Single-Dose Versus Fractionated Radioimmunotherapy of Human Colon Carcinoma Xenografts Using 131I-Labeled Multivalent CC49 Single-Chain Fvs1

Vol. 7, 175–184, January 2001 Clinical Cancer Research 175

Single-Dose versus Fractionated Radioimmunotherapy of Human Colon Carcinoma Xenografts Using 131I-labeled Multivalent CC49 Single-chain Fvs1

Apollina Goel, Sam Augustine, labeled systemic toxicity was observed in any treatment Janina Baranowska-Kortylewicz, David Colcher, groups. The results show that radioimmunotherapy delivery for sc(Fv) and [sc(Fv) ] in a fractionated schedule clearly Barbara J. M. Booth, Gabriela Pavlinkova, 2 2 2 2 presented a therapeutic advantage over single administration. Margaret Tempero, and Surinder K. Batra The treatment group receiving tetravalent scFv showed a sta- Departments of Biochemistry and Molecular Biology [A. G., tistically significant prolonged survival with both single and S. K. B.], Pathology and Microbiology [S. A., B. J. M. B., G. P., fractionated administrations suggesting a promising prospect S. K. B.], and Radiation Oncology [J. B-K.], College of Pharmacy [S. A.], Eppley Institute for Research in Cancer and Allied Diseases of this reagent for cancer therapy and diagnosis in MAb-based [S. K. B.], University of Nebraska Medical Center, Omaha, Nebraska radiopharmaceuticals. 68198; Coulter Pharmaceutical Incorporated, San Francisco, California 94080 [D. C.]; and University of California San Francisco Cancer Center, San Francisco, California 94115 [M. T.] INTRODUCTION RIT3 is a rapidly developing therapeutic modality for the treatment of a wide variety of carcinomas (1). Numerous anti- ABSTRACT body-radionuclide combinations have been evaluated in clinical The prospects of radiolabeled antibodies in cancer detec- studies (2–9). RIT has yielded complete responses in hemato- tion and therapy remain promising. However, efforts to logical diseases like Hodgkin and non-Hodgkin lymphoma (10, achieve cures, especially of solid tumors, with the systemic 11); however, for solid tumors only a partial clinical response administration of radiolabeled monoclonal antibodies (MAbs) has been observed (12–14). Methods for improving the thera- have met with limited success. Using genetic engineering tech- peutic index (tumor:normal tissue ratio) remain to be optimized niques, MAbs have been tailored to improve the therapeutic for the development of more effective RIT for solid tumors (15). index (tumor:normal tissue ratio) in clinical radioimmuno- Most of the radioimmunoconjugates studied to date involve therapy. In the present study, we investigated the potential of the use of whole immunoglobulins, particularly IgG. Intact ϳ tetravalent {[sc(Fv)2]2} and divalent [sc(Fv)2] single chain Fvs MAbs (Mr 150,000) remain in circulation for an extended time of MAb CC49 for therapy in athymic mice bearing s.c. LS- and can therefore increase the radiation dose delivered to normal 174T human colon carcinoma xenografts. Mice received 1000 tissues (16). Moreover, the poor tumor penetration of MAbs ␮ 131 131 Ci of I-labeled [sc(Fv)2]2 or I-labeled sc(Fv)2, either as a results in the localization of only 0.0001–0.01%ID/g of tumor single injection on day 6 or as four injections (250 ␮Ci each) on (17). Many solid tumors are relatively radioresistant; therefore, days 6, 7, 8, and 9; the day of tumor implantation was taken as RIT of such tumors requires high radiation doses, which can day 0. The median survival for the control group was 26 days. result in significant nonspecific uptake by normal tissue leading Comparisons of single and fractionated therapeutic regimens to toxicity, especially to the hematopoietic system (18). Because showed median survival as 32 (P < 0.001) and 53 days (P < the size of the antibody-based molecule relative to the renal

0.0001), respectively for [sc(Fv)2]2 and 26 (P > 0.5) and 38 days threshold for first-pass clearance is a major factor in determin- (P < 0.0001), respectively for sc(Fv)2 when compared with the ing the residence time of the radioimmunoconjugate in circula- Ј Ј control groups. The time for the quadrupling of tumor volume tion, a range of IgG formats, like F(ab )2, Fab , or Fab, have (for single and fractionated therapeutic treatments were: 9.0 ؎ been investigated for therapeutic potential at preclinical (19–21 ؎ ؎ 0.8 and 21.1 2.9 days respectively for sc(Fv)2; 16.6 1.9 and and clinical levels (5, 22, 23). Decreasing the size of the anti- ؎ ؎ 32.9 2.7 days respectively for [sc(Fv)2]2; and 8.3 0.7 and body molecule increased both the degree of penetration and the days respectively for the control group. No 131I- clearance rate from the blood pool; however, these molecules 0.6 ؎ 8.4 were difficult to generate at a large scale with clinical-grade purity and did not provide a significant increment in quantitative tumor retention as an intact antibody (24). Received 7/26/00; revised 10/18/00; accepted 10/18/00. The advent of molecular biology has enabled the designing The costs of publication of this article were defrayed in part by the and purification in ample abundance of small, high-affinity, payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by grants from the United States Department of Energy (DE-FG02-95ER62024) and NIH (P5O CA72712 and RO1 CA78590). 3 The abbreviations used are: RIT, radioimmunotherapy; scFv, single 2 To whom requests for reprints should be addressed, at Department of chain Fv; sc(Fv)2, covalent divalent scFv; [sc(Fv)2]2, noncovalent tet- Biochemistry and Molecular Biology, Eppley Institute for Research in ravalent scFv; HPLC, high performance liquid chromatography; MAbs, Cancer and Allied Diseases, University of Nebraska Medical Center, monoclonal antibodies; SPR, surface plasmon resonance; BSM, bovine 984525 Nebraska Medical Center, Omaha, NE 68198-4525. Phone: submaxillary gland mucin; %ID/g, % of injected dose/g; RI, radiolocal-

(402) 559-5455; Fax: (402) 559-6650; E-mail: [email protected]. ization index; VL, variable light chain; VH, variable heavy chain.

Downloaded from clincancerres.aacrjournals.org on September 25, 2021. © 2001 American Association for Cancer Research. 176 Therapeutic Efficacy of 131I-labeled Multivalent CC49 scFvs for Colon Carcinoma

multivalent antibody-based molecules as carriers for radionu- Hercules, CA). The elution was monitored by an in-line UV

clides (25). ScFvs are recombinant proteins composed of a VL detector at 280 nm.

amino acid sequence of an immunoglobulin tethered to a VH The immunoreactivity of scFvs was analyzed by a solid sequence by a designed peptide (26, 27). Compared with an phase competition ELISA using BSM (Sigma Chemical Co., St. intact antibody, scFvs can bind to a tumor cell in a more Louis, MO) as the antigen (36). The binding affinities of

homogeneous distribution (28, 29). Such fragments lead to a sc(Fv)2, [sc(Fv)2]2, and CC49 IgG for BSM were determined by higher tumor:normal tissue ratio, but the percentage of injected SPR measurements using an upgraded version of BIAcore 1000 dose delivered to the tumor is usually poor because of their (Pharmacia Biosensor, Uppsala, Sweden) as described previ- monovalent nature and faster removal from the circulatory sys- ously (36, 44). The kinetic rate constants (kon and koff) were tem. Moreover, the high renal accretion of these small mole- measured, and the equilibrium association constant (KA) and cules can lead to severe nephrotoxicity at therapeutic doses (24). equilibrium dissociation constant (KD) were derived. To increase the functional affinity of scFvs, the valency of scFvs Radioiodination of scFvs. The scFv forms were labeled 125 131 has been increased by connecting them together by either non- with either Na IorNa I using 1,3,4,6-tetrachloro-3␣,6␣- covalent or covalent interactions (28–30). The divalent scFvs diphenylglycoluril (IodoGen; Pierce Chemical Co., Rockford, have shown improved avidity and efficacy for tumor targeting at IL) as the oxidant (45). For therapeutic labeling, iodinations 131 preclinical levels (31–36). were carried out with 15–20 mCi of Na I (NEN, Boston, CC49 is a second-generation murine MAb showing high MA)/mg of scFvs in 0.1 M sodium phosphate buffer (pH 7.2) affinity for the tumor-associated glycoprotein 72 (37). CC49 with IodoGen (250 ␮g/mg of protein) and at a protein concen- MAb is under clinical trials for radiation-mediated therapy of tration of 2–3 mg/ml. Unincorporated radioiodine was separated ovarian, colorectal, breast, and prostate carcinoma (4, 38–41). from the labeled protein by size exclusion chromatography In the present study, we have investigated for the first time the using a Sephadex G25 column (Pharmacia, Piscataway, NJ). The specific activity of 125I- and 131I-labeled scFv molecules therapeutic potential of divalent [sc(Fv)2] and tetravalent was about 3–9 mCi/mg. The radiochemical purity of all of the {[sc(Fv)2]2} CC49 scFvs under single and dose-fractionation schedules in athymic mice bearing human colon carcinoma radiolabeled scFvs was Ն98% as confirmed by ITLC. xenografts. Fractionated therapeutic treatment was found to be Characterization of Radiolabeled scFvs. Radiolabeled proteins were analyzed on SDS-PAGE gels, and the radioactiv- clearly superior to single administration for both sc(Fv)2 and ity associated with the protein was measured using the Image- [sc(Fv)2]2. We believe that the multivalent CC49 scFvs hold potential toward the generation of optimum tumor-targeting Quant software of the PhosphorImager (Molecular Dynamics, reagents in radionuclide-mediated therapy. Sunnyvale, CA). Analytical size-exclusion HPLC was performed as described above. Fractions (1 ml) of the radiolabeled protein were collected, and the radioactivity was determined in MATERIALS AND METHODS a Packard Minaxi Auto-Gamma 5000 gamma counter (Meriden, Protein Expression and Purification. For functional CT). The immunoreactivity of radiolabeled CC49 scFv forms expression of the divalent and tetravalent CC49 scFvs, the was assessed by RIA where BSM (surrogate for TAG-72 anti-

construct (VL-linker-VH-linker-VL-linker-VH-His6) was cloned gen) and BSA (negative control) were attached to a solid-phase in the yeast shuttle vector, pPICZ␣A (Invitrogen, Carlsbad, CA) matrix (Reacti-Gel HW-65F; Pierce; Ref. 36). and transformed into competent Pichia pastoris KM71 cells Animals and Tumor Model. Female athymic mice (nu/ (his4arg4aox1⌬::ARG4) as described earlier (36). Upon expres- nu; 4–6 weeks of age) were used for the in vivo studies (Charles sion, 20–30% of divalent scFvs were found to associate as River, Wilmington, MA). The human colon carcinoma LS-174T tetravalent and/or higher aggregated forms. ScFvs were purified cell line (American Type Culture Collection, Rockville, MD) by immobilized metal affinity chromatography using the che- was implanted s.c. (4 ϫ 106), and the mice were used 6 days lating resin Ni2ϩ-nitrilotriacetic acid Superflow (Qiagen Inc., (tumor volume, ϳ250–300 mm3) after the injection of cells. Valencia, CA; Ref. 36). A Superdex 200 column (1.6 ϫ 60 cm; Mice were kept in microisolator cages and fed ad libitum Pharmacia Biotech., Piscataway, NJ) was used to separate di- pathogen-free mouse diet and water. The procedures used were valent and tetravalent scFvs. Protein concentrations were deter- in accordance with the USPHS Guidelines for the Care and Use mined by the method of Lowry et al. (42). CC49 IgG used for of Laboratory Animals and were also approved by the Univer- control studies was purified on Protein G Sepharose Fast Flow sity of Nebraska Medical Center IACUC. Potassium iodide resin (Pharmacia Biotech.) and dialyzed into HEPES saline (0.001%) was given in the drinking water for 3 days before and buffer [10 mM HEPES, 150 mM NaCl, (pH 7.4)]. terminated 3 days after the administration of the radioiodinated Characterization of Purified Multivalent scFvs. The scFv. This blocked the thyroid radioiodine uptake that enabled purity of scFvs was assessed by SDS-PAGE and HPLC size the excretion of free iodine out from the animal body without exclusion chromatography. SDS-PAGE was performed accord- contributing substantially to the whole animal dose or therapeu- ing to the method of Laemmli (43) under reducing and nonre- tic dose to the tumor. ducing conditions. The gels were stained with Coomassie Blue Biodistribution and Pharmacokinetics Studies. Dual- R-250. HPLC analyses were done on TSK G2000SW and TSK label biodistribution studies were performed in LS-174T human G3000SW (Toso Haas, Tokyo, Japan) size-exclusion columns xenograft-bearing mice after a simultaneous i.v. injection via the 125 ␮ 131 connected in series using 67 mM sodium phosphate buffer (pH tail vein of I-labeled sc(Fv)2 (5 Ci) and I-labeled [sc(Fv)2]2 125 131 6.8), 100 mM KCl as the mobile phase. The columns were (2.5 ␮Ci) or I-labeled CC49 IgG (5 ␮Ci) and I-labeled ␮ calibrated using the Gel Filtration Calibration Kit (Bio-Rad, [sc(Fv)2]2 (2.5 Ci) as described earlier (35, 36). For the whole-

body retention studies, mice bearing the LS-174T xenografts (three/group) received injections via the tail vein with 1.5 ␮Ci of radioiodinated scFvs. Each scFv was evaluated separately. The whole-body radioactivity was determined at various times after injection in a custom-built NaI crystal. The blood clearance studies were performed as described previously (35, 36). Therapy Studies with 131I-labeled scFvs. For the therapy studies, mice bearing established LS-174T tumors were used. The animals were randomized, the initial body weight was recorded, and tumor volume was measured by caliper. Two therapy regimens were used: (a) a single i.v. dose of 1000 ␮Ci 131 ϭ ϭ of I-labeled sc(Fv)2 (n 10) or [sc(Fv)2]2 (n 10) administered on day 6; and (b) four i.v. doses of 250 ␮Ci of 131I- ϭ ϭ labeled sc(Fv)2 (n 10) or [sc(Fv)2]2 (n 10) on days 6, 7, 8, and 9. For the control groups (n ϭ 5), mice received injections i.v. with PBS (pH 7.4) either as a single administration on day 6 or as four injections on days 6, 7, 8, and 9. The tumor growth was monitored twice a week by measuring the tumor in two dimensions where volume ϭ (length of short axis in mm)2 ϫ Fig. 1 HPLC size-exclusion profiles of radioiodinated CC49 [sc(Fv) ] (length of long axis in mm)/2, as described earlier (35). The 2 2 and sc(Fv)2. Samples were analyzed using TSK G2000SW and TSK body weight of mice was recorded twice a week. Mice were G3000SW size-exclusion columns connected in series. The [sc(Fv)2]2 ᭜ Ⅺ ϳ euthanized when the short axis of the tumor was Ն12 mm, ( ) and sc(Fv)2 ( ) were eluted as single peaks of Mr 120,000 and tumor ulceration was detected, or the animals lost Ն20% of their 60,000, respectively. original body weight. Radiation Dosimetry. The radiation-absorbed dose delivered to the tumors and normal organs such as kidneys and ϫ 4 Ϫ1 Ϫ1 ϭ ϫ Ϫ4 Ϫ1 ϭ ϫ 8 liver was calculated according to the Medical Internal Radiation 9.1 10 M s , koff 8.9 10 s , and KA 1.0 10 Ϫ1 ϭ ϫ 4 Ϫ1 Ϫ1 ϭ ϫ Dose committee of the American Society of Nuclear Medicine M for [sc(Fv)2]2; and kon 2.2 10 M s , koff 8.0 Ϫ4 Ϫ1 ϭ ϫ 7 Ϫ1 (46, 47). For kidneys (average weight, ϳ0.15 g), electron/␤ 10 s , and KA 2.8 10 M for sc(Fv)2. were only included in the mean absorbed dose. For all of the After radioiodination, the integrity of proteins stored at 4°C other organs and tumors, the self-absorbed dose was calculated. was ascertained daily by SDS-PAGE and HPLC for at least 4 The mouse organ-cumulated activity (␮Ci ϫ h) was calculated days. On SDS-PAGE, both sc(Fv)2 and [sc(Fv)2]2 migrated as by integrating time-activity curves derived from the biodistri- Mr 58,000 under nonreducing and reducing conditions and were bution data after the first injection of 131I-labeled scFv. The data found to be stable when stored at 4°C in 1% mouse serum. were expressed in ␮Ci/g and was not corrected for decay. It was HPLC analysis indicated that Ն95% of the radioactivity was assumed that radionuclide localizes immediately, i.e.,nolag associated with the protein peak for both sc(Fv)2 and [sc(Fv)2]2 131 time, in the organ of interest. It was further assumed that the (Fig. 1). However, the amount of free I increased to 12% (72 effective half-lives do not change with each consecutive injec- h after labeling) and was corrected for therapeutic administration of the protein in the fractionated administration scheme. For tions. The immunoreactivity of radiolabeled scFvs by solid calculation of the cumulative tumor radiation doses, the absorp- phase RIA was 85–95% (0.8–1.5% nonspecific binding). At ϭ 72 h after labeling, a decrease of ϳ15 and 20% in the immu- tion phase times were Tabs 2.2 h and 2.6 h, for dimer and tetramer, respectively. The tumor was assumed not to grow noreactivity was observed for sc(Fv)2 and [sc(Fv)2]2, respec- during the treatment period. tively. Statistical Analysis. For comparing time with tumor Pharmacokinetics and Biodistribution of Radioiodi- quadrupling between various groups of mice, the data were nated scFvs. Blood clearance curves showed elimination half- fitted to estimate the slope of the growth curve. The Wilcoxon lives of 80, 170, and 330 min for sc(Fv)2, [sc(Fv)2]2, and CC49 signed rank test was used to generate the two-tailed Ps. The IgG. Whole-body clearance studies confirmed the rapid elimi- survival fraction of each treatment group was evaluated accord- nation of scFv fragments. At 48 h after administration, ϳ95% of ing to the method of Kaplan and Meier. The survival curves both radioiodinated scFvs cleared from the body, whereas the were compared, and a Logrank test was used to generate the Ps clearance was only 75% for CC49 IgG. The tumor localization using the GraphPad Prism, Version 2.01 (GraphPad Software of [sc(Fv)2]2 was 2-fold higher than sc(Fv)2 beginning at 4 h Inc., San Diego, CA). after injection (Fig. 2). Uptake at earlier time points (i.e. Ͻ4h) was similar for all of the proteins. At 24 h after administration, Ϯ Ϯ the %ID/g in tumor was 10.5 1.1 for [sc(Fv)2]2 and 5.1 0.7 RESULTS for sc(Fv)2. The maximum tumor uptake of [sc(Fv)2]2 occurred Characterization of Divalent and Tetravalent CC49 between 6 and 16 h. CC49 IgG showed 28.4 Ϯ 1.7%ID/g in Ն scFvs. After purification, sc(Fv)2 and [sc(Fv)2]2 were 95% tumor at 24 h after administration. However, in blood CC49 IgG pure as indicated by SDS-PAGE and HPLC analyses. The showed 12.5 Ϯ 0.9%ID/g as compared with 0.3 Ϯ 0.1 and 0.2 Ϯ ϭ binding parameters derived from the SPR studies were: kon 0.1%ID/g for [sc(Fv)2]2 and sc(Fv)2, respectively. RIs (%ID/g

Downloaded from clincancerres.aacrjournals.org on September 25, 2021. © 2001 American Association for Cancer Research. 178 Therapeutic Efficacy of 131I-labeled Multivalent CC49 scFvs for Colon Carcinoma

Fig. 2 Biodistribution of radioiodinated Œ F CC49 IgG ( ), sc(Fv)2 ( ), and [sc(Fv)2]2 (f) in groups of athymic mice bearing LS- 174T human colon carcinoma xenografts. The results are %ID/g of tissue Ϯ SD; n ϭ 6.

Ϯ Ϯ of tumor divided by %ID/g of normal tissue) of the major organs 2.9 days for sc(Fv)2, 32.9 2.7 days for [sc(Fv)2]2, and 8.4 were determined for CC49 IgG, sc(Fv)2, and [sc(Fv)2]2. For 0.6 days for the control group (Table 1). Survival analysis

well-perfused organs such as the liver and the spleen, the tumor: showed a significant tumor growth inhibition for sc(Fv)2 and Ͻ liver and tumor:spleen ratios were 2.3:1 and 2.6:1 for CC49 IgG, [sc(Fv)2]2 with the median survival time as 38 (P 0.0001) and 8.1:1 and 8.2:1 for sc(Fv)2, and 3.9:1 and 10.4:1 for [sc(Fv)2]2 53 days (P Ͻ 0.0001), respectively, as compared with the at 24 h after administration. control group (Fig. 4). Mice treated with fractionated doses Therapeutic Study with Single-dose Administration of showed a Յ8% loss in body weight, in contrast to the mice that Radioiodinated scFvs. The rate of tumor growth in groups received injections with a single dose of 1000 ␮Ci, which receiving [sc(Fv)2]2 was statistically different from that in the showed a Ն10% body weight loss from 20 day onward (Fig. 5). control animals (P ϭ 0.03) but not for sc(Fv) (P Ͼ 0.05; Fig. 2 In tumors, the absorbed radiation doses for sc(Fv)2 and 3). The group administered with [sc(Fv) ] showed tumor re- 2 2 [sc(Fv)2]2 were 38.2 and 168.9 rads, respectively, for each gression for 14–16 days as compared with the control group, protein and were 4-fold higher than doses after a single admin- which showed approximately a 6–8-fold increase in tumor istration (Table 2). The cumulative activity in the liver and volume (Fig. 3). The times for tumor-quadrupling volume were kidneys also increased but was well below the threshold levels. Ϯ Ϯ 9.0 0.8 and 16.6 1.9 days for sc(Fv)2 and [sc(Fv)2]2, respectively (Table 1). The time for tumor-quadrupling volume for the control group was 8.3 Ϯ 0.7 days (Table 1). A compar- DISCUSSION ison of survival curves with the control group for single dosing RIT using MAbs against tumor-associated antigens has showed the median survival time as 26 (P Ͼ 0.5) and 32 days shown limited clinical success for the treatment of solid tumors Ͻ (P 0.001) for the sc(Fv)2 and [sc(Fv)2]2, respectively (Fig. 4). (15) attributable mainly to the intrinsic characteristics of solid No apparent signs of systemic radiotoxicity were detected. An- tumors such as poor radiosensitivity, heterogeneous expression imals did not lose Ͼ20% of their body weight with 1000 ␮Ci as of antigens, relatively poor tumor vasculature, elevated intersti- a single dose (Fig. 5). The cumulative radiation doses delivered tial pressure, and tumor necrosis (48, 49). Achieving optimal

to the tumor for bolus sc(Fv)2 and [sc(Fv)2]2 were 8.5 and 36.0 adsorbed dose in solid tumors therefore requires the systemic rads, respectively (Table 2). Compared with sc(Fv)2, the higher delivery of high administered activities that result in incidental uptakes of [sc(Fv)2]2 in the liver and kidneys resulted in a radiotoxicity to the bone marrow and organs involved in the 2–3-fold increase in the absorbed radiation dose. catabolism of the radiopharmaceuticals such as the kidneys and Therapeutic Study with Dose Fractionation of Radioio- the liver (12, 14). Some of the approaches that have been dinated scFvs. Mice receiving fractionated therapeutic doses developed for increasing the therapeutic index of radiolabeled exhibited a statistically significant difference in tumor growth MAbs include: using high affinity antibodies (50); administer- Ն ϭ for both sc(Fv)2 (P 0.05) and [sc(Fv)2]2 (P 0.03) from the ing multiple doses of radiolabeled MAbs (51–53); predosing untreated control group (Fig. 3). For both therapeutic regimens, with an unlabeled antibody before the radiolabeled antibody (10, tumors regressed for 24–28 days. During the same time, the 54); using a “cocktail” of MAbs rather than a single radiolabeled control group showed approximately a Ͼ18-fold increase in antibody (55); and using combined modalities treatments (15). tumor volume or was removed from the study (Fig. 3). The Alternatively, recombinant antibody-based molecules with times for tumor-quadrupling volume were calculated as 21.1 Ϯ high affinity, functional avidity, and optimal size are being

Fig. 3 The effect of administration of (A) a single dose of 1000 ␮Ci and (B) four dose fractions of 250 ␮Ci each of 131I- labeled sc(Fv)2 or [sc(Fv)2]2 on growth of LS-174T xenograft in athymic mice. Each line represents the relative increase in tumor volume in an individual mouse.

engineered and evaluated for the improved RIT of solid tumors ity and in vivo tumor targeting (36). Upon expression, 20–30%

(35, 56–59). However, only a few studies have investigated the of sc(Fv)2 was found to associate as tetravalent and/or higher tumor localization of trivalent antigen-binding antibody con- molecular weight aggregated forms. The tetravalent scFv Ј structs (Fab )3 (60–62) and tetravalent IgG dimers (63, 64) in {[sc(Fv)2]2} revealed a 3–4-fold higher KA than sc(Fv)2 in SPR xenografted mice. studies. Also, [sc(Fv)2]2 showed a 2–3-fold slower koff as com- We have previously compared the therapeutic efficacy of pared with CC49 IgG, indicating a stronger association with the 131I-labeled noncovalent scFv dimers and intact IgG of MAb antigen because of a higher functional affinity of the molecule. CC49 in athymic mice bearing human colon carcinoma xe- The gain in avidity due to tetravalency, along with a lower nografts. The maximum tolerated dose for IgG was 500 ␮Ci and molecular weight than intact IgG, suggests this recombinant ␮ 131 could be escalated to 1500 Ci with I-labeled sc(Fv)2. Even scFv as a good candidate for tumor targeting. 131 with the lowest dose of I-labeled sc(Fv)2, a statistically In biodistribution studies, [sc(Fv)2]2 exhibited an approx- significant prolonged survival time was observed as compared imately 2-fold increase in tumor localization over sc(Fv)2 fol- with the control (P ϭ 0.036; Ref. 35). For CC49 IgG, single lowing 4 h after administration. Although tumor uptake of CC49 administration of 250 or 500 ␮Ci resulted in complete tumor IgG was significantly higher at later time points (16 h onwards), regression in 30 and 60% of mice, respectively (35). Reduced the nonspecific retention of radioactivity was found to be ele- tumor growth in 80–100% of mice has been reported earlier vated in the major organs studied because of the long circulation with 131I-labeled CC49 IgG (50). Although IgG has shown half-life of radiolabeled IgG (65). The tumor:blood ratio for promising preclinical results, the clinical performance of intact sc(Fv)2 and [sc(Fv)2]2 was detected as 1.5- and 15-fold higher, antibody for cure of solid tumors has been limited (38, 40, 41). respectively, than CC49 IgG at 24 h after administration. Blood

This is primarily due to slower clearance of IgG from the blood clearance studies showed the elimination half-life of sc(Fv)2 and pool that limits the maximum tolerated dose and dose escalation [sc(Fv)2]2 as approximately 4- and 2-fold faster than intact essential for the treatment of solid tumors. CC49 IgG. The tumor:liver and tumor:spleen ratios for 131I-

A covalent dimeric CC49 scFv [sc(Fv)2] was subsequently labeled sc(Fv)2 were found to be lower than the values observed 131 constructed and characterized for in vitro antigen-binding affin- for I-labeled sc(Fv)2 earlier (35). The increased RI of non-

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131 Table 1 Therapeutic efficacy of I-labeled CC49 sc(Fv)2 and [sc(Fv)2]2 on the growth of LS174-T human colon carcinoma xenografts The time for tumor-quadrupling volume for each group was determined with single and/or fractionated radioimmunotherapeutic administrations. The average tumor size at day 6 for the group was used as the initial value for calculating the relative tumor quadrupling time. Therapeutic Mean time for quadrupling reagent Dose and scheduling of tumor volume (days) ␮ Ϯ sc(Fv)2 1000 Ci at day 6 9.0 0.8 ␮ ϫ Ϯ sc(Fv)2 250 Ci 4 at days 6, 7, 8, and 9 21.1 2.9 ␮ Ϯ [sc(Fv)2]2 1000 Ci at day 6 16.6 1.9 ␮ ϫ Ϯ [sc(Fv)2]2 250 Ci 4 at days 6, 7, 8, and 9 32.9 2.7 Control Single injection of 200 ␮l PBS at day 6 8.3 Ϯ 0.7 Control 200 ␮l PBS ϫ 4 at days 6, 7, 8, and 9 8.4 Ϯ 0.6

dimer provided less effective therapy, probably because of the larger and more aggressive tumors (250–300 mm3 at approximately day 6). However, with tetravalent scFv, a single therapeutic treatment of 1000 ␮Ci showed a 2-fold slower tumor volume-quadrupling time than the untreated group, with the median survival time of the group increased by ϳ1.3-fold. No significant systemic toxicity was recorded for the single administration of 1000 ␮Ci attributable mainly to the fast blood clearance properties of multivalent scFvs (ϳ95% were cleared out in 48 h). With radiolabeled IgGs, both a specific (targeting the tumor-associated antigen) and a nonspecific (emission properties of the radionuclide) components can be associated with tumor regression. However, the therapeutic advantage attained by the specific antigen binding is significant over the nonspecific cy- Fig. 4 Survival analysis of athymic mice bearing LS-174T human totoxic properties of 131I (50). Therefore, in the present study, colon carcinoma xenografts versus time after a single administration of ␮ f ᭜ ␮ PBS (pH 7.4) was used as the negative control rather than an 1000 Ci of sc(Fv)2 ( ) or [sc(Fv)2]2 ( ) or four injections of 250 Ci each of sc(Fv) (Ⅺ) or [sc(Fv) ] (᭛). The control group (F) received irrelevant scFv. Also, the reference control of CC49 IgG was not 2 2 2 131 injections with PBS (pH 7.4). Arrow represents the day of initiation of included because the maximum tolerated dose for I-labeled therapeutic regimen, i.e. day 6. CC49 IgG was 500 ␮Ci for single administration (35). Fractionated therapy at medium dose levels has been sug- gested to provide effective RIT (51, 53, 69). Therefore, a dose- fractionation study was performed to directly compare the ad-

covalent sc(Fv)2 resulted primarily because of its faster clear- vantages of RIT involving dose fractionation over single ance from the blood than covalent sc(Fv)2 (36). For [sc(Fv)2]2, administration. Fractionated doses were administered every 24 h the RI for tumor:liver ratio was found lower compared with that as whole-body clearance studies demonstrated that Ͼ90% of the

for sc(Fv)2 due to the faster clearance of the sc(Fv)2 from the radioiodinated sc(Fv)2 and [sc(Fv)2]2 was cleared from the blood. The liver might be the possible site for elimination as also animal body. Also, at 24 h after administration the specific

evidenced by a lower kidney uptake of [sc(Fv)2]2 (Mr 120,000). accumulation of scFvs in the tumors was detected. For both

High tumor localization with low nonspecific retention suggests sc(Fv)2 and [sc(Fv)2]2, dose fractionation yielded slower tumor that these multivalent scFvs may be preferred choices for RIT of growth with the tumor-quadrupling time about 2.5- and 4-fold solid tumors. slower than the control group, respectively. In our study, the We have performed a direct comparison of the radioimmu- groups that received fractionated therapy of radioiodinated

notherapeutic efficacy of dimeric and tetrameric CC49 scFvs sc(Fv)2 and [sc(Fv)2]2 showed a statistically significant length- under both single and fractionated regimens. The outcome of ening of the median survival time, which was ϳ1.6- and ϳ2.4- RIT strongly depends upon the initial size of the tumor because fold longer than in the untreated group. As a reference, IgG was the uptake of radiolabeled antibody decreases exponentially not included in the dose-fractionation experiment because intact with the tumor size thereby reducing the tumor dose (66). IgG remains in circulation for a much longer period. At 24 h, the Impressive therapeutic results have been documented in animals %ID/g in blood was Ͼ10 for IgG and could have lead to with small or micrometastatic tumors (51, 67, 68) and in patients potential bone-marrow toxicity upon multiple administrations. with small tumor burdens (13, 14). In the present study, only In previous dose-fractionation studies with IgG, usually the larger tumors were used to extrapolate the situation found in a therapeutic regimen consisted of two or three administrations of clinical setting with higher tumor burdens. We have reported 300 ␮Ci of 131I-labeled B72.3/CC49 IgG at either a 7-day previously that the therapy with 131I-labeled noncovalent dimers interval (51) or a 3-day interval (53). gave a tumor regression lasting for ϳ16 days at 1500-␮Ci Besides the molecular size and functional affinity of the radiation dose (35). The treatment with 1000 ␮Ci of covalent antibody fragment, the therapeutic efficacy of a radioimmuno-

Table 2 Radiation dose estimates of 131I-labeled CC49 scFvs in LS-174T xenograft-bearing mice Cumulative radiation doses (rads) Therapeutic schedule Tumora Liver Kidneys ␮ sc(Fv)2, 1000 Ci 8.5 0.9 1.3 ␮ ϫ sc(Fv)2, 250 Ci 4 38.2 3.7 5.3 ␮ [sc(Fv)2]2, 1000 Ci 36.0 3.0 2.9 ␮ ϫ [sc(Fv)2]2, 250 Ci 4 168.9 12.2 11.5 a Tumor doses are for 1 g nodule.

Although in the present study only a partial tumor regres- Fig. 5 Toxicity of radioiodinated scFvs as monitored by the percentage change in body weight. Animals were given either a single administra- sion occurred, a definitive advantage of multivalency and dose ␮ f ᭜ tion of 1000 Ci of sc(Fv)2 ( ) or [sc(Fv)2]2 ( ) or four injections of fractionation was noticed. There are increasing reports where ␮ Ⅺ ᭛ F 250 Ci each of sc(Fv)2 ( ) or [sc(Fv)2]2 ( ). The control group ( ) either a partial or a complete ablation of xenografted solid received PBS (pH 7.4). Arrow represents day 6. tumors have been shown using RIT in conjunction with chemotherapy (75), radiotherapy (76, 77), or blood flow-modifying agents (78). Recently Behr et al. (79) demonstrated that ␣-emit- conjugate can depend on tumor vasculature, the total antibody ters like 213Bi hold advantage over conventional low linear protein dose, and the radiation dose administered (70–72). energy transfer radionuclides like 90Y in curing solid tumors Moreover, for fractionated RIT, morphological and physiolog- with antitumor FabЈ fragments. Also, pretargeting RIT ap- ical changes in the tumor blood vessels occur after the first proaches that use either radiolabeled bivalent haptens with administration of the radiolabeled antibody. This can signifi- bispecific antibody (80) or biotin-streptavidin as receptor-ligand cantly alter the localization of the subsequent dose of antibody pair (81) have shown improved cure rates. Studies are under (73). Adams et al. (72) performed a study to compare the effect way to try further dose escalation and use of pretargeting ap- of dose escalation (50 ␮g to 1000 ␮g) and repeated i.v. admin- proaches to improve the therapeutic efficacy of the multivalent istrations on the tumor localization of divalent scFvs. They CC49 scFvs. demonstrate that a highly specific localization can be main- In summary, we investigated the potential of the geneti- tained with multiple i.v. bolus injections of divalent scFv ad- cally engineered, multivalent scFvs of MAb CC49 as candidates ministered 24 h apart. We believe that the increased therapeutic for RIT of colon carcinoma. Multiple administrations of the effect seen in the present study by fractionating 1000 ␮Ci radiolabeled modality showed higher tumor:normal tissue ratios dosage to four doses of 250 ␮Ci each, given 24 h apart, helps in in a shorter time without normal tissue toxicity, leading to a overcoming the rapid clearance of the scFvs and in maintaining statistically significant reduction in tumor progression and pro- a better tumor localization. Nevertheless, the fractionated doses longed median survival times. Subsequent studies may combine cannot be considered as additive because the irradiation-induced the advantages of multivalency and pharmacokinetic properties vascular changes can be significant for the tumor. of divalent and tetravalent scFvs with dose-fractionation admin- The tumor dosimetry showed: single administration of istration to determine an optimum therapeutic treatment and

[sc(Fv)2]2 resulted in 4-fold higher radiation dose as compared improved diagnosis of cancer.

with sc(Fv)2; the radiation dose with fractionated therapeutic scheduling of sc(Fv)2 was equivalent to single administration of ACKNOWLEDGMENTS [sc(Fv)2]2; and dose fractionation of [sc(Fv)2]2 further increased We thank K. Devish, J. Jokerst, H. Conway, and Erik Moore for (5-fold) the tumor radiation absorbed dose. No radiation-related expert technical assistance. We acknowledge the Molecular Biology problems are anticipated on important target organs like the Core Lab for sequencing studies, the Molecular Interaction Facility for liver and kidneys based on the radiation dose calculations. BIAcore studies, and Kristi L. W. Berger, communications specialist One of the major concerns of therapeutic strategies based and editor, Eppley Institute, University of Nebraska Medical Center, for on multiple dose scheduling is the development of human anti- editorial assistance. The monovalent CC49 scFv construct was a gen- mouse antibodies. ScFvs should have reduced immunogenicity erous gift from the National Cancer Institute Laboratory of Tumor Immunology and Biology and the Dow Chemical Company. because they do not contain CH2 and CH3 domains of intact immunoglobulins, or the CH1orCL domains (responsible for Ј Ј REFERENCES antiallotype responses) found in Fab or F(ab )2 fragments. We have recently developed a hu/muCC49 scFv containing the 1. DeNardo, S. J., Kroger, L. A., and DeNardo, G. L. A new era for human subgroup IV germ-line V and variable region of the radiolabeled antibodies in cancer? Curr. Opin. Immunol., 11: 563–569, L 1999. murine CC49 heavy chain (74). In vivo tumor-targeting studies 2. Cheung, N. K., Kushner, B. H., Yeh, S. D. J., and Larson, S. M. 3F8 showed similar biodistribution and pharmacokinetic properties monoclonal antibody treatment of patients with stage 4 neuroblastoma: of the shuffled and completely murine scFv (74) with possible a Phase II study. Int. J. Oncol., 12: 1299–1306, 1998. implications in the reduction of human antimouse antibody 3. Matthews, D. C., Appelbaum, F. R., Eary, J. F., Fisher, D. R., responses in patients. Durack, L. D., Hui, T. E., Martin, P. J., Mitchell, D., Press, O. W., Storb,

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Apollina Goel, Sam Augustine, Janina Baranowska-Kortylewicz, et al.

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