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 .