Approaches to improved antibody- and peptide-mediated targeting for imaging and therapy of cancer

Journal of Controlled Release, 28 ( 1994) 167-l 73 0 1994 Elsevier Science B.V. All rights reserved 0168-3659/94/$07.00

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COREL 0088 1

XSDZO168-3659(93)E0065-N

Approaches to improved antibody- and peptide-mediated targeting for imaging and therapy of cancer Alan R. Fritzberg, Paul L. Beaumier,

Becky J. Bottino and John M. Reno

NeoRx Corporation, Seattle, WA, USA (Received 4 March 1993; accepted in revised form 10 May 1993 )

Antibodies with their ability to selectivity bind antigens have been of great interest in targeting radiation, drugs and toxins to tumors. Limited success in delivery of radioactivity has been enjoyed with conventional attachment to antibodies. This is due to slow tumor targeting processes and slow disappearance from blood and variable uptake and disappearance from the excretory organs, liver and kidney. Preliminary studies in animal models and patients have shown promise in increasing the tumor to blood exposure ratio by pretargeting antibody followed by small molecule delivery of radioactivity using a molecular capture system. Efficient capture of the small molecule radioactivity carrier by tumor localized antibody and rapid clearance and excretion of the untargeted radioactivity decreases the background problem for imaging and lowers marrow toxicity for radioimmunotherapy. Small peptide ligands that bind to receptor bearing tumors offer similar advantages. Key words: Antibody-mediated Molecular capture

targeting; Peptide-mediated

Introduction Radiopharmaceuticals are unusual ‘drugs’ in that they lose potency due to physical decay while localizing following administration. In the case of imaging agents, photon flux decreases the signal strength and the signal-to-noise ratio decreases. For a radiotherapeutic agent, since efftcacy is proportional to its radiation dose rate, physical decay reduces the ‘potency’ of the conjugate as time post-administration increases. Importantly, toxicity to marrow and non-target organs is a result of exposure during the targeting Correspondence to: Alan R. Fritzberg, NeoRx Corporation, 4 10 W. Harrison, Seattle, WA 98 119, USA.

targeting; Radioactivity;

Cancer therapy;

process. Therefore, to be maximally effective, the ideal controlled delivery vehicle for either a diagnostic or therapeutic radiopharmaceutical should accumulate in target rapidly and clear from normal tissues quickly. Furthermore, the radiopharmaceutical packaged as an easily formulated ‘kit’ would enjoy the broadest end-user acceptance. For the last 15 years, monoclonal antibodies (MAbs) and their fragments have been applied to targeted radioisotope delivery for diagnosis and therapy of cancer. Basic research has defined the features and limitations of antibodybased delivery vehicles and laid the foundation for their application in targeted delivery. After systemic administration, antibody is diluted into

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a plasma volume of several liters, substantially reducing local concentration supplying tumor. Since antibodies access tumor by passive diffusion, the local concentration at the tumor, antibody affinity and antigen density parameters dictate the fraction bound [ 1,2]. While longer circulating whole IgG antibody results in the highest delivery to tumor, clinically demonstrated levels targeted in human tumors have been disappointing, with peak tumor concentrations of only approximately 0.005% injected dose per g administered (%ID/g ) or only 5 pg per 100 mg administered. Combined with this limitation, dose-limiting myelosuppression is observed in dose escalation protocols. For example, with ‘86Re-labelled MAb and F( ab’ )2, a maximum bone marrow tolerable dose of 120 mCi/m* (ca. 2 15 mCi total dose) and 150 mCi/ m* (ca. 270 mCi total dose), respectively was determined [ 3 1. At this level in the whole IgG case, 0.005% of 215 mCi results in 11 pCi/g to tumor or about 900 rad. This radiation dose is clearly below levels needed to predictably treat solid tumors. Since uptake is variable from tumor to tumor, and is typically heterogeneous in its tumor distribution, actual doses in some tumors and in viable tumor tissue may be much higher. Thus, anecdotal responses in patients have been seen in solid tumors [ 3,4]. With the low absolute levels of dose localized in tumor and the low therapeutic ratio of tumor to bone marrow seen, it is becoming clear that systemic radioimmunotherapy of solid tumors with radiolabelled antibody, even with autologous marrow transplantation, is unlikely to be effective as a result of insufficient radiation delivered to tumor and dose-limiting myelosuppression. Although it would be very desirable for a MAb-based delivery vehicle to selectively target a higher percentage of the injected dose, it seems unlikely that major increases in localization levels are likely. We have compared the tumor targeting and non-tumor biodistribution and pharmacokinetits of a variety of antibody forms in order to determine the optimal targeting vehicle for delivery of radiation. The data from animal and

clinical studies suggest approaches to improving the delivery of radiation to tumors. Materials

and Methods

Monoclonal antibodies and other carriers NR-LU- 10, a murine monoclonal antibody isy&, iS SpeCifiC for a 40 kDa @yCOprOtein antigen associated with carcinomas [ 5,6 1. Other forms of NR-LU- 10 studied include the dCH2 deletion mutant, an engineered divalent molecule lacking the second domain in the Fc portion [ 7 ] ; a F( ab’ )* fragment produced from whole antibody by standard enzymatic pepsin digestion; and the Fab fragment produced from whole antibody by standard enzymatic papain digestion. A small peptide and biotin were evaluated only for blood disappearance pharmacokinetics using the piodophenyl (PIP) radioiodinated moiety as radiolabel [ 8 1. Otype

Blood disappearance half life measurement Groups of three BALB/c mice 6-8 weeks old, 22-25 g, were injected i.v. with ‘251-labelled test material, and up to eight serial 1O-p1 blood samples were collected in duplicate from the retroorbital plexus using microcapillary pipets. The percent injected dose was calculated using a mouse blood volume of 8% of body weight, the volume injected from the weighed syringe, and the count rate in an injectate standard. Disappearance kinetics were determined using RSTRIP, a nonlinear least squares data litting pharmacokinetic analysis software (Micro Math Scientific Software, Salt Lake City, UT). Percent injected dose vs. time data were fitted to a biexponential curve. LS-180 xenograft nude mouse model LS-1 SO, a human adenocarcinoma cell line, was propagated in tissue culture using defined media [ 91. Female, athymic, nude mice (Simonsen, Gilroy, CA) 18-22 g, were eartagged, housed five per cage in an AALAC accredited,

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controlled environment barrier facility and maintained on autoclaved chow and water. Xenografts emerged in 7- 10 days with 100% take rate from the subcutaneous implantation of 5 x 1O6 cells in the left side midline.

30 25 I-

1 I

0

24

T I

Biodistribution of labelled antibodies NR-LU- 10 and other targeting molecules were labeled with ‘251 using the p-iodophenyl (PIP) moiety labeling method [ 8 ] and used to assess biodistribution properties. Timepoints were selected to define the distribution timecourse and adjusted to individual antibody form half-life. Doses were selected empirically to optimize tumor localization in the LS- 180 xenograft system: 50 fig Mab, 20 pg dCH2, 20 pg F( ab’ )* and doses of 10 pg of Fab, an (octreotide analogue), 0.5 pg peptide and 5 pg of biotin were administered for the blood pharmocokinetic studies. For the sake of comparison, Fv data was taken from the literature [ lo]. Total counts were calculated from a dilution standard and the weight of the injectate. Groups of four mice/timepoint were weighed, bled via the retroorbital plexus and sacrificed by cervical dislocation. Blood samples and tumor were weighed and counted with standards in a y-scintillation well counter (Packard Instrument Co., Laguna Hills, CA) setting the window from 25 to 80 keV. Data analysis using LOTUS software was used to calculate percent injected dose per g.

Results The tumor targeting and circulation pharmacokinetics of various forms of IgG antibody and fragments were compared in an animal model. Thus, whole IgG ( 150 kDa), a CH2 deletion mutant(130kDa),F(ab’),(lOOkDa)andFab(50 kDa) were radioiodinated and administered intravenously to mice bearing LS- 180 colon carcinoma tumors. The tumor targeting kinetics are shown in Fig. 1. Whole antibody IgG reached the highest percent dose per g tumor and showed the best tumor retention. In fact, when corrected for tumor growth during the study, little loss on a

V-VMAB

48

72

96

TIME(HR) 0-0~~~2 A-LIF(AB')Z

120 O-OFAB

Fig. 1. Tumor time course in nude mice (n = 4/timepoint with S.D. error bar) implanted with LS-180 human adenocarcinoma xenografts. Proteins were labeled with PIP-‘251 and injected i.v.

0

24

48 TIME

V-VMAB

0-0~~~2

(HR) ~-~F(AB~)z

0-OFA

Fig. 2. Time course for longer lived carrier proteins labeled with PIP-‘25I injected iv. Serial, duplicate blood samples were taken from the retroorbital plexus in groups of three BALB/ c mice.

percent injected dose is seen. Noteworthy is ongoing/continued accretion into tumor over a 24h period. The fragments all show more rapid rates of tumor uptake with the smallest form, Fab, peaking earliest. The dCH2, while reaching high tumor uptake, showed less tumor retention. F (ab’ ) 2 and Fab fragments peaked at lower levels and were poorly retained compared to whole IgG. Blood disappearance comparative data are shown in Fig. 2. Whole IgG clearly had the long-

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est residence time in blood, giving it the highest background signal and bone marrow toxicity as a radiotherapeutic. The KHZ, F (ab’ ) z and Fab are each cleared by liver or kidney mechanisms. The ratio of areas under the curve ( AUC) for tumor and blood correspond roughly to relative radiation exposure and were 7: 1 for IgG, 6: 1 for KHZ, 5: 1 for F(ab’), and 14: 1 for Fab. Thus, significant therapeutic advantage for tumor targeting was not gained by the use of antibody fragments except for the Fab fragment. While a higher therapeutic ratio was obtained, the 50 kDa Fab fragment showed substantial renal retention and thus would deliver a large radiation burden to the kidneys applied as a radiotherapeutic. However, for imaging, the rapid targeting and background disappearance of the Fab fragment allows the use of the ideal imaging radionuclide, 99mTc, with its 6-h half-life [ 111. With the need to improve the tumor to blood therapeutic ratio, new strategies involving small molecule targeting vehicles of radioactivity are being investigated. Thus, we have compared the blood disappearance of several smaller representative radiolabelled agents: namely Fab, Fv, a small peptide, and biotin relative to whole IgG (Fig. 3 ). All smaller forms disappear much more rapidly than IgG, with Fab and Fv intermediate

iIT

_

0

1

I

i

4

3

TABLE 1 Blood half-lives of various targeting entities determined BALB/c mice

in

Carrier

.v,

c+HL/h

p-HL/h

Biotin/Oligopeptide Fv Fab F(ab’ jz

- 1000 25 000 50 000 105 000 130 000 160 000 900 000

0.06 0.1 0.3 2.9 0.7 3.4 1.7

1.0 2.7 6.8 17.5 14.1 81.5 16.2

ACH2 IgG IgM

to the very rapidly clearing small peptide or biotin derivative. Pharmacokinetic parameters for the blood compartment behavior of the various targeting entities are shown in Table 1. A large range is seen that at face value appears to correspond to molecular size and the renal threshold for glomerular filtration of protein (about 50-60 kDa) [ 12 1. Thus, at levels above this threshold, renal excretion is restricted and disappearance is governed by hepatic uptake or plasma instability leading to smaller fragments (e.g., F(ab’ )2 to Fab’ ) which are then small enough to be excreted via glomerular filtration. The small molecular weight carriers are very rapidly cleared as they are sufficiently small as to not be retarded in the Iiltration process. These comparisons of blood disappearance and tumor targeting provide a basis for assessing nontarget uptake or radiation burden relative to tumor target uptake or retention. Conventional radiolabelled antibody fragments provide opportunities to match physical half-life with targeting and background clearance, but in general do not radically improve the ratio of tumor AUC to blood (marrow ) AUC.

Discussion

TIME (HR) O-OFAB

V---V

F’d

0-OPEPTIDE

0 -

0 BlOilN

Fig. 3. Time course for shorter lived targeting vehicles labeled with PIP-‘25I injected i.v. Serial, duplicate blood samples were taken from the retroorbital plexus in groups of three BALB/c mice. Whole MAb is included for reference.

In the overall effort to utilize antibodies to target radiation for imaging and treatment of tumors, significant progress has been made, but limitations with conventional approaches have been seen. A large number of antibodies have

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been generated to tumor antigens, evaluated in preclinical models and in patients and have most recently made their way through the production and regulatory labyrinth. Thus, much is known about the utility of many tumor antigens as targets for cancer, the antibodies developed to such antigens, and factors involved in tumor targeting, including homogeneity of tumor uptake, retention in tumor, and other factors important to pharmaceutical development. Patient studies, however, have shown that while high sensitivities for the detection of tumor can be attained, uptake in solid tumors is variable and typically ranges from 0.00 1 to 0.0 1% ID/g [ 131. While these values appear low, achieving uniformity at 0.01% ID/g would give about 35 ,&i/g of ls6Re delivered via a 1: 1 ReMab ratio (3 Ci/mg specific activity) at 100 mg antibody IgG dose. This level of radioactivity deposits 3000 rad, assuming effective retention of the radioactivity for the duration of the 3.7day physical half-life. Doubling uptake to 0.02% ID/g or a 2 : 1 Re-Mab at 0.0 1% ID/g would provide 6000 rad or a level suggested as useful by Mach [ 14 1. If higher energy Yttrium-90 is considered, only 33 pCi/g tumor would be needed to give 6000 rad. These levels of delivery are achieved occasionally and anecdotal responses in solid tumors have been seen [ 3 1. However, targeting radiotherapy by the conventional approach, that is, radiomoieties covalently coupled to protein, does not appear to be able to consistently deliver the needed levels without intolerable bone marrow toxicity. Thus, alternative approaches are needed and are under investigation. Bone marrow support Replacement of bone marrow cells or bone marrow support is a straight forward approach, as radioimmunoconjugates have produced little or no toxicity to other organs. Autologous bone marrow transplantation is being used to increase dose levels in clinical studies, and the recent availability of colony-stimulating factors suggests their use as alternatives to marrow harvesting and reinfusion. However, these procedures are associated with significant mortality and

morbidity. Although they do allow more radioactivity to be injected beyond the level of marrow toxicity, a second organ of toxicity or unacceptable whole body exposure will be reached. These techniques simply do not address the fundamental problem inherent in conventional RIT. Pretargeting

approach

In the pretargeting approach, cold non-radiolabelled antibody is administered first to target the tumor cells. Without attached radioactivity, many dosing regimens are possible and as much time as necessary can be taken to achieve optimal tumor uptake. Further, antibody mass can be increased to optimize penetration and homogeneity [ 151. Next, a clearing step allows removal of circulating antibody and avoids the antibody in circulation as target. Finally, a small molecule bearing the radioactivity is delivered using a molecular capture mechanism. This offers the opportunity to use a system that involves more efficient capture than conventional radiolabelled antibody: tumor antigen binding, as well as the rapid blood clearance kinetics of the small molecule radioactive carrier. In effect, the radioactivity delivery process is decoupled from the slow antibody-targeting process. Thus, target to background imaging potential is raised and marrow exposure lowered. Molecular capture systems utilized include anti-hapten [ 16-181 avidin-biotin [19-211 or sense-antisense polynucleotides [ 22 1. Clinical studies have been encouraging with the avidin-biotin capture system studied in 20 patients by Paganelli et al. [ 23 1. The amount estimated in tumor at 2- and 3-h post injection was 0.012 ? 0.0060% ID/g, comparable to levels reached with conventional antibody targeting in 24-48 h. At the same period, blood was IO-fold lower. Although the kidney was 0.012% ID/g, comparable to tumor, various approaches can be taken to reduce renal reabsorption or secretion processes that mediate kidney uptake and retention. Tumor targeting peptides Several peptides and small proteins have been evaluated for tumor uptake, selectivity and po-

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tential to deliver radioactivity. These include the somatostatin analog octreotide [ 24,25 1, epiderma1 growth factor (EGF) [ 261 and melanocyte stimulating hormone (MSH) [ 271. In these cases, high affinity binding to receptors which are found in increased numbers on tumor cells is seen. The use of these naturally occuring peptides as targeting agents would seem to have many advantages, including lack of immunogenicity, but it remains to be seen whether the differential number of receptors on target cells and normal cells is enough to achieve sufficient selectivity. This is especially important for therapy applications where toxic entities carried by these ligands are meant to kill the targeted cells. Another concern is the affinity of the binding to the expressed receptor, as this factor can directly affect retention. Internalization and subsequent catabolism could easily result in the loss of the radioisotope from the targeted cell as well. However, all of these peptides or small proteins, e.g., EGF are rapidly cleared from the blood, providing decreased marrow exposure. Tumor uptake is rapid with clear visualization shown in animals and in patients with 1231 [28] or “‘In-octreotide. At this time, detection of a variety of tumors has been demonstrated with either radioiodine or ’ ’ ’ ‘In. The uptake seen in some tumors suggests potential for peptide mediated delivery of radiotherapeutic radionuclides.

Summary Conventional approaches to delivery of radioactivity to tumors using antibodies appears to be limited in terms of amount targeted and diagnostic and therapeutic ratio of tumor to blood that can be obtained. By creating an antibodybased receptor using pretargeting of the antibody, subsequent administration of the radioactivity attached to a small molecule allows the tumor to blood ratio to be increased due to efficient capture of the small molecule and rapid clearance of nontargeted radioactivity. Similarly, radiolabelled peptide ligands captured by receptors on tumor cells provide a high tumor to blood ratio. These approaches promise the potential of

targeting therapeutically effective amounts of radiation without bone marrow or otherwise significant toxicity.

Acknowledgements The authors wish to acknowledge the contributions of the following people: Donald Axworthy and Gina Engrissei for radiolabelling and animal studies, Robert Mclntyre for antibody fragmentation and protein purification, and Denise Carlson for manuscript word processing.

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