Purpose: Immunoglobulins are catabolized in the hepatocytes, primarily by cathepsins. The liver becomes the likely dose-limiting tissue for radiometals, like 90Y, in radioimmunoconjugates (RICs) used for radioimmunotherapy in combination with bone marrow support. To assess whether in vitro cathepsin-degradable peptide linkers between the chelated radiometal and the antibody decreased hepatic radiation dose, cumulated activity was used as a surrogate for radiation dose.

Experimental design: Four different cathepsin-degradable peptides used to link 1,4,7,10-tetraazacyclododecane-N,N′,N″,N‴-tetraacetic acid-chelated 111In to two different monoclonal antibodies were studied in athymic mouse models of human breast cancer or lymphoma. Measured concentrations of activity during 5 days were used to reflect pharmacokinetic behavior for normal tissues and tumor. With the use of linear regression to fit a monoexponential decay function, cumulated activities in the liver and xenografts were calculated.

Results: The pharmacokinetic behavior of the cathepsin-degradable peptide-linked RICs was similar to that for the 2-iminothiolane (2IT) nondegradable linked RICs except for the liver. The liver cumulated activities of peptide-linked RICs were significantly decreased from those of the corresponding 2IT-linked RICs, varying between reductions of 59 and 68%. Cumulated activities of peptide-linked RICs in the xenografts were as great as those of 2IT RICs, so that the therapeutic indices (tumor:liver cumulated activity ratios) were substantially better for cathepsin-degradable peptide-linked RICs.

Conclusions: Cathepsin-degradable peptides used to link chelated radiometals to antibodies reduce liver radiation dose and improve the therapeutic index for radioimmunotherapy given in combination with bone marrow support.

90Y is an attractive radionuclide for RIT3 because it provides greater tumor retention and has more energetic β emissions than 131I (1, 2, 3). Bifunctional chelating agents used in radiometal-labeled RICs have a metal chelating group at one end and a reactive functional group, capable of binding to proteins, at the other end. The macrocyclic chelator, DOTA, binds yttrium with extraordinary stability. DOTA also binds indium with considerable stability (4, 5), so that DOTA chelates of γ-emitting 111In can be used to trace the pharmacokinetics of corresponding 90Y-RICs. Protein amino groups may be conveniently conjugated to bifunctional macrocyclic chelating agents by 2IT to permit addition of a radiometal to the conjugate. A bifunctional bromoacetamidobenzyl derivative of DOTA, BAD, has been conjugated to RICs via 2IT (4, 6). However, 90Y clearance from the liver of mice given 90Y-2IT-BAD RICs has been appreciably slower than that of corresponding radioiodinated Abs (2, 3, 7). Elimination of radioactivity from the liver involves both enzymatic liberation of low-molecular-weight fragments from the targeting Ab and intracellular processing of these fragments (8, 9, 10, 11, 12). One strategy to increase liver clearance is to insert a degradable linker between the targeting Ab and the chelated radiometal.

Degradable linkers explored for reducing radiation to normal tissues from RIT include esters, thioethers, disulfides, amides, and hydrocarbon chains (8, 10, 11, 13, 14, 15). Peptide linkers are preferred because of their stability in the circulation (8, 14, 15). Simple tetrapeptides have been shown to be degraded by endoproteases in vitro at enzyme levels present in vivo(8, 16, 17).

Because of evidence that intrahepatocyte cathepsins are responsible for most of the protein metabolism in hepatic lysosomes, peptide linkages susceptible to cathepsins were created to facilitate excretion of the radiochelate in RICs (17). The chelated peptide linker, DOTA-GGGF-ITC, was designed to produce upon degradation a radiochelate complex that facilitated clearance from hepatocytes (17). This glycine and phenylalanine peptide linkage was degraded by cathepsin B in vitro. 90Y-DOTA was conjugated to the antiadenocarcinoma MAb, L6 (ChL6), via GGGF and compared in mice with HBT3477 human breast adenocarcinoma xenografts to 90Y-BAD conjugated to ChL6 via the stable linker 2IT (18). Lower concentrations of 111In and 90Y in the liver were observed for -DOTA-GGGF-ChL6 than for 2IT-BAD-ChL6; -DOTA-GGGF-ChL6 provided a tumor to liver radiation dose ratio of 35 to 1 in patients (19, 20).

Recently, Peterson and Meares (8, 21) have used one-bead, one-peptide combinatorial libraries (22, 23, 24, 25) to generate additional peptide substrates for cathepsin. The peptides GFQGVQFAGF, GFGSVQFAGF, and GFGSTFFAGF demonstrated more rapid cathepsin degradation than the prototype peptide GGGF in vitro(8).

To examine the properties of 111In (and 90Y)-DOTA-peptide-MAbs in vivo, pharmacokinetic studies on RICs with these four cathepsin-degradable peptides were conducted in athymic mice with human breast cancer or lymphoma xenografts. Analogous studies were conducted with 111In (and 90Y)-2IT-BAD-MAbs for comparison. DOTA-peptide RICs are novel because the DOTA chelator binds these radiometals with exceptional stability, so that the peptide linker when degraded in the liver releases the chelated radiometal (Fig. 1), thereby reducing the radiation dose to the liver and improving the tumor-liver TI. Data documenting the improved TI of DOTA-peptide RICs are shown here.

Mouse Models.

HBT 3477, a human breast adenocarcinoma cell line obtained from Bristol-Myers Squibb Pharmaceutical Research Institute (Seattle, WA), was grown in Iscove’s medium (Life Technologies, Inc., Gaithersburg, MD). Raji, a human Burkitt’s lymphoma cell line obtained from the American Type Culture Collection (Manassas, VA), was grown in RPMI 1640. Female athymic BALB/c-nu/nu mice (Harlan Sprague Dawley, Frederick, MD, or Simonson Co., Gilroy, CA), ∼6 weeks old, were maintained according to University of California animal care guidelines, on a normal diet, ad libitum. For Lym-1 studies, mice were given 400 rads external beam radiation 3–7 days before implantation. HBT or Raji cells, 2.5–5.0 × 106, harvested in logarithmic phase, were implanted s.c. into the abdominal wall of each mouse. Studies for each RIC were initiated ∼3 weeks after implantation, when tumors were 50–500 mm3. A total of 198 mice were included in the study; there were between 11 and 25 mice in each RIC group.

Abs.

ChL6 (Bristol-Myers Squibb Pharmaceutical Research Institute), an Ab chimera consisting of a human IgG1 constant region and the Fab′ region of mouse MAb L6, reacts with an integral membrane glycoprotein highly expressed on human breast, colon, ovary, and lung carcinomas (26, 27). Lym-1 (Techniclone, Inc., Tustin, CA), an IgG2a MAb generated in mice immunized with human Burkitt’s lymphoma cell nuclei, recognizes a cell surface 31- to 35-kDa antigen on malignant B cells and reacts with >80% of human B cell non-Hodgkin’s lymphoma (28).

RICs.

Various DOTA-peptide linkers were provided by C. F. Meares and J. Peterson (Table 1; Fig. 2). Methods for preparing all of the RICs have been described in detail previously (17, 29, 30, 31). Briefly, RICs of 2IT-BAD-ChL6 and DOTA-peptides of Lym-1 (p000, p0001, p005, and p013) were prepared by combining the MAb with the respective DOTA linker to produce an IC to which either 111In or 90Y was added to produce the RIC. To produce 2IT-BAD-Lym-1 and DOTA-peptide (p000)-ChL6 RICs, the chelated linker was prelabeled with 111In or 90Y, and then the MAb was added. When prechelation was used, both the intermediate chelated radiometal and the final RICs were scavenged with a molar excess of EDTA or diethylenetriaminepentaacetic acid. When an intermediate IC was prepared before addition of 111In or 90Y, only the final RIC was scavenged with a molar excess of ETDA or diethylenetriaminepentaacetic acid (to remove loosely bound 111In or 90Y). RICs were purified and transferred to buffered saline, using column-gel filtration chromatography.

Quality Assurance.

RICs were examined by HPLC, cellulose acetate electrophoresis, TLC, and live cell and solid phase radioimmunoassays (5). HPLC (Beckman 332; Beckman, San Ramon, CA) was performed using a molecular sieving column (Beckman SEC-3000 or TSK-3000) eluted in 0.1 m sodium phosphate-0.1 m potassium sulfate-0.025% (w/v) sodium azide, pH 7.1, at a flow rate of 1.0 ml/min. RICs were detected by UV absorbance at 280 nm (Beckman 166 detector) and by radioactivity (Beckman 170 detector). Cellulose acetate electrophoresis (Gelman Sciences, Inc., Ann Arbor, MI) was performed using 0.05 m sodium barbital buffer, pH 8.6, and a current of 5 mA/strip. Samples were electrophoresed for 11 and 45 min. TLC of RICs was performed using silica-coated plates (DC-Plastikfolien Kieselgel 60 F254; EM Science, Cherry Hill, NJ). The mobile phase was a 1:1 mixture of equal volumes of 10% (w/v) aqueous ammonium acetate and methanol. TLCs were scanned for radioactivity using a radioactive gel scanner (AMBIS Systems, San Diego, CA) or cut at Rf 0.3 and counted in a gamma well counter. Immunoreactivity of all RICs was assessed by cell binding and solid phase radioimmunoassays, as described previously (32, 33). 125I-ChL6 or 125I-Lym-1, lightly labeled and previously shown to be indistinguishable from unlabeled Ab, were assayed as reference standards.

By these assays for immunochemical integrity, at least 93% of the radioactivity in all RICs was in monomeric form. The absolute immunoreactivities of the RICs in live cell assays were at least 60%, and at least 85% of that of a 125I-labeled reference standard RIC. Similarly, all of the RICs had high immunoreactivity in solid phase assays, except for those linked by p013. Whereas the live cell immunoreactivity of this RIC was high, the solid phase immunoreactivities were substantially reduced (see “Discussion”).

Pharmacokinetics.

RIC (100 μl; 15–40 μCi) was injected into the tail vein of each mouse. The radioactive dose was measured using a dose calibrator (CRC-12; Capintec, Inc., Pittsburgh, PA) and confirmed by counting the mouse immediately after injection, using two opposed, isoresponsive sodium iodide detectors (Picker Nuclear, North Haven, CT) calibrated against appropriate standards for volume and geometry effects. To determine total body clearances, mice were counted immediately and serially for 5 days after injection using the isoresponsive sodium iodide detector system. The counts were decay corrected and expressed as % ID. Blood clearances were determined by collecting blood samples from the tail veins of mice serially for 5 days after injection and counting them in a gamma well counter. Decay-corrected radioactivity in the blood was expressed as % ID, using a weight-based theoretical blood volume. Pharmacokinetic data for other tissues were obtained by sacrificing mice at 1, 3, and 5 days after injection; removing and weighing the tissues; and counting them in a gamma well counter. The concentration in each organ was expressed as % ID/g. Using standards of comparable volume and geometry for blood and organs, all 90Y sample counts were corrected for attenuation in addition to decay.

Cumulated Activity.

The cumulated activity h, the sum of all radioactive decays in a tissue (h), during the time interval of interest was obtained from the sequential measurements of the radioactivity Ah(t) in the liver or xenograft. Because of the monoexponential physical decay of short-lived 111In or 90Y, tissue clearance was assumed to be monoexponential, so that the expected activity concentration at time t could be represented as Aλexp(−λt), where A represents the activity at time t = 0 and λ represents the rate of decrease in activity concentration with time. The cumulated activity was obtained by fitting the data with this monoexponential function using nonlinear regression analysis, SAS procedure NLIN, applied to the activity concentrations.4 Based on the monoexponential model, the cumulated activity is A:λ and is estimated using the estimated values for A and λ. The SE of this ratio was estimated from the SE estimates of A and λ. An extension of Fieller’s theorem was used to provide an approximation for a SE of the ratio of two random variables based on the standard errors of the two variables and their correlation, and assuming that each was approximately normally distributed (34). Because a monoexponential function was not appropriate for the data for 111In-2IT-BAD-Lym-1, a trapezoidal function, assuming the physical decay of 111In after the last observation, was used. Cumulated activity concentrations in units of microcuries h/g per unit of administered radioactivity (μCi-h/g/μCi) were obtained by adjusting cumulated activity for tissue mass and for administered radioactivity. To estimate the TI, the ratio of estimated activity of the tumor to that of the liver for each RIC was computed.

Study Design.

When 111In- and 90Y-p000-linked ChL6 RICs were found to be effective for decreasing liver cumulated activity, when compared with the corresponding nondegradable 2IT-RICs, the p000 peptide linker was used as a standard for comparison with subsequently chosen decapeptide linkers. Three decapeptide linkers were selected from the combinatorial library because of intense fluorescence that exceeded that of p000. Two of these peptides, GFQGVQFAGF and GFGSVQFAGF, were cleaved by cathepsin B; the third peptide, GFGSTFFAGF, was cleaved by cathepsin D and also by cathepsin B but to a lesser degree (Table 1).

Statistical Methods.

The calculation of the cumulated activity and its standard error are described above. To compare the cumulated activity between 2IT-BAD and p000, the assumption was made that the estimates were approximately normally distributed, and a comparison was made by taking the difference in the estimates (2IT-BAD and p000) and dividing the difference by the square root of the squares of the standard errors for the two cumulated activity estimates. This provides a Z score which can be compared with standard normal distribution tables. Results were considered statistically significant if the P for the two-sided test was [l]0.05. Although the assumptions would not be expected to hold exactly, the results for the differences in liver were sufficiently different, that the conclusions seem robust.

All of the RICs were remarkably stable in human serum or plasma, showing loss of radiometals from the RICs of <0.4%/day in vitro (Figs. 3 and 4). Additionally, body and blood clearances in mice of RICs of the same MAb were similar (Fig. 5). By way of example, the activities that remained in the body were 70, 85, 81, 78, and 79% of the injected dose for 111In-2IT, -p000, -p001, -p005, and -p013, respectively, and 21, 31, 25, 21 and 21%, respectively, in the blood at 48 h after injection of RICs of Lym-1.

Comparing 2IT-BAD RICs with DOTA-peptide RICs of the same MAb, the most striking differences were those for the liver activity concentrations and cumulated activities (Figs. 6,7,8). Cumulated activities of DOTA-peptide RICs were consistently and significantly less than those of the 2IT-BAD RICs of the same MAb. The greatest percentage of decrease (68%) occurred when the liver cumulated activity of 111In-DOTA-p005-Lym-1 was compared with 111In-2IT-BAD-Lym-1 (5.1 versus 15.9 μCi-h/g/μCi; P < 0.01) and the least percentage of decrease (59%) occurred when 111In-DOTA-p000-ChL6 was compared with 111In-DOTA-2IT-BAD-ChL6 (3.6 versus 8.9 μCi-h/g/μCi; P < 0.01). Liver cumulated activities for other peptide-linked MAbs were correspondingly decreased when compared with the 2IT-BAD analogue, whether labeled with 111In or 90Y. However, liver cumulated activities for RICs with library peptide linkers (p001, p005, and p013) were not further decreased from that of the p000 peptide RIC of Lym-1 MAb.

Importantly, activity concentrations and cumulated activities in the xenografts were always as great for peptide-linked RICs when compared with the 2IT-BAD RICs of the same MAb (Figs. 6 and 8). Although xenograft cumulated activities for peptide-linked RICs were higher than those of 2IT-BAD RICs, none of the differences was statistically significant. Because the liver cumulated activities for the peptide RICs were consistently and significantly reduced when compared with the 2IT-BAD RICs, whereas the xenograft cumulated activities were maintained, the tumor:liver cumulated activity ratios, TIs, for all peptide RICs, whether labeled with 111In or 90Y, were substantially improved (Fig. 9). The TIs for 111In-p000-linked ChL6 and Lym-1 were 1.64 and 3.17, respectively, whereas those for 2IT-BAD-ChL6 and Lym-1 were 0.67 and 0.47, respectively (TI for yttrium-labeled-2IT-BAD-Lym-1 was 0.31). Similarly, the TI of 90Y-p000-linked ChL6 was almost twice that of 2IT-BAD-ChL6. Concentrations over time of RICs in the kidney and other normal tissues were not greatly different for RICs of the same MAb.

An optimal linker for RICs for RIT should: (a) link the targeting Ab to the radiometal carrier without impairing the functionality of either; (b) be stable in the circulation; and (c) degrade under specified conditions to expedite clearance of the radiometal from normal tissues but not from the tumor. Degradable linkers may increase the specificity of the targeted therapy beyond that provided by the preferential distribution of the carrier MAb. When delivered on intact RICs, radiometals provide prolonged tumor residence but, as Sharkey et al.(1) reported, large fractions of radiometals are retained in the liver. The effect of slow clearance of 90Y from the liver is a lower TI. To accelerate clearance of radiometals from the liver, RICs with cathepsin-degradable peptide linkers between the Ab and the radioactive carrier have been designed to liberate low molecular weight DOTA-chelated radiometal species. These peptide linkers are intended to allow differential removal of the radiometal from the RIC when it is internalized in hepatocytes but not when it is bound to surface antigens of malignant cells. Cathepsins required for degradation of the linker are abundant in the hepatocytes where MAbs are metabolized but less likely to be available and active on the surface of the malignant cells. This paper describes linkers for RIT, in which the cytotoxic agent was a radiometal and peptide linkers were selected for cathepsin-degradation.

Cathepsins generally refer to a group of lysosomal proteases that are active within acidic environments. As lysosomal proteases, cathepsins play an important role in protein degradation. The characteristic lysosomal location and acidic pH are important features of cathepsins. The lysosomal protease, cathepsin D, for example, typically functions as a major enzyme in intracellular protein degradation. As a protease secreted from prostate carcinoma cell lines, cathepsin D has been reported to degrade proteins when in the proper environment (35, 36).

A peptide originally developed for prodrug delivery (37) was evaluated by Meares, DeNardo, and coworkers to accelerate clearance of radiometals from the liver (16, 38). Ala-Leu-Ala-Leu, inserted between 111In-benzyl-EDTA and mouse antilymphoma MAb Lym-1, demonstrated rapid degradation by cathepsin B in vitro but did not accelerate clearance from the liver of mice (16). DOTA-GGGF-ITC was designed to produce a cleaved radiochelate complex with a net charge of zero to allow more rapid clearance from liver cells (17). The peptide linkage between glycine and phenylalanine was shown to be hydrolyzed by cathepsin B in vitro. In mice bearing HBT3477 human breast adenocarcinoma xenografts, lower concentrations of 90Y in liver were observed for 90Y-DOTA-GGGF-ChL6 than for 90Y-2IT-BAD-ChL6, reducing radiation dose to liver by nearly one-half (18). 111In/90Y-DOTA-GGGF-ChL6 provided a TI for tumor to liver radiation of 35:1 in a clinical trial, (19, 20). Recently, Peterson and Meares (8, 21) used the one-bead, one-peptide combinatorial library approach (22, 23, 24, 25) to develop peptide substrates for cathepsin. In the combinatorial strategy, 94 different peptide sequences were built on bead supports by incorporating nine amino acids in four variable positions, such that each bead contained multiple copies of a unique sequence. The peptides GFQGVQFAGF and GFGSVQFAGF were more rapidly digested in vitro than the prototype peptide, GGGF. A third peptide, GFGSTFFAGF, was rapidly digested by cathepsin D.

To examine the properties of RICs having these cathepsin-degradable peptide linkers, 111In-, or 90Y-DOTA-peptide MAbs or -2IT-BAD MAbs were injected into athymic mice bearing human cancer xenografts. The prototype peptide linker, GGGF, and the other three peptide linkers, when incorporated in 111In or 90Y DOTA chelated ChL6 (anti adenocarcinoma) or Lym-1 (antilymphoma) RICs significantly decreased liver cumulated activities. However, the library peptides did not further reduce liver cumulated activity when compared with GGGF. These results suggest that the process of proteolytic degradation by two abundant native hepatic proteases is not rate limiting in terms of ultimately clearing radioactive metabolites from the liver. Xenograft concentrations of 111In- and 90Y-DOTA-peptide RICs were not significantly different from those of the corresponding 2IT-BAD RIC.

Xenograft:liver TIs of 111In- and 90Y-DOTA-peptide MAbs were much more favorable (Fig. 9). Because a variety of malignancies have been shown to have activated enzymes extracellularly, as well as intracellularly, an important observation from our work is that xenograft activity concentrations and cumulated activities were maintained for four cathepsin-degradable peptides on two different MAbs, ChL6 and Lym-1, in two aggressive human malignancies, an adenocarcinoma and a lymphoma. This suggests that our rationale for the cathepsin-degradable peptide linkers has merit, given that the expected differential effects in the hepatocyte and in the tumor have been documented. It was hypothesized that peptide linkers on MAbs selected because they are retained on the surface membrane of malignant cells would not be digested in the malignancy. Interestingly, the only peptide of the group that was degradable by cathepsin D did show evidence suggesting that the effect of malignant cell enzymes must be considered in the selection of degradable peptides. Immunoreactivity was determined for all of the chimeric L6 and Lym-1 RICs using radioimmunoassays in both cell culture and solid phase. As reported in “Materials and Methods,” immunoreactivity of all of the RICs was high. This was true for peptide p013 and the other RICs when assayed in live cell culture. However, the immunoreactivity of p013 RIC, when determined in solid phase assays using malignant cell extracts, was substantially reduced, presumably because enzymes were released and activated during the malignant cell extraction process.

As indicated above, three of the linkers evaluated here were derived from a peptide library with 94 or 6561 permutations. This small peptide library deliberately excluded charged amino acids. It may be possible to identify peptide substrates with higher efficiency and specificity by increasing the diversity and length of the peptides in the library. A random heptapeptide library, XXXXXXX (wherein X = a combination of the 20 eukaryotic amino acids plus 10 additional unnatural l-amino acids), has been synthesized and screened against cathepsin D. Two of the brightest peptide beads were selected and sequenced. Interestingly, these two peptide beads contained unnatural amino acids at the cleavage site. Their amino acid sequences were: T-H-Hyp-V-N-Nal2*Nle, Aib-E-F-M-A-F*Phg (∗ represents the cleavage site, Hyp = l-hydroxyproline, Nal2 = l-naphthalalanine-2, Aib = l-aminoisobutyric acid, Nle = l-norleucine, Phg = l-phenylglycine). Work is under way in our laboratory to evaluate the susceptibility and specificity of these peptide substrates to degradation by cathepsins. Although the combinatorial approach may yet prove to be a useful tool for generating metabolizable peptide linkages in vitro, better understanding of in vivo cleavage and residualization is needed. RICs are metabolized in lysosomes of both tumor and liver cells. Thus, the key to exploiting the linker strategy may well lie as much in the differences between these two cell types as in the molecular features of the linker that impart hepatic degradation. Additional knowledge of the mechanisms for hepatocyte internalization and intracellular traffic of radiolabeled MAbs and activities in various organelles should allow more specific libraries for generating more effective linkers. It is even likely that other mechanisms limit the amount of improvement that can be achieved using these peptide linkers.

In summary, 111In/90Y-DOTA-peptide RICs have novel properties for RIT that are superior to those of the corresponding 2IT-BAD RICs. Studies in vitro and in mice and preliminary studies in patients corroborate the potential of these new RICs for RIT. Peptide linkers designed for degradation in liver cells substantially increased TI in preclinical models. In pilot trials of these 111In- and 90Y-DOTA-peptide-MAbs in patients, the effectiveness of the GGGF peptide linker has been borne out (19, 39). Thus far, library peptides have not led to further reductions in liver radiation dose when compared with the GGGF peptide linker.

1

Presented at the “Ninth Conference on Cancer Therapy with Antibodies and Immunoconjugates,” October 24–26, 2002, Princeton, NJ. Supported by grants from the National Cancer Institute (PO1 CA47829 and UO1 CA61641) and by Department of Energy Grant DE-FG03-84ER60233).

3

The abbreviations used are: Ab, antibody; MAb, monoclonal anti-body; ChL6, chimeric mouse-human L6 monoclonal antibody; BAD, 2-[p-(bromoacetamido)benzyl]-1,4,7,10-tetraazocyclododecane-N,N,N,N‴-tetraacetic acid; DOTA, 1,4,7,10-tetraazacyclododecane-N,N,N,N‴-tetraacetic acid; DOTA-GGGF-ITC, DOTA-glycylglycyl-glycyl-l-(p-isothiocyanato)phenylalanine amide; 2IT, 2-iminothiolane; IC, immunoconjugate; ID, injected dose; RIC, radioimmunoconjugate; RIT, radioimmunotherapy; HPLC, high-performance liquid chromatography; TI, therapeutic index.

4

SAS OnlineDoc, Version 8, February 2000, SAS Institute, Inc., Cary, NC.

Fig. 1.

Cathepsin degradation of the peptide so that the chelated radiometal is released into the circulation to be cleared by the kidneys in the urine.

Fig. 1.

Cathepsin degradation of the peptide so that the chelated radiometal is released into the circulation to be cleared by the kidneys in the urine.

Close modal
Fig. 2.

2IT-BAD-MAb and DOTA-p000-MAb ICs with three and four carboxyl arms, respectively.

Fig. 2.

2IT-BAD-MAb and DOTA-p000-MAb ICs with three and four carboxyl arms, respectively.

Close modal
Fig. 3.

Plasma stability of 111In-DOTA-p000-Lym-1 in vitro. Molecular sieving HPLC revealed that almost all of the 111In eluted in a volume corresponding to monomeric Ab. The initial profile is that of the RIC followed by HPLC of plasma samples over time. There is the slightest suggestion of a species of smaller molecular weight at later time points.

Fig. 3.

Plasma stability of 111In-DOTA-p000-Lym-1 in vitro. Molecular sieving HPLC revealed that almost all of the 111In eluted in a volume corresponding to monomeric Ab. The initial profile is that of the RIC followed by HPLC of plasma samples over time. There is the slightest suggestion of a species of smaller molecular weight at later time points.

Close modal
Fig. 4.

Stability of 111In- and 90Y-DOTA-p000-Lym-1 and -2IT-BAD-Lym-1 in human plasma in vitro. All of the RICs demonstrated remarkable stability.

Fig. 4.

Stability of 111In- and 90Y-DOTA-p000-Lym-1 and -2IT-BAD-Lym-1 in human plasma in vitro. All of the RICs demonstrated remarkable stability.

Close modal
Fig. 5.

Body and blood clearances in mice given 111In-2IT-BAD-Lym-1 or -DOTA-peptide-Lym-1. Clearances for the RICs were similar.

Fig. 5.

Body and blood clearances in mice given 111In-2IT-BAD-Lym-1 or -DOTA-peptide-Lym-1. Clearances for the RICs were similar.

Close modal
Fig. 6.

Activity concentrations of 111In in liver, tumor, and kidney in mice given 111In-2IT-BAD-ChL6 (top) and 111In-DOTA-p000-ChL6 (bottom), designed for degradation by cathepsin B. Activity concentrations of 111In in tumor and kidney were similar for both RICs. Liver activity concentrations of 111In-DOTA-p000-ChL6 were substantially lower than those of 111In-2IT-BAD-ChL6, so that its liver cumulated activity was much less. Bars, SD.

Fig. 6.

Activity concentrations of 111In in liver, tumor, and kidney in mice given 111In-2IT-BAD-ChL6 (top) and 111In-DOTA-p000-ChL6 (bottom), designed for degradation by cathepsin B. Activity concentrations of 111In in tumor and kidney were similar for both RICs. Liver activity concentrations of 111In-DOTA-p000-ChL6 were substantially lower than those of 111In-2IT-BAD-ChL6, so that its liver cumulated activity was much less. Bars, SD.

Close modal
Fig. 7.

Whole body autoradiographs of mice given 111In-DOTA-p013-Lym-1, 100 μCi i.v. Whole body autoradiographs at 1 (A) and 3 (B) days showed maximum 111In in the abdominal Raji lymphoma xenografts (straight arrows). 111In in the liver decreased by day 3 from that present on day 1 (curvilinear arrows). Peptide linker p013 was chosen to illustrate clearance of liver activity and retention of xenograft activity even though this peptide was less stable when exposed to malignant cell enzymes (see “Discussion”).

Fig. 7.

Whole body autoradiographs of mice given 111In-DOTA-p013-Lym-1, 100 μCi i.v. Whole body autoradiographs at 1 (A) and 3 (B) days showed maximum 111In in the abdominal Raji lymphoma xenografts (straight arrows). 111In in the liver decreased by day 3 from that present on day 1 (curvilinear arrows). Peptide linker p013 was chosen to illustrate clearance of liver activity and retention of xenograft activity even though this peptide was less stable when exposed to malignant cell enzymes (see “Discussion”).

Close modal
Fig. 8.

Cumulated activities (μCi-h/g/μCi) for 111In-2IT-BAD and peptide-linked RICs for liver (□) and tumor (▪). When compared with 2IT-BAD RICs, liver cumulated activities for DOTA-peptide RICs were consistently reduced for both MAbs, whereas the tumor cumulated activities were maintained. Cumulated activities were obtained using nonlinear regression to analyze monoexponential activity concentrations obtained from the pharmacokinetic studies (error bars represent SE).

Fig. 8.

Cumulated activities (μCi-h/g/μCi) for 111In-2IT-BAD and peptide-linked RICs for liver (□) and tumor (▪). When compared with 2IT-BAD RICs, liver cumulated activities for DOTA-peptide RICs were consistently reduced for both MAbs, whereas the tumor cumulated activities were maintained. Cumulated activities were obtained using nonlinear regression to analyze monoexponential activity concentrations obtained from the pharmacokinetic studies (error bars represent SE).

Close modal
Fig. 9.

Therapeutic indices (tumor:liver ratios of cumulated activities) were substantially better for the DOTA-peptide-linked RICs when compared with the 2IT-BAD-linked RICs. TIs, when reflected as relative cumulated activities for tumor and the dose-limiting normal tissue, liver, are surrogates for relative radiation doses which in turn can be used as surrogates for relative tumor to dose-limiting normal tissue (liver) cytotoxicity.

Fig. 9.

Therapeutic indices (tumor:liver ratios of cumulated activities) were substantially better for the DOTA-peptide-linked RICs when compared with the 2IT-BAD-linked RICs. TIs, when reflected as relative cumulated activities for tumor and the dose-limiting normal tissue, liver, are surrogates for relative radiation doses which in turn can be used as surrogates for relative tumor to dose-limiting normal tissue (liver) cytotoxicity.

Close modal
Table 1

Nomenclature, amino acid sequence, and cathepsin cleavage in vitro for peptide linkers

LinkerAmino acid sequenceDevelopmentEnzyme cleavage in vitro
p000DOTA-GGGFPrototypeCathepsin B
p001 DOTA-GFQGVQFAGF 9-aa Cathepsin B 
p005 DOTA-GFGSVQFAGF combinational Cathepsin B 
p013 DOTA-GFGSTFFAGF library Cathepsins D and B 
LinkerAmino acid sequenceDevelopmentEnzyme cleavage in vitro
p000DOTA-GGGFPrototypeCathepsin B
p001 DOTA-GFQGVQFAGF 9-aa Cathepsin B 
p005 DOTA-GFGSVQFAGF combinational Cathepsin B 
p013 DOTA-GFGSTFFAGF library Cathepsins D and B 

We thank Claude Meares, Ph.D., University of California, Davis, for providing the peptide linkers and for other significant contributions to this research. We also thank Drs. Douglas Grenier, Oliver Renn, and Min Li of the Department of Chemistry; and Dave Kukis, M.S., and Dr. Ninh Doan of the Division of Hematology/Oncology, for their significant contributions to this research. Without these contributions over years, this research would not have been possible.

1
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