bladder cancer is the fourth most common malignancy in men and the ninth most common in women , with an estimated incidence of 67,160 and 13,750 estimated deaths ( 2007 ) in the united states .
transitional cell carcinoma ( tcc ) refers to those bladder cancer tumors derived from urothelial tissue , more than 90% of which originate in the urinary bladder and present as nonmuscle - invasive disease in the majority of patients at diagnosis.1 of those patients who respond to current standard of care , approximately two - thirds will have recurrence , and 10%20% of recurrent tumors will have progressed to muscle - invasive disease.2 typically , treatment for high - risk , nonmuscle - invasive bladder cancer ( nmibc ) is transurethral resection of the bladder tumor and adjuvant therapy with bacillus calmette  guerin ( bcg ) .
although bcg treatment can reduce the risk of recurrence and progression , its use is limited by the adverse effect profile and intolerance that occurs in 20% of patients.36 epithelial cell adhesion molecule ( epcam ) is overexpressed in many carcinomas relative to their normal tissue counterparts , as is the case in tcc.7,8 in addition , epcam expression increases as these cancers progress from lower to higher grades.811 together , these features make epcam a clinically relevant antigen for targeted therapy in bladder cancer .
vb4 - 845 is a recombinant fusion protein that targets epcam - positive cancer cells .
it consists of an anti - epcam humanized single - chain variable fragment ( scfv ) linked to a truncated form of pseudomonas exotoxin a ( eta252608 ) that lacks the cell - binding domain.12 once bound to epcam on the surface of carcinoma cells , vb4 - 845 is internalized , whereupon the exotoxin portion of the fusion protein induces apoptosis.13,14 one concern of targeted therapies has been the toxicity associated with systemic administration of this class of drug.15 moreover , repeated use of therapeutics comprising foreign proteins is limited by their immunogenicity . therefore , it is desirable to develop therapies designed for local administration , thereby increasing the clinical benefit of these treatments while minimizing any drug - related effects . accordingly , locoregional delivery of eta - conjugated antibodies has been demonstrated to be well tolerated and clinically effective in patients with glioblastoma multiforme , erbb2-expressing breast tumors , and squamous cell carcinoma of the head and neck.1618 nonclinical studies showed a significant reduction in toxicity with locally administered vb4 - 845 .
similarly , local injections of vb4 - 845 were well tolerated in cynomolgus monkeys with adverse events ( aes ) being mild and easily managed.19 in addition to the strong nonclinical safety profile , vb4 - 845 exhibits highly potent activity against epcam - expressing tumor cell lines and has been shown to localize to epcam - positive tumor xenografts.12 based on these preclinical results , a phase i dose - escalation trial was performed using vb4 - 845 as an intravesical therapy in bcg - refractory and bcg - intolerant patients with grade 2 or 3 nmibc .
only patients 18 years of age or older with immunohistochemically confirmed epcam - positive grade 2 or 3 nmibc ( ta , t1 , in situ carcinoma [ tis ] ) , either refractory to ( recurrence within 2 years following at least one complete cycle of bcg therapy ) or intolerant of bcg therapy , were eligible for this study .
other key inclusion criteria were adequate renal ( serum creatinine 1.5  the upper limit of normal ( uln ) or creatinine clearance 60 mls / min ) , hepatic ( alanine aminotransferase and aspartate aminotransferase 2.5  uln and bilirubin levels 1.5  uln ) , and hematological ( granulocytes 1500/l , platelets 100,000/l , and hemoglobin > 8 g / dl ) function .
women of child - bearing potential , and all men , must have agreed to use adequate contraception prior to and for the duration of the study .
key exclusion criteria included patients with muscle - invasive tumors , nodal involvement , or distant metastases ; patients with a history of upper tract tcc , adenocarcinoma , or squamous cell carcinoma of the bladder ; and patients with disease involving the prostatic ducts or stroma . moreover , excluded were patients with a history of pelvic malignancy , hydronephrosis , or clinically significant abnormalities of the upper urinary tract and those who had undergone bcg therapy within 6 weeks prior to the start of vb4 - 845 dosing .
written informed consent was obtained from each participant before any study - related activity was performed .
this study was conducted according to section c.05.010 of division 5 of the food and drug regulations of the government of canada , ich harmonized tripartite guidelines for good clinical practice , the declaration of helsinki ( 2002 ) , and all local laws and regulations concerning clinical studies and the protection of study patients .
regulatory clearance for the study was obtained from the biologics and genetic therapies directorate of health canada in canada .
the study was an open - label , multicenter , dose - escalating trial of intravesically administered vb4 - 845 .
eight dose levels were initially evaluated , starting at 0.1 mg once weekly for 6 consecutive weeks and escalating through 0.2 , 0.33 , 0.66 , 1.32 , 2.64 , 5.28 , and 10.56 mg / dose .
the maximum tolerated dose ( mtd ) was not reached ; therefore , an additional escalation through 13.73 , 17.85 , 23.20 , and 30.16 mg was undertaken .
each dose was administered to the bladder through a catheter and held for 2 h prior to voiding .
safety data from each dose cohort was evaluated after 3 weeks of treatment before proceeding to the next dose cohort .
dose - limiting toxicity ( dlt ) was defined as the occurrence of treatment - related aes , including intractable cystitis persisting for more than 1 week associated with severe pain , urgency , and/or frequency not relieved by measures considered to be routine standard of care according to each clinical site ; significant hematuria leading to clot obstruction ; grade 4 flu - like symptoms ; grade 3 or higher hematological toxicity ( if it is a two grade increase from baseline ) ; or any other grade 3 nonhematological toxicity ( with the exception of alopecia ) . if two patients experienced a dlt at any dose , that dose would be defined as the mtd .
specific additional evaluations were performed depending on the timing of the visit , as described below .
pretreatment screening evaluations were conducted within 4 weeks of the baseline visit and included medical history , pregnancy test , 12-lead electrocardiogram , and assessment of karnofsky performance status .
immunohistochemistry on snap - frozen tissue samples was performed at a single facility to determine whether tumors were epcam positive .
in addition to the evaluations performed at every study visit , patients were monitored for aes for 3 h following drug administration on days 1 , 8 , 15 , 22 , 29 , and 36 .
the toxicity grade of any ae was classified according to the national cancer institute common toxicity criteria , version 3 .
aes were considered to be treatment related by the investigators if they were possibly , probably , or definitely related to vb4 - 845 administration . on day 2
additional assessments performed included pregnancy test , 12-lead electrocardiogram , and karnofsky performance status . to evaluate tumor response ,
a biopsy was required if the cystoscopy revealed lesions suspicious for malignancy , or cytology results were suspicious or positive for malignancy .
blood samples for pharmacokinetic analysis were taken on day 1 prior to and 1 , 2 , and 3 h postdosing .
predose samples were also taken on days 8 , 15 , 22 , 29 , and 36 .
for each sample , 3 ml of venous blood was collected into a tube containing lithium heparinate and placed on ice .
vb4 - 845 plasma levels were measured using a glp - validated , mts - based , potency assay.18 the assay detects intact vb4 - 845 by its ability to kill the epcam - positive cell line cal-27 , where the measured ic50 is directly proportional to the concentration of intact drug .
the assay was shown to have a lower limit of detection of 14 pg / ml .
blood samples for assessing humoral immune reactivity to vb4 - 845 were taken prior to dosing on days 1 , 8 , 15 , 22 , 29 , 36 , and at the final study visit .
samples were collected as for the pharmacokinetic analysis , and antibody titers to the scfv and eta252608 portions of the fusion protein ( human antihuman antibodies ( haha ) and human anti - pseudomonas antibodies ( hapa ) ) were measured using an enzyme - linked immunosorbent assay.18 responses were expressed as the geometric mean of measurable titers at each time point .
tumor response was assessed on evaluable patients 46 weeks following the last dose of vb4 - 845 .
the evaluation was based on cytology , cystoscopy , and , if required , biopsy .
complete response was defined as nonpositive urinary cytology and either normal cystoscopy or abnormal cystoscopy with negative biopsy .
only patients 18 years of age or older with immunohistochemically confirmed epcam - positive grade 2 or 3 nmibc ( ta , t1 , in situ carcinoma [ tis ] ) , either refractory to ( recurrence within 2 years following at least one complete cycle of bcg therapy ) or intolerant of bcg therapy , were eligible for this study .
other key inclusion criteria were adequate renal ( serum creatinine 1.5  the upper limit of normal ( uln ) or creatinine clearance 60 mls / min ) , hepatic ( alanine aminotransferase and aspartate aminotransferase 2.5  uln and bilirubin levels 1.5  uln ) , and hematological ( granulocytes 1500/l , platelets 100,000/l , and hemoglobin > 8 g / dl ) function .
women of child - bearing potential , and all men , must have agreed to use adequate contraception prior to and for the duration of the study .
key exclusion criteria included patients with muscle - invasive tumors , nodal involvement , or distant metastases ; patients with a history of upper tract tcc , adenocarcinoma , or squamous cell carcinoma of the bladder ; and patients with disease involving the prostatic ducts or stroma . moreover , excluded were patients with a history of pelvic malignancy , hydronephrosis , or clinically significant abnormalities of the upper urinary tract and those who had undergone bcg therapy within 6 weeks prior to the start of vb4 - 845 dosing .
written informed consent was obtained from each participant before any study - related activity was performed .
this study was conducted according to section c.05.010 of division 5 of the food and drug regulations of the government of canada , ich harmonized tripartite guidelines for good clinical practice , the declaration of helsinki ( 2002 ) , and all local laws and regulations concerning clinical studies and the protection of study patients .
regulatory clearance for the study was obtained from the biologics and genetic therapies directorate of health canada in canada .
the study was an open - label , multicenter , dose - escalating trial of intravesically administered vb4 - 845 .
eight dose levels were initially evaluated , starting at 0.1 mg once weekly for 6 consecutive weeks and escalating through 0.2 , 0.33 , 0.66 , 1.32 , 2.64 , 5.28 , and 10.56 mg / dose .
the maximum tolerated dose ( mtd ) was not reached ; therefore , an additional escalation through 13.73 , 17.85 , 23.20 , and 30.16 mg was undertaken .
each dose was administered to the bladder through a catheter and held for 2 h prior to voiding .
safety data from each dose cohort was evaluated after 3 weeks of treatment before proceeding to the next dose cohort .
dose - limiting toxicity ( dlt ) was defined as the occurrence of treatment - related aes , including intractable cystitis persisting for more than 1 week associated with severe pain , urgency , and/or frequency not relieved by measures considered to be routine standard of care according to each clinical site ; significant hematuria leading to clot obstruction ; grade 4 flu - like symptoms ; grade 3 or higher hematological toxicity ( if it is a two grade increase from baseline ) ; or any other grade 3 nonhematological toxicity ( with the exception of alopecia ) . if two patients experienced a dlt at any dose , that dose would be defined as the mtd
specific additional evaluations were performed depending on the timing of the visit , as described below .
pretreatment screening evaluations were conducted within 4 weeks of the baseline visit and included medical history , pregnancy test , 12-lead electrocardiogram , and assessment of karnofsky performance status .
immunohistochemistry on snap - frozen tissue samples was performed at a single facility to determine whether tumors were epcam positive .
in addition to the evaluations performed at every study visit , patients were monitored for aes for 3 h following drug administration on days 1 , 8 , 15 , 22 , 29 , and 36 .
the toxicity grade of any ae was classified according to the national cancer institute common toxicity criteria , version 3 .
aes were considered to be treatment related by the investigators if they were possibly , probably , or definitely related to vb4 - 845 administration . on day 2
additional assessments performed included pregnancy test , 12-lead electrocardiogram , and karnofsky performance status . to evaluate tumor response , cystoscopy / photography and cytology were performed .
a biopsy was required if the cystoscopy revealed lesions suspicious for malignancy , or cytology results were suspicious or positive for malignancy .
blood samples for pharmacokinetic analysis were taken on day 1 prior to and 1 , 2 , and 3 h postdosing .
predose samples were also taken on days 8 , 15 , 22 , 29 , and 36 .
for each sample , 3 ml of venous blood was collected into a tube containing lithium heparinate and placed on ice .
vb4 - 845 plasma levels were measured using a glp - validated , mts - based , potency assay.18 the assay detects intact vb4 - 845 by its ability to kill the epcam - positive cell line cal-27 , where the measured ic50 is directly proportional to the concentration of intact drug .
the assay was shown to have a lower limit of detection of 14 pg / ml .
blood samples for assessing humoral immune reactivity to vb4 - 845 were taken prior to dosing on days 1 , 8 , 15 , 22 , 29 , 36 , and at the final study visit .
samples were collected as for the pharmacokinetic analysis , and antibody titers to the scfv and eta252608 portions of the fusion protein ( human antihuman antibodies ( haha ) and human anti - pseudomonas antibodies ( hapa ) ) were measured using an enzyme - linked immunosorbent assay.18 responses were expressed as the geometric mean of measurable titers at each time point .
tumor response was assessed on evaluable patients 46 weeks following the last dose of vb4 - 845 .
the evaluation was based on cytology , cystoscopy , and , if required , biopsy .
complete response was defined as nonpositive urinary cytology and either normal cystoscopy or abnormal cystoscopy with negative biopsy .
all participants were caucasian with a median patient age of 69 ; 78% were men and 22% were women ( table 1 ) .
all patients evaluated for efficacy ( 61/64 ) had previously received bcg therapy , with 95% of the patients having had two or more bladder cancer recurrences . based on their previous bcg treatment history ,
only five patients were considered to have been enrolled as bcg intolerant rather than bcg refractory .
all patients had grade 2 or 3 , stage ta or t1 , tcc , and/or tis , and , except for two cases , all papillary tumors had been previously resected 728 days prior to the first dose of study drug . of note
, as this study was carried out prior to the widespread adoption of re - transurethral resection ( re - tur ) of tat1 , high - grade nmibc in clinical practice , and its inclusion in standard treatment guidelines , re - tur of t1 tumors was not performed .
all tumors showed immunohistological evidence of epcam - positive membrane staining ( figure 1 ) .
risk factors for progression present in the patient population are summarized in table 2 . at baseline ,
98% of patients had at least one factor associated with disease progression , and 64% of these individuals had three or more risk factors .
vb4 - 845 was administered to the bladder via catheter once a week for 6 consecutive weeks , followed by 46 weeks without treatment .
an mtd was not determined from the initial dose escalation as no dlt was reported ; therefore , additional dose cohorts were added as described above , and , again , all patients received all six scheduled doses without any dlt .
forty - one of the 64 patients ( 64% ) experienced an ae during the course of the study .
twenty - one ( 33% ) patients experienced only aes that were assessed by the investigator as unrelated to study treatment . classifying the aes as unrelated to the study drug was decided after considering the temporal relationship of the onset of the event to the administration of the study drug , whether the event could be explained by concomitant medications or concurrent disease , the response to withholding the study drug , and the response to rechallenge with the study drug .
these included , but were not limited to , urinary tract infection , incontinence , nocturia , pharyngolaryngeal pain , and bladder spasm .
the remaining 20 patients ( 31% ) experienced aes judged to be related to vb4 - 845 administration .
the most common treatment - related aes were dysuria and hematuria ( table 4 ) .
systemic aes included fatigue , fever and chills , loss of appetite , myalgia , dizziness , and nausea .
the frequency of treatment - related aes did not increase with dose escalation ( figure 2 ) .
all treatment - related aes were grade 1 or 2 , with the exception of one grade 3 occurrence of hematuria that was reported as possibly related to vb4 - 845 administration .
this event was not reported as a dlt as the investigator judged it to be related to aspirin use , and the patient experienced no aes with subsequent vb4 - 845 dosing .
there were no cases where the occurrence of aes required discontinuation of treatment , and no patient experienced a serious ae related to the administration of vb4 - 845 .
there was one patient death due to cardiac failure , which occurred 3 weeks after the last dose of vb4 - 845 .
this death was assessed by the investigator as unrelated to study treatment and attributed to long - standing coronary artery disease , previous myocardial infarction , and hypertension .
postinstillation plasma levels of vb4 - 845 were measured in 63 patients , except at the final visit where samples were provided by 61 patients . in almost all patients ,
vb4 - 845 plasma levels were below the limit of detection of the assay ( 14 pg / ml ) at all time points examined .
vb4 - 845 was detectable in only two patients : one had levels of 19 and 17 pg / ml at 1 h after vb4 - 845 instillation and on day 8 prior to dosing , respectively , and the other had a level of 18 pg / ml at the 1 h time point .
the immunogenicity of vb4 - 845 was examined by analyzing hapa and haha titers in blood samples taken at specified time points during the trial ( table 5 ) .
hapa response was more vigorous , as patients developed a measurable titer earlier ; the majority of patients exhibited hapa by day 29 , with 77% ( 47/61 ) having a measurable titer at final visit . in contrast , only 16% ( 10/61 ) of patients had haha by the end of the study .
hapa titers were also generally higher , with a mean maximum titer of 20,512 versus 3373 for haha responses .
a comparison of mean titers of the hapa and haha responses measured in samples taken on the final visit showed no significant difference between responders and nonresponders ( data not shown ) .
the absence of any apparent relationship between antibody titer and response to treatment suggests that the immune response was not detrimental to clinical outcome .
sixty - one patients were considered to be evaluable for efficacy ; two patients were excluded from the efficacy analysis due to an absence of bcg treatment prior to the study , and there was one study - unrelated death for whom no final tumor assessment was obtained .
complete response based on tumor classification at baseline and dose group is summarized in table 6 .
overall , a complete response was achieved by 39% ( 24/61 ) of the patients . of the patients with tis ,
29% achieved a complete response , while complete responses were observed in 44% and 43% of the patients with t1 and ta , respectively .
of the five patients classified as bcg intolerant , only one ( 20% ) had a complete response .
given the limited number of patients per dose cohort , it was not possible to make definitive individual dose comparisons . in order to examine a potential dose response ,
patients were classified into three dose groups : ( 0.1 to < 1.0 mg ) = lowest dose group ; ( 1.0 to < 10.0 mg ) = middle dose group ; and ( 10.0 mg ) = highest dose group .
a comparison of the response rates in the lowest dose group versus the combined middle and highest dose group revealed a statistically significant p - value of 0.0418 .
all participants were caucasian with a median patient age of 69 ; 78% were men and 22% were women ( table 1 ) .
all patients evaluated for efficacy ( 61/64 ) had previously received bcg therapy , with 95% of the patients having had two or more bladder cancer recurrences . based on their previous bcg treatment history ,
only five patients were considered to have been enrolled as bcg intolerant rather than bcg refractory .
all patients had grade 2 or 3 , stage ta or t1 , tcc , and/or tis , and , except for two cases , all papillary tumors had been previously resected 728 days prior to the first dose of study drug . of note
, as this study was carried out prior to the widespread adoption of re - transurethral resection ( re - tur ) of tat1 , high - grade nmibc in clinical practice , and its inclusion in standard treatment guidelines , re - tur of t1 tumors was not performed .
all tumors showed immunohistological evidence of epcam - positive membrane staining ( figure 1 ) .
risk factors for progression present in the patient population are summarized in table 2 . at baseline ,
98% of patients had at least one factor associated with disease progression , and 64% of these individuals had three or more risk factors .
vb4 - 845 was administered to the bladder via catheter once a week for 6 consecutive weeks , followed by 46 weeks without treatment .
an mtd was not determined from the initial dose escalation as no dlt was reported ; therefore , additional dose cohorts were added as described above , and , again , all patients received all six scheduled doses without any dlt .
forty - one of the 64 patients ( 64% ) experienced an ae during the course of the study .
twenty - one ( 33% ) patients experienced only aes that were assessed by the investigator as unrelated to study treatment . classifying the aes as unrelated to the study drug was decided after considering the temporal relationship of the onset of the event to the administration of the study drug , whether the event could be explained by concomitant medications or concurrent disease , the response to withholding the study drug , and the response to rechallenge with the study drug .
these included , but were not limited to , urinary tract infection , incontinence , nocturia , pharyngolaryngeal pain , and bladder spasm .
the remaining 20 patients ( 31% ) experienced aes judged to be related to vb4 - 845 administration .
the most common treatment - related aes were dysuria and hematuria ( table 4 ) .
systemic aes included fatigue , fever and chills , loss of appetite , myalgia , dizziness , and nausea .
the frequency of treatment - related aes did not increase with dose escalation ( figure 2 ) .
all treatment - related aes were grade 1 or 2 , with the exception of one grade 3 occurrence of hematuria that was reported as possibly related to vb4 - 845 administration .
this event was not reported as a dlt as the investigator judged it to be related to aspirin use , and the patient experienced no aes with subsequent vb4 - 845 dosing .
there were no cases where the occurrence of aes required discontinuation of treatment , and no patient experienced a serious ae related to the administration of vb4 - 845 .
there was one patient death due to cardiac failure , which occurred 3 weeks after the last dose of vb4 - 845 .
this death was assessed by the investigator as unrelated to study treatment and attributed to long - standing coronary artery disease , previous myocardial infarction , and hypertension .
postinstillation plasma levels of vb4 - 845 were measured in 63 patients , except at the final visit where samples were provided by 61 patients . in almost all patients ,
vb4 - 845 plasma levels were below the limit of detection of the assay ( 14 pg / ml ) at all time points examined .
vb4 - 845 was detectable in only two patients : one had levels of 19 and 17 pg / ml at 1 h after vb4 - 845 instillation and on day 8 prior to dosing , respectively , and the other had a level of 18 pg / ml at the 1 h time point .
the immunogenicity of vb4 - 845 was examined by analyzing hapa and haha titers in blood samples taken at specified time points during the trial ( table 5 ) .
hapa response was more vigorous , as patients developed a measurable titer earlier ; the majority of patients exhibited hapa by day 29 , with 77% ( 47/61 ) having a measurable titer at final visit . in contrast , only 16% ( 10/61 ) of patients had haha by the end of the study .
hapa titers were also generally higher , with a mean maximum titer of 20,512 versus 3373 for haha responses .
a comparison of mean titers of the hapa and haha responses measured in samples taken on the final visit showed no significant difference between responders and nonresponders ( data not shown ) .
the absence of any apparent relationship between antibody titer and response to treatment suggests that the immune response was not detrimental to clinical outcome .
sixty - one patients were considered to be evaluable for efficacy ; two patients were excluded from the efficacy analysis due to an absence of bcg treatment prior to the study , and there was one study - unrelated death for whom no final tumor assessment was obtained .
complete response based on tumor classification at baseline and dose group is summarized in table 6 .
overall , a complete response was achieved by 39% ( 24/61 ) of the patients . of the patients with tis ,
29% achieved a complete response , while complete responses were observed in 44% and 43% of the patients with t1 and ta , respectively .
of the five patients classified as bcg intolerant , only one ( 20% ) had a complete response .
given the limited number of patients per dose cohort , it was not possible to make definitive individual dose comparisons . in order to examine a potential dose response
, patients were classified into three dose groups : ( 0.1 to < 1.0 mg ) = lowest dose group ; ( 1.0 to < 10.0 mg ) = middle dose group ; and ( 10.0 mg ) = highest dose group .
a comparison of the response rates in the lowest dose group versus the combined middle and highest dose group revealed a statistically significant p - value of 0.0418 .
tcc is the most common cancer of the bladder , and most patients present with nonmuscle - invasive disease .
currently , bcg is the standard of care for this malignancy . despite its well - documented therapeutic benefit ,
bcg therapy is delayed or discontinued in a large proportion of patients due to associated toxicity . in rare instances
, patients can develop severe conditions associated with bcg infection , in some cases life - threatening , rendering bcg a nonviable treatment option .
in addition , for those patients who fail to respond to bcg therapy or experience early disease recurrence , alternative therapeutic options are limited . given the potential for intolerance and the high rate of recurrence in patients with nmibc , alternative treatments are under investigation .
targeted therapies limit drug exposure to only diseased cells , thereby minimizing drug - related toxicities .
we have described a phase i study of vb4 - 845 , an anti - epcam scfv 
eta , administered intravesically to high - risk patients . of the 75 patients initially screened for participation , 74 ( 99% ) showed epcam - positive disease .
this high rate of epcam expression suggests that epcam represents a significant antigen for vb4 - 845-targeted therapy in patients with advanced nmibc .
although not performed in this study , an examination of the expression levels of epcam in biopsy samples obtained from patients with residual disease following vb4 - 845 treatment might be informative and will be considered for future studies .
aes experienced with vb4 - 845 were mild and easily managed , and no ae required the discontinuation of treatment , even in the highest dose cohort . although the presence of pre - existing lower urinary tract symptoms may make a determination of the frequency of aes difficult , what is evident is that the relatively mild toxicities experienced with vb4 - 845 are in direct contrast to other intravesical therapies where local and systemic toxicities can be treatment limiting .
vb4 - 845 is a targeted therapy that exerts its effect via specific interaction with the cell surface antigen epcam .
although overexpressed on carcinoma cells , epcam expression is lower on normal tissue and limited to the basal layers rather than luminal surfaces .
these features could minimize drug - related toxicities and may explain why related aes were predominantly grade 1 and 2 .
it is also interesting to note that failure to reach an mtd for vb4 - 845 over the dose range tested and the apparent absence of any relationship between the appearance of aes and dose are in keeping with its excellent safety profile .
pharmacokinetic analyses indicated that vb4 - 845 was not systemically absorbed and remained within the bladder until elimination by voiding .
this is anticipated , given the integrity of the bladder and the molecular size of vb4 - 845 ( ~69 kda ) .
vb4 - 845 was shown to be immunogenic with the toxin portion eliciting the more vigorous response .
given the humanization of the scfv antibody fragment and entirely foreign nature of the bacterial toxin , the more intense immune response against the toxin was expected .
a similar pattern of immune response was observed with lmb-2 , a scfv  eta immunotoxin , whereby more patients exhibited a stronger response to the eta moiety than to the scfv portion.20 despite the observed immunogenicity , the development of an immune response was not considered to have any bearing on clinical outcome , as there was no apparent relationship between antibody titer and response to treatment .
rather , the presence of circulating antibodies to vb4 - 845 may , in fact , limit any systemic exposure that may occur by rapidly clearing any drug that may enter circulation . together , the specificity of vb4 - 845 and its lack of systemic exposure translate to a very acceptable safety profile .
although the phase i trial was primarily designed to determine the mtd , tumor response was assessed as a single efficacy endpoint at 3 months to elucidate any observable trends .
although dosing was not optimized for therapeutic benefit , complete responses were observed across all tumor stages and dose groupings .
although it is not possible to draw definitive conclusions between individual dose and response to treatment , when examined according to dose groups , the data are suggestive of an increased response with higher doses .
although no direct examination of receptor occupancy has been performed , the dose range of vb4 - 845 administered far exceeds the drug concentration required to achieve > 99% cell killing in epcam - positive bladder tumor cell lines . therefore , it is possible that the tumor response may be saturated at the higher doses .
it should be noted that the short duration of follow - up is a significant limitation with respect to assessing efficacy ; thus , efficacy results reported in this study are considered preliminary and serve only to support further clinical trials with longer follow - up assessments .
intravesical therapy with the anti - epcam fusion protein vb4 - 845 , for the treatment of nmibc was extremely well tolerated .
aes were generally mild and very manageable with no patients experiencing any serious aes related to vb4 - 845 therapy .
the lack of detectable postinstillation plasma levels in the vast majority of patients indicates that the drug is effectively held within the bladder and not systemically absorbed .
although most patients developed antibody titers , the data obtained in this study showed no evidence of detrimental effects on clinical outcome associated with the immune response .
preliminary efficacy results from this high - risk , often treatment - refractory , patient population suggest that vb4 - 845 can safely inhibit tumor growth in patients with epcam - positive , high - grade nmibc .
 purposea phase i dose - escalation study was performed to determine the maximum tolerated dose ( mtd ) of the immunotoxin vb4 - 845 in patients with nonmuscle - invasive bladder cancer ( nmibc ) refractory to or intolerant of bacillus calmette  
 guerin ( bcg ) . 
 secondary objectives included evaluation of the safety , tolerability , pharmacokinetics , immunogenicity , and efficacy of vb4-845.patients and methodssixty - four patients with grade 2 or 3 , stage ta or t1 transitional cell carcinoma or in situ carcinoma , either refractory to or intolerant of bcg therapy , were enrolled . 
 treatment was administered in ascending dose cohorts ranging from 0.1 to 30.16 mg . after receiving weekly instillations of vb4 - 845 to the bladder via catheter for 6 consecutive weeks , 
 patients were followed for 46 weeks post - therapy and assessed at week 12.resultsan mtd was not determined , as a dose - limiting toxicity was not identified over the dose range tested . 
 vb4 - 845 therapy was safe and well tolerated with most adverse events reported as mild ; as a result , no patients were removed from the study in response to toxicity . by the end of the study 
 , the majority of patients had developed antibodies to the exotoxin portion of vb4 - 845 . 
 a complete response was achieved in 39% of patients at the 12-week time point.conclusionsvb4-845 dosed on a weekly basis for 6 weeks was very well tolerated at all dose levels . although an mtd was not determined at the doses administered , vb4 - 845 showed evidence of an antitumor effect that warrants further clinical investigation for the treatment of nmibc in this patient population . 