Patent Application: US-201514626071-A

Abstract:
an mri contrast material includes tumor - targeting metal - loaded ferritin nanoparticles constructed with genetically modified ferritin coupled to a target - specific agent . ferritin derived from archaeoglobus flulgidus forms hollow nanocages surrounding paramagnetic or superparamagnetic metal core , storing a significantly greater quantity of iron or other paramagnetic or superparamagnetic metal than natural ferritins , and is conjugated via a short linker with a monoclonal antibody against a cell surface antigen overexpressed by a cancer , to selectively and efficiently attach to tumor cells to enhance mri contrast . significant t 2 contrast with diminished t 1 effect was observed owing to the heterogeneous nanoconjugate distribution when bound to cells . in a treatment method , after imaging , an external stimulus heats the cell - bound agent to release the metal and selectively destroy the targeted cells . the enhanced imaging and release of toxic metal ions provides simultaneous early detection and treatment .

Description:
the invention will be understood from the following description of an exemplary embodiment and measurement results obtained therewith , together with discussion of the observed binding , magnetic and imaging characteristics reported below and their use in imaging , diagnosing and treating tissue conditions such as cancer . briefly , the invention provides a new mri contrast agent , namely cell - targeting ferritin cage nanoparticles loaded with iron or other magnetic or paramagnetic metal . the invention also provides diagnostic and treatment methods using the contrast agent . initially we describe in detail the preparation of an iron - loaded , cancer - targeting ferritin nanoparticle contrast agent and its properties . the ferritin used in the present study is a genetically engineered ferritin obtained from archaeoglobus fulgidus . cloning , expression and purification were performed following the methods previously described in sana et al . ( 2010 ). enrichment of the ferritin with iron ( iii ) ions and the analysis of iron loading were achieved by following the methods reported in liu et al . 2003 ; glahn et al . 1995 ; and bonomi and pagani 1986 . the process was repeated three times , and the average value for the number of iron ( iii ) ion per each ferritin was determined to be 6 , 700 . it was observed that iron loading beyond 7000 fe / cage resulted in some difficulty in maintaining monodispersion in suspension , with precipitation possible due to aggregation . generation and characterization of the necl - 5 specific mouse igg monoclonal antibody ( mab 324 . 5 ) has been described previously ( hixson et al . 1986 ; faris et al . 1990 ; lim et al . 1996 ). to prepare the contrast agent , the two components , mab necl - 5 and fe ( iii )- enriched ferritin , were tethered by a convergent method . fig1 schematically illustrates preparation of the ferritin / mab necl - 5 nanoconjugate ferritin material 6 used in the examples herein . this diagram also illustrates the relative length and size of the components , ferritin ( about 12 . 5 nm in diameter ), antibody ( about 10 nm in length ) and the linker ( about 1 nm ) shown in the schema 1 - 6 of fig1 . briefly , lysine residues of 1 , mab ( necl - 5 ) were reacted with sata ( n - succinimidyl s - acetylthioacetate , 4 equiv . thermoscientific ) in a hepes buffer solution ( ph 7 . 5 ), which result in 3 , a thioacetyl acetamide elongation . separately , lysine residues of 2 , fe ( iii )- enriched ferritin were treated with sulfo - smcc ( succinimidyl 4 -[ n - maleimidomethyl ] cyclohexane - 1 - carboxylate , 4 equiv . thermoscientific ) in the same buffer solution to yield 5 , which in turn , reacted with 4 , the deacetylated thiol form of 3 . the reaction of 4 with 5 proceeded in the presence of edta in order to suppress the disulfide formation between two 4 molecules . the desired conjugate 6 was obtained and isolated by a size exclusion chromatography ( sec ) superdex 200 10 / 300 gl column ( ge healthcare , buckinghamshire , uk ). transformed rat pec were maintained in a 1 : 1 mixture of rpmi 1640 ( gibco , carlsbad , calif .) and mcdb 153 ( sigma - aldrich , st . louis , mo .) supplemented with sodium bicarbonate ( 1 . 9 g / l ), sodium pyruvate ( 0 . 5 %), fetal bovine serum ( fbs ) ( 5 %, hyclone , logan , utah ), epidermal growth factor ( 0 . 02 μg / ml , bd biosciences , san jose , calif . ), bovine pituitary extract ( 5 μg / ml , bd biosciences ), dexamethasone ( 2 mm in 95 % etoh ), glutamine ( 1 %), gentamycin ( 0 . 1 mg / ml , gibco ), its ( 0 . 25 %, bd biosciences ), forskolin ( 2 . 5 μg / ml , calbiochem , san diego , calif .) and normocin and incubated at 37 ° c . in a 5 % co 2 humidified atmosphere . cells were grown to approximately 75 - 80 % confluence , and were trypsinized and washed in hanks balanced salt solution ( hbss ; sigma - aldrich ). cell suspensions were incubated in the presence or absence of necl - 5 nanoconjugate in 1 × pbs supplemented with 0 . 5 % bsa at 4 ° c . for 1 hr with gentle rotation . following two 5 min washes in hbss , cells suspensions were mixed 1 : 1 with 1 % seaplaque low melting temperature agarose ( lonza , rockland , me .) in 2 ml conical vials for subsequent imaging . cell preparations in 2 ml vials ( along with an undosed control cell sample ) were scanned using the same procedure as for the uniform dispersion gel samples with relaxation rates and relaxivities calculated in the same manner . each cell pellet contained approximately 2 × 10 7 cells . a targeted nanoconjugate version of the ferritin construct was prepared for in vitro testing as shown in fig1 by binding a monoclonal antibody targeting the necl - 5 glycoprotein , expressed by many epithelial carcinomas . transformed rat prostate epithelial cells ( 2 . 0 × 10 7 cells per sample ) were incubated with the targeted form of the ferritin nanoconjugate at three dose levels : 50 , 100 , and 200 μg conjugate per ml . after the incubation ( 37 ° c ., 45 minutes ), the samples were washed and centrifuged for three cycles . all of the washes including unbound conjugates were collected and analyzed for iron content using the bathophenanthroline disulfonic acid / sodium dithionate method described earlier ( bonomi and pagani 1986 ). for mr relaxivity measurements , iron loaded ferritin cages loaded to 6700 fe / cage were uniformly dispersed in 1 % agarose gel at concentrations of 1 , 2 , 5 , 10 , 20 , 50 , 100 , 200 , 500 and 1000 nm . corresponding phantoms were prepared using natural horse ferritin . the gels were contained in 1 . 5 ml vials for scanning . scans were acquired using a 3 tesla siemens tim trio system . a 32 - channel head resonator was used for signal receive . field shimming to second order was performed prior to acquisition of mapping scans . the ferritin vials , along with controls ( agarose gel alone ) were placed horizontally in a holder within the head resonator . tomographic images 2 mm thick were acquired of the vials in cross - section with an in - plane resolution of 0 . 4 mm . for estimation of t 2 a multi - spin echo sequence was used with a repetition time of 1500 ms and 12 echo times ranging from 10 ms to 120 ms in 10 ms steps . in addition , gradient echo images were acquired to give an indication of susceptibility contrast ( tr = 1500 ms , te = 4 - 24 ms , six echoes ). inversion recovery was used for estimation of t 1 with a repetition time of 4000 ms and 12 inversion times ranging from 100 ms to 2400 ms . relaxation time maps were formed by fitting signal intensity vs echo time ( or inversion time ) to the relevant signal equations using three - parameter nonlinear least squares fitting routines ( matlab ). relaxivity was determined using a linear fit for relaxation rate vs ferritin concentration . fig2 is a composite indirect immunofluorescence and electron micrograph image showing the tumor targeting activity and imageability of the nanoconjugate material 6 of fig1 . indirect immunofluorescence imaging demonstrated strong reactivity of the ferritin / mab necl - 5 nanoconjugate ( fig2 , top left ) against transformed necl - 5 positive rat prostate epithelial cells that was comparable to anti - necl - 5 antibody alone ( fig2 , top right ). furthermore , transmission electron microscopy ( tem ) showed that the nanoconjugate binds to the rat prostate epithelial cells in a manner comparable to gold conjugated anti - necl - 5 antibody ( fig2 , lower panel ). these in vitro studies indicate that conjugation of the modified ferritin cage to anti - necl - 5 antibody did not affect the targeting specificity or reactivity of the antibody against the necl - 5 antigen . mri imaging of phantoms made evident that contrast effects of all three weightings ( t 1 , t 2 , and t 2 *) were visible when the ferritins were evenly distributed in an agarose gel ( fig3 a ). fig3 a shows cross section images of the uniformly distributed ferritin ( 7000 fe / cage ) samples , wherein the top shows inversion recovery with inversion time = 1200 ms , the middle shows spin echo image with te = 20 ms , and the bottom shows gradient echo image with te = 16 ms . fig3 b shows corresponding relaxation time maps for t 1 ( top ), t 2 ( middle ) and t 2 * ( bottom ). color scales on the right side are relaxation time in seconds . in all of the frames , the top row is the horse ferritin samples at concentrations of 1 , 10 , 100 , and 1000 nm ( left to right ); the middle row consists of the engineered ferritin samples at the same concentrations and the bottom row contains gel only without ferritin . for the t 2 and t 2 * weightings , contrast is evident at the shortest echo times ( 10 ms and 4 ms , respectively ). the horse ferritin , which is here taken as indicative of endogenous ferritin or a conventional natural ferritin , did not show any significant contrast in the images , although a slight effect was noted in the t 2 and t 2 * maps ( fig3 b ) while t 1 effect is negligible . relaxivity ( r 1 , r 2 ) was calculated as the slope of the line resulting from a linear fit of relaxation rate vs concentration . the values for the ferritin loaded to 6700 fe / cage were r 1 = 1290 mm − 1 s − 1 and r 2 = 5742 mm − 1 s − 1 . these values were significantly higher than those obtained from the horse ferritin ( r 1 = 0 . 674 mm − 1 s − 1 , n = 95 . 54 mm − 1 s − 1 ). this result compares favorably to commercial superparamagnetic iron oxide nanoparticle ( spion ) imaging preparations as well as micelle - contained fept variants ( taylor et al . 2011 ). fig4 a and 4b show relaxation time maps of tissue bound nanoconjugate . fig4 a illustrates t 2 and fig4 b illustrates t 2 * relaxation time maps of the nanoconjugate when bound to target rat prostate epithelial cells . it was observed that the t 1 effect was negligible , whereas in the uniformly distributed case ( fig3 b ) the t 1 effect was clearly seen . this may relate to the heterogeneous particle distribution resulting in static dephasing ( bowen et al . 2002 ). mean relaxation time values were determined for regions of interest taken from the center 80 pixels of the in vitro sample images , and are shown in table 1 . the entries are mean ± standard deviation of relaxation times for the in vitro study . circular regions of interest ( 100 pixels ) were taken from the center of the vials . sa denotes soft agar . fig5 is a plot of the conjugate retention vs dose for the in vitro preparation . the clear linear dependence indicates that receptor saturation was not reached even at the highest dose , and that greater binding is possible for this preparation with doses beyond 200 μg / ml . assay results for the in - vitro preparation of iron per cell are shown in table 2 . the iron concentrations were estimated based on the volume of the cell pellets , number of cells per pellet and quantity of iron per cell . for the t 2 * values determined in table i , fig6 shows the corresponding signal intensity and contrast curves to illustrate the optimum echo times based on the doses . contrast is defined as the difference between the signal intensity curve at each concentration subtracted from the control . it was observed that as the dose level increases ( and t 2 *) decreases , that peak contrast increases , and the echo time corresponding to peak contrast decreases . the echo times for peak contrast vs . the control occur at 25 ms ( 50 μg / ml ), 23 ms ( 100 μg / ml ) and 19 ms ( 200 μg / ml ). these magnitudes imply that for an image ( pixel ) signal - to - noise ratio of 20 in the baseline image , the contrast change will be detectable with a dose of 20 μg / ml for the in vitro preparation described above . that dose would correspond to approximately 0 . 62 μg / cell iron loading . the high ratio of r 2 */ r 2 is indicative of static dephasing ( bowen et al . 2002 ) resulting from local accumulations of particles as opposed to uniform distribution . dependence of t 1 and t 2 in the presence of superparamagnetic nanoparticles has been described for uniform distribution using modified forms of the solomon - bloembergen - morgan equations ( koenig et al . 1995 ; bulte et al . 1999 ). these calculations predicted superparamagnetic particles as having a much smaller effect on t 1 than on t 2 owing to the large magnetic moment . this observation was confirmed in the uniform distribution measurements and may be the result of diffusion of associated water molecules through the ferritin channels ( aime et al . 2002 ). with respect to r 2 and r2 *, compartmentalization causes the assumptions behind the quantum solution to fail , an effect previously described in cell - based studies ( weissleder et al . 1997 ; majmudar et al . 1989 ). compartmentalization is also accompanied by a substantial increase in the ratio r 2 */ r 2 which is not predicted by the quantum solution . the quantum solution assumes the extreme motional narrowing condition , in which water diffusion between superparamagnetic particles is occurring on a time scale significantly shorter than the peak frequency offset and identical values for r 2 and r 2 * are predicted . compartmentalization of the particles results in bulk susceptibility producing local field inhomogeneities that render the assumption invalid . monte carlo simulations of water diffusing through local dipolar fields however , have been successfully employed in predicting the relationship between r 2 and r 2 * for the case of particle compartmentalization ( weisskoff et al . 1994 ; muller et al . 1991 ; hardy and hendelman 1989 ; fisel et al . 1991 ; majmudar and gore 1988 ). changes in t 2 and t 2 * were clearly distinguished in the in vitro preparation at a concentration ( in the cell pellet ) of 103 nmol . the minimum detectable concentration for the agent depends on a number of factors including cell density , magnetic field shim conditions in the region of the tissue binding the agent , the scan type ( spin vs gradient echo ) and scan parameters ( repetition time , echo time , and geometric factors affecting signal to noise ratio ). as seen from the binding assay ( fig5 , table 2 ) it appears likely that concentrations in excess of 400 nm can be produced in this in vitro preparation or an in vivo case with similar cell density , which would thus result in a very substantial contrast effect . the foregoing experimental results establish the effective targeting and imaging of a specific protein by a ferritin construct , and quantification of the relevant mri imaging and dosing parameters in an in vitro experimental model . in the study reported by sana et al . ( 2010 ), a clear t 1 effect was observed at a field strength of 3 tesla , the same field strength used in this study . this was verified in examples herein with the preparation in which ferritin particles were uniformly distributed in agarose gel . the lack of t 1 effect in the in vitro experiment may be the result of a reduced ability for free water to access the channels of the bound ferritin . if this is the case , use of the modified ferritin as a t 1 agent appears to be restricted to cases where the particles are maintained in an unbound state such that free water access to the ferritin channels is maximized . one example would be application as a blood pool agent for angiography studies where passage out of the microvasculature into the interstitial space is not desired . in such an application , a targeting ligand would not be required . rat high passage pec ( p93 ) cells and soft agar infiltrating ( sai )- selected prostate epithelial cells ( pec ) were tumorigenic when injected into immunodeficient beige / nude mice . tumor size was evaluated at four weeks post - injection for the high passage cells , and three weeks for the sai - derived pec tumors . sai - derived tumors showed a shorter latency period than high passage derived tumors , and the average weight of removed tumors at the time of sacrifice was 0 . 2 grams ( n = 3 , 4 weeks ) and 0 . 76 grams ( n = 5 , 3 weeks ), for high pass and sai injected cells , respectively . indirect immunofluorescence imaging and western blotting each demonstrated that high passage ( p102 ) and sai - selected rat prc expressed high levels of the cell surface glycoprotein necl - 5 . to evaluate the ferritin - based contrast agent , in vivo mri imaging of immunodeficient mice previously injected with pec sai cells was performed at 4 and at 24 hours after injection of anti - necl - 5 / ferritin or ferritin alone , and was compared to baseline values taken before the ferritin injections . the nanoconjugate targeted tumor showed significant reduction of t 2 signal at 4 hours post - injection , and a substantially lesser reduction of t 2 at 24 hours , while the control , and regions of muscle tissue in both sets of mice were not substantially affected by either the targeted or the non - targeting ferritin . example 2 thus extends the results to in vivo application of an anti - necl - 5 / ferritin nanoconjugate for imaging rat prostate epithelial cell tumors , and shows a time - dependent but dramatic difference in mri response and imaging characteristics . methods of imaging therefore advantageously include or are preceded by a preliminary time series dose / response sequence of measurements to acquire mri characteristic data to optimize the interval between administration of the agent and imaging of the tumor . in accordance with a further aspect of the invention the metal - filled ferritin cages , once bound to the target tissue , are caused to release the paramagnetic or superparamagnetic metal contents from their core . this process may be initiated or accelerated by heating , for example by applying a quickly - alternating magnetic field to generate heat , or by applying focused ultrasound to heat the particles and open pores of the ferritin cages . the high valence metal ions thus released from the core of the ferritin cages result in a locally toxic concentration of metal ions . thus , imaging allows the treating physician to coordinate the excitation of the tumor - bound agent and release of the ferritin - caged metal to treat the tumor . the enhanced imaging characteristics enable earlier detection than would otherwise be possible , increasing the effectiveness of such a localized toxic treatment . the development of targeted imaging contrast agents with high specificity is an important step in the advancement of cancer diagnostics . yet the diagnostic indicators for some cancers are relatively non - specific . for example , prostate cancer diagnosis relies on the use of prostate specific antigen ( psa ) as a prostate tumor marker that has also served as a target for functionalized nanoparticle detection studies ( taylor et al . 2011 ). however , it was recently found that benign prostatic hyperplasia ( bph ) also produces psa , so that basing a diagnosis on psa results in over - diagnosis and leads to unnecessary treatment ( chou et al . 2011 ). in accordance with the present invention , by targeting cd155 , the human homologue of rat necl - 5 , this diagnostic ambiguity would be eliminated . in examples herein we have demonstrated targeting of a ferritin - based metal complex to necl - 5 in a transformed rat prostate epithelial cell line model . a clear effect was seen for changes in t 2 and t 2 * as would be reflected in spin echo and gradient echo imaging , respectively . the agent produced a visible effect ( compared to a control ) at a concentration of 102 nm fe in the in vitro study along with an indication of the feasibility of binding to produce a concentration in excess of 400 nm . this is believed to be the first description of use of the modified ferritin complex as a contrast agent for targeting of a specific protein in an in vitro experimental model . as shown here , the in vitro data indicates that the modified ferritin conjugate has utility as both a t 2 and t 2 * contrast agent when conjugated to an antibody of interest for targeting and imaging antigen - specific tissues . the antigen - specific tissues may be cancer cells or other diseased cells that express a specific cell surface molecule . many such molecules have been characterized and associated with specific cancers or tissue pathologies ; the antibody employed for targeting the ferritin nanoparticles may be an antibody to such a characterizing molecule , or may be an antibody to a relevant portion thereof . in other embodiments , rather than the ferritin being conjugated to an antibody , equivalent specificity and effective accumulation and concentration at the relevant cells can be expected if the ferritin is clothed with the epitope , or active portion of the antibody responsible for binding . for example , the entire ferritin - epitope construct may be genetically engineered as a fusion protein . furthermore , the targeted surface molecules may be a molecule that is specific to a highly invasive cell line , so that mri images reveal specific information as to tumor type . example 2 reports in vivo results imaging highly invasive tumors grown from soft agar infiltrating prostate epithelial cells . by specifically identifying surface markers and employing targeting antibodies for such cells , the techniques of the invention significantly advance early detection and treatment . the magnitude of the relevant magnetic resonance parameters described above further indicates that other targeting functionalities — such as cloaking the ferritin in a targeting functionalized phospholipid or nanoemulsion as the delivery vehicle — can also be applied to advantage to achieve for in vivo delivery to tumor sites . a targeted nanoemulsion for in vivo use is compounded to allow the agent to circulate in the bloodstream sufficiently many times to accumulate specifically at the targeted tissue . once the relevant t 2 and t 2 * values are determined , further baseline studies may be performed for a given targeting agent and target cell line to determine the optimum interval required after administering the ferritin nanoparticles for effective tissue binding to occur , so that diagnostic imaging and / or metal ion release therapy can be efficiently performed without taking multiple or comparative sets of before / after mri scans . comparison of pre - and post - administration mri image data indicate tumorous regions of ferritin accumulation , and imaging protocols that display the difference will provide high contrast , tumor - specific imaging . for example , since the t 1 effect in example 1 was seen only when particles were uniformly suspended and unbound , so detection of a tumor would be revealed by t 2 and t 2 * weighting . once a baseline scan is acquired of the suspect region , tumor presence is revealed by reduction of t 2 and t 2 * relative to the baseline scan when the contrast agent has been administered . coupling a tumor - targeting agent ( e . g ., an antibody ) to the nanoparticle ferritin contrast agent in the present invention assures that the agent binds to the relevant tissue with high efficiency and specificity , so that while a dose / response relationship governs the image , only very small amounts of the contrast agent are needed for diagnostic imaging . the foregoing describes a tissue - targeting nanoparticle mri contrast agent and confirmatory measurements and observations that confirm its improved imaging characteristics , as well as its utility in methods of diagnosis and of treatment of specific diseased tissue or cancer conditions . the invention and illustrative methods being thus described , further variations and modifications will occur to those skilled in the art , and all such variations and modifications are understood to be within the scope of the invention and claims appended hereto . aime s , frullano l , crich s g , 2002 . compartmentalization of a gadolinium complex in the apoferritin cavity : a route to obtain high relaxivity contrast agents for magnetic resonance imaging . angew . chem . int . ed . 41 : 1017 - 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