity of tubbh derivatives . 

the cytotoxicity of 
 free tubbh ( circles ) , the nontargeted l1-tubbh ( triangles ) , the targeted 
 crl - l1-tubbh ( squares ) , and the targeted crl - l1-tubbh in the presence 
 of 100-fold greater concentration competing crl - l1 ( diamonds ) conjugates 
 in hek 293 cells transfected with cck2r when pulsed for 2 h at 37 
 c , washed with culture medium three times , and then incubated 
 for an additional 66 h. cells were incubated with h - thymidine 
 for 4 h and washed three times .
after the final wash , the cells were 
 dissolved in 0.5 ml of 0.25 m naoh .
viability of the cells was assessed 
 by analyzing the incorporation of h - thymidine .
error bars 
 represent standard deviation . to determine the efficacy of crl - l1-tubbh in vivo
, we followed 
 the same treatment regimen used for davbh conjugates except that tubbh 
 conjugates were injected intravenously .
crl - l1-tubbh showed efficient 
 antitumor activity , eliminating all detectable malignant lesions from 
 all five mice ( figure 6a ) and prolonged survival 
 ( si , figure 9 ) . as expected , nontargeted 
 l1-tubbh showed no difference from the saline - treated control group 
 when an identical concentration as that of the crl - l1-tubbh targeted 
 conjugate was tested .
moreover , when crl - l1-tubbh was competed with 
 100-fold excess of crl - l1 , the antitumor effect of the targeted tubbh 
 was negated ( figure 6a ) .
this indicates that 
 the antitumor activity observed in the crl - l1-tubbh group was entirely 
 receptor mediated .
importantly , the crl - l1-tubbh - treated mice displayed 
 no visible signs of gross toxicity , and no weight loss was observed 
 ( figure 6c ) . in vivo therapeutic efficacy and gross 
 toxicity of tubbh conjugates . 

( a , b ) volume of tumors and ( c , d ) percent weight change in nu / nu mice subcutaneously injected with 5.0  10 cck2r - transfected hek 293 cells ( cck2r(+ ) cells ) in 50% hc 
 matrigel ( a , c ) or 1.0  10 kb cells ( cck2r( ) 
 cells ) in 50% hc matrigel on day 0 .
mice were randomized to different 
 treatment groups ( n = 5 ) and began treatment on day 
 17 , when the tumor volume was 100 mm .
saline ( diamonds ) , 
 nontargeted l1-tubbh ( triangles ) , targeted crl - l1-tubbh ( squares ) , 
 and targeted crl - l1-tubbh in the presence of 100-fold greater concentration 
 of competing crl - l1 ( circles ) conjugates were administered via lateral 
 vein injection following a tiw schedule for 3 weeks .
finally , to more thoroughly establish the necessity of receptor - mediated 
 targeting for crl - l1-tubbh efficacy , the targeted conjugate was tested 
 using the same protocol in a cck2r - negative kb xenograft model . in 
 vitro crl - l1-tubbh conjugate yielded an ic50 value of 269 
 nm ( si , figure 9 ) , similar to the ic50 values observed with the nontargeted and competed targeted 
 conjugates in the cck2r - expressing hek cells .
as shown in figure 6b , crl - l1-tubbh showed no difference in tumor growth 
 from the vehicle control .
because free tubulyisn b hydrazide is known 
 to be cytotoxic against kb cells , the 
 lack of reduction in tumor volume in the kb xenograft must derive 
 from the absence of cck2r and not from any intrinsic resistance to 
 tubbh . again
, none of the mice in this study exhibited any signs of 
 gross toxicity , and no substantial weight loss was observed ( figure 6d ) .
conjugation of cytotoxic 
 drugs to ligands that selectively target 
 cancer cells has emerged as a promising method to both improve drug 
 efficacy and reduce drug toxicity .
although only a few low molecular 
 weight ligand - targeted cancer drugs have been tested in vivo to date , 
 results suggest that the potential to design such targeted therapeutics 
 for most human malignancies is promising .
folate receptor - targeted 
 chemotherapeutic agents have demonstrated the capacity to treat malignancies 
 of the ovary , lung , kidney , breast , and endometrium , and psma - targeted drugs have shown promise for treating cancers 
 of the prostate .
lhrh receptor- and v3-targeted therapeutics can expand this 
 list to include cancers of the ovary and breast , but to date , few ligands are available to target cancers of the 
 gi tract . in this study , we attempted to obtain a ligand that would 
 deliver attached drugs to gastroinstestinal malignancies .
on the basis 
 of literature demonstrating overexpression of cck2r in gastrointestinal 
 stromal tumors and colon , stomach , esophageal , and pancreatic cancers , the crl conjugates described in this paper should have the potential 
 to address this deficiency .
one of the more interesting results 
 to emerge from this study was 
 the high specificity displayed by both crl - l1-davbh and crl - l1-tubbh 
 for cck2r - expressing cancer cells .
crl , davbh , and tubbh were all 
 found to be very hydrophobic , predicting that any conjugate of the 
 ligand to a drug would be extremely lipophilic , resulting in a strong 
 proclivity to associate nonspecifically with all cell membranes .
thus , 
 to increase hydrophilicity , a water - soluble linker ( l1 ) was utilized 
 to conjugate crl to the cytotoxic drugs .
cck2r - dependent binding of 
 crl - l1-davbh and crl - l1-tubbh was established by the absence of toxicity 
 when ( i ) crl was deleted , ( ii ) conjugate binding was competed with 
 excess ligand , or ( iii ) cck2r was absent from the targeted cancer 
 cell .
drug 
 conjugates can be rendered tumor specific by the judicious choice 
 of a hydrophilic linker due to the linker impeding the diffusion of 
 the hydrophobic drug through the cell membranes , thus reducing nonspecific activity .
however , this effect 
 is somewhat concentration dependent as some conjugates can diffuse 
 into cells , and therefore at high concentrations , nonreceptor mediated 
 activity can occur . in the case of linking crl to davbh and tubbh , 

the insertion of a bulky water - soluble linker between the ligand and 
 drug converted an otherwise nonspecific drug conjugate into a highly 
 receptor - targeted drug conjugate at physiologically relevant concentrations .
our crl - tubbh conjugates were also tested against xenograft tumors 
 similar to those exposed to the davbh conjugates , except an additional 
 treatment group was included where crl - l1-tubbh was competed with 
 excess crl - l1 .
similar to the davbh conjugates , the nontargeted l1-tubbh 
 conjugate did not appear to have any effect on tumor growth .
the lack 
 of efficacy of l1-conjugated cytotoxic compounds in vivo is most likely 
 due to the hydrophilic linker , which does not allow for the cytotoxic 
 compound to readily diffuse through the cell membrane , whereas the 
 targeted crl - l1-davbh and crl - l1-tubbh both showed efficacy in vivo .
somewhat surprisingly , the crl - l1-davbh conjugate did not regress 
 tumor volume even though similar concentrations of davbh conjugated 
 to folate or dupa did exhibit tumor regression .
this result is most likely due to a lower 
 number of cck2r receptors present on the cell surface as compared 
 to folate or psma .
this resulting lack of tumor regression in vivo 
 encouraged the testing of a more potent cytotoxic agent with a similar 
 mechanism of action .
when the more potent crl - l1-tubbh conjugate was 
 tested , not only do it halt tumor growth , but it even regressed the 
 tumor to an indistinguishable level .
importantly , the crl - l1-tubbh 
 treatment group that was competed with crl - l1 showed no effect on 
 inhibiting tumor growth , similar to both the vehicle control and the 
 nontargeted conjugate . to further assess the necessity of receptor - mediated 
 targeting , we tested xenografts of the receptor - negative kb cell line 
 with the targeted crl - l1-tubbh conjugate . unlike the cck2r - positive 
 hek 293 xenografts ,
the kb xenografts showed neither regression nor 
 a delay of tumor growth when treated with the conjugate . taken together , 
 these results strongly support the necessity of receptor - mediated 
 targeting with these conjugates for any in vivo efficacy . in 
 summary
, the current study demonstrates that cck2r - targeting 
 ligands can be used to deliver drugs selectively to cck2r - positive 
 tumors to generate a dramatic receptor - specific antitumor effect without 
 observable gross toxicity to healthy tissues .
because current treatments 
 for colon , lung , pancreatic , and related cancers commonly cause hair 
 loss , bone marrow suppression , weight loss , and so forth , the prospects 
 for developing a less toxic treatment for these malignant diseases 
 offer some optimism for the management of these cancers in the future .
 as the delivery of selectively targeted 
 cytotoxic agents via antibodies 
 or small molecule ligands to malignancies has begun to show promise 
 in the clinic , the need to identify and validate additional cellular 
 targets for specific therapeutic delivery is critical . 
 although a 
 multitude of cancers have been targeted using the folate receptor , 
 psma , bombesin receptor , somatostatin receptor , lhrh , and v3 , there is a notable lack of specific small 
 molecule ligand / receptor pairs to cellular targets found within cancers 
 of the gi tract . 
 because of the selective gi tract expression of the 
 cholecystokinin 2 receptor ( cck2r ) , we undertook the creation of conjugates 
 that would deliver microtubule - disrupting drugs to malignancies through 
 the specific targeting of cck2r via a high affinity small molecule 
 ligand . 
 the cytotoxic activity of these conjugates were shown to be 
 receptor mediated in vitro and in vivo with xenograft mouse models 
 exhibiting delayed growth or regression of tumors that expressed cck2r . 
 
 overall , this work demonstrates that ligands to cck2r can be used 
 to create selectively targeted therapeutic conjugates . 
Introduction
Results
Discussion