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Whereas bivalent fragments have been widely used for radio-immunotherapy, no systematic study has been published on the therapeutic performance of monovalent conjugates in vivo. The aim of our study was, therefore, to determine the therapeutic performance of (131)I-labeled Fab as compared to bivalent conjugates and to analyze factors that influence dose-limiting organ toxicity and anti-tumor efficacy. The maximum tolerated doses (MTDs) and dose-limiting organ toxicities of the (131)I-labeled anti-CEA antibody MN-14 [IgG, F(ab')2 and Fab] were determined in nude mice bearing s.c. human colon cancer xenografts. Mice were treated with or without bone marrow transplantation (BMT) or inhibition of the renal accretion of antibody fragments by D-lysine or combinations thereof. Toxicity and tumor growth were monitored. Radiation dosimetry was calculated from biodistribution data. With all 3 (131)I-labeled immunoconjugates [IgG, F(ab')2 and Fab], the red marrow was the only dose-limiting organ; MTDs were 260 microCi for IgG, 1,200 microCi for F(ab')2 and 3 mCi for Fab, corresponding to blood doses of 17 Gy, 9 Gy and 4 Gy, respectively. However, initial dose rates were 10 times higher with Fab as compared to IgG and 3 times higher as compared to F(ab')2. The MTD of all 3 immunoconjugates was increased by BMT by approximately 30%. In accordance with renal doses below 10 Gy, no signs of nephrotoxicity were observed. Despite lower absorbed tumor doses, at equitoxic dosing, Fab fragments were more effective at controlling tumor growth than the respective bivalent fragment or IgG, probably due to higher intratumoral dose rates. Our data indicate that the improved anti-tumor effectiveness of antibody fragments as compared to IgG and the higher myelotoxicity at comparably lower red marrow doses are most likely due to the higher initial dose rates observed with antibody fragments.

In order to optimize radioimmunotherapy (RAIT) as a cancer-treatment modality, it is necessary to select the appropriate radionuclide and antibody carrier. We evaluated the therapeutic potential of a single cycle of Mu-9 anti-CSAp monoclonal antibody (MAb) labeled with 3 different radionuclides, 131I, 90Y and 188Re. Intact antibodies and bivalent fragments with different blood clearance kinetics, normal organ distribution and varying tumor accretion and retention are also evaluated. Efficacy of treatment for large and small tumor burden was assessed in nude mice bearing s.c. GW-39 human colonic-carcinoma xenografts or intrapulmonary micrometastatic GW-39 colonies at the maximal tolerated dose of each agent. The magnitude and duration of myelosuppression associated with each radioantibody was considered by monitoring peripheral blood counts, marrow colony-forming unit activity and hematopoietic tissue weight. Radiation-dose estimates were calculated based on the kinetics of antibody accretion and elimination from tumor and normal tissues, and the results were correlated with tumoricidal activity and dose-limiting toxicity results. These studies, therefore, represent a detailed analysis, in a well-defined experimental tumor system, of several parameters (antibody form, radioisotope, tumor size) influencing the overall outcome of RAIT using equitoxic doses. It was found that myelosuppression is the primary dose-limiting toxicity for all radioantibodies except 90Y-F(ab')2, even though the different agents showed varied organ distribution. In a single-cycle treatment schedule of Mu-9 MAb, the 131I-labeled IgG is the radioimmunoconjugate of choice for the treatment of s.c. and intrapulmonary growth of the GW-39 human colonic-carcinoma xenograft in nude mice.

The goal of our studies was to determine whether administration of IL-1/GM-CSF to mice could reduce radio-antibody-induced myelosuppression and allow either dose escalation of radio-antibody using 131I, 90Y or 188Re conjugated to either intact antibody or bivalent fragments or more frequent dosing with 131I-IgG. Survival, peripheral blood counts, hematopoietic tissue weight and number of marrow CFCs were used to determine the ability to dose-intensify with a single dose or to reduce the spacing between doses. In this report, we show that in the absence of cytokines, 2 cycles of 131I-IgG spaced at 28, 35, 42 and 49 days resulted in 100%, 100%, 40% and 0% lethality, respectively. In contrast, cytokine intervention reduced lethality to 45%, 20%, 0% and 0% at the same time intervals between doses. Thus, the use of cytokines permits at least a 1 week earlier redosing of 131I-IgG. Cytokine intervention also has reduced the magnitude of myelosuppression, as measured by neutropenia and thrombocytopenia, thus permitting intensification of single doses of radio-iodinated intact antibodies, bivalent fragments and 90Y-IgG by at least 30%, 50% and 25%, respectively. However, cytokines were not effective at permitting dose escalation of either 90Y-F(ab')2 or 188Re-IgG. Further optimization of the dose schedule of cytokine administration needs to be explored for these 2 nuclide-antibody forms.

CD22 antibodies (Abs) bound to B-cell lymphomas are known to be internalized and catabolized rapidly. Therefore, it would be expected that use of CD22 as a target for radioimmunotherapy should be enhanced by the use of "residualizing" radiolabels, which are trapped within the cell after catabolism of the Ab to which they had been conjugated. Our study was intended to evaluate this hypothesis using Ab LL2. In initial experiments, we found that LL2 binding was strongly temperature dependent, with approximately 15-fold greater binding at 37 degrees C than at 0 degrees C. A series of experiments suggested that this difference is due to a conformational change in the antigen at low temperature, so that the LL2 epitope is partially blocked. In vitro, residualizing labels-including 125I-dilactitol tyramine and 111In-DTPA-were retained by cells much longer than a conventional iodine label. In vivo, residualizing labels also showed a marked advantage in terms of uptake by Ramos B-cell lymphoma xenografts in nude mice. However, the absolute Ab uptake by xenografts was quite low, in comparison with results obtained with many carcinoma xenografts, which appears to be due in part to vascular properties of the B-cell lymphoma xenografts.

The hemoregulatory peptide, pGlu-Glu-Asp-Cys-Lys (pEEDCK or HP5b), has been shown to reversibly inhibit the proliferation of bone-marrow progenitor cells, and has been reported to protect mice from the myelotoxicity associated with ara-C, a chemotherapeutic agent. We undertook to use this reagent to reduce radioimmunotherapy(RAIT)-associated bone-marrow toxicity by suppressing hematopoiesis during the critical period when bone marrow is exposed to radiation. The reported studies optimize the use of HP5b to reduce the duration of neutropenia and thrombocytopenia. We found that 3.6 micrograms/day of HP5b administered through a continuous 7d mini-osmotic pump, together with a bolus dose of 3.6 micrograms 3 hours before the radioantibody dose, gave the best effect, as measured by neutrophil counts on day 28 post RAIT. With sub-lethal doses of RAIT, the period of neutropenia was reduced by 2 weeks, and there appeared to be more rapid recovery of morphologically mature myeloid cells. The peptide, however, does not appear to alleviate the lymphotoxicity associated with RAIT. No adverse effects have been noted from continuous infusions of the peptide. In the past, we reported that cytokines (IL-I/GM-CSF) are not marrow-restorative when given after RAIT. However, an additive effect is observed when HP5b infusions are combined with post-RAIT cytokine administration, suggesting that a significant pool of bone-marrow progenitor cells remains when HP5b is co-administered with RAIT. Thus, HP5b is an alternate approach to reducing myelotoxicity, and may be used in combination with cytokines to further reduce the duration of myelosuppression.

Localization of radiolabeled antibodies in the perivascular space of tumors resulted in morphological changes in blood vessel structure and physiological changes in tumor vessel function. Vessel diameter decreased by day 14 and was associated with a significant decline in vascular volume (VV). Upon recovery of VV, the basement membrane surrounding the endothelium had thickened. Tumor vascular permeability (VP) decreased within 7 days of treatment and remained suppressed throughout the 42-day observation period of our study. The decline in VP, which could be visualized by fluorescent microscopy of FITC-dextran extravasation, was dose-related and could be quantified at doses as low as 800 cGy. The radioantibody-induced 50-80% decline in tumor VP was observed in 3 human colonic xenografts (GW-39, LSI74T and MOSER). Decreases in VP have been observed for proteins ranging in size from 20 to 150 kDa. Similar effects on VP were noted when low protein doses (10-30 micrograms), which resulted in heterogeneous antibody distribution, were used or with high protein doses (400-750 micrograms), which resulted in more uniform penetration of antibody. If 188Re or 90Y, radiometals with higher beta-energy and longer path lengths, were substituted for 131I, a similar decrease in VP was observed. The radioimmunotherapy (RAIT)-induced decrease in tumor VP resulted in a 90% decline in accretion of a second dose of radioantibody. The 10% of the second dose that was taken up by the tumor targeted many already non-viable tumor regions and some, but not all, viable tumor cell clusters.

We evaluated the accretion of 131I-labeled NP-4 anticarcinoembryonic antigen (CEA) into 4 size-matched human colonic carcinomas grown s.c. in nude mice. Antibody uptake for LS174T and GW-39 tumors was relatively high (19 to 23% ID/g on day 3), whereas moderate uptake was seen in the Moser tumor (7.5% on day 3) and low uptake was detected in the GS-2 tumor (1.8% on day 3). Blood clearance of radioantibody was twice as fast in mice with GS-2 tumors than in mice with GW-39, LS174T or Moser tumors. Seven physiological parameters that might influence radioantibody accretion were evaluated in order to better understand the differences in observed tumor targeting: vascular volume, blood flow rate, vascular permeability, tumor antigen content, serum antigen content and complexation of radioantibody, intratumoral antigen distribution, and intracellular antigen distribution. Although marked variability in vascular physiology, antigen content and antibody complexation of the 4 tumors grown in the same host and site existed, it was insufficient to explain the differences in antibody uptake. However, intra-tumoral distribution of antigen, and sub-cellular accessibility of antigen for radioantibody were important considerations. GS-2 tumors are well differentiated and have polarized cells. CEA in GS-2 is largely inaccessible to radioantibody; most of the antigen is located in the lumen of the glands or on the apical surface of gland cells and most of the antibody distributes to the stromal region on the basolateral surface. The low antibody targeting in GS-2 could therefore be explained by restricted intra-tumor accessibility of antibody. Scatchard analysis of NP-4 binding to Moser cells under non-internalizing and internalizing conditions revealed that 90% of the antigen is found within the cell, unavailable to bind with the NP-4 antibody, which is slow to internalize. In contrast, CEA in LS174T cells was almost entirely accessible. The reduced antibody targeting to Moser xenografts might therefore, be explained by restricted antibody accessibility at the cellular level.

A syngeneic anti-idiotype monoclonal antibody (MAb) (CM-11) directed against an anti-carcinoembryonic antigen (CEA) murine MAb (NP-4) was evaluated as a second antibody (SA) to promote the rapid clearance of radiolabeled NP-4 from the blood. Initial studies confirmed that CM-11 IgG removed 131I-NP-4 IgG from the blood as effectively as a polyclonal donkey anti-goat IgG removed 131I-goat IgG. However, use of an F(ab')2 in place of either the NP-4 or CM-11 IgG was not as effective in removing primary radiolabeled antibody, despite the formation of high-molecular-weight complexes. In accordance with previous results, the timing and dose of the SA injection was critical for optimizing tumor uptake and improving tumor/non-tumor ratios. In nude mice bearing GW-39 human colonic tumor xenografts, a delay in the injection of CM-11 by 48 hr after injection of radiolabeled NP-4 was optimal, since this allowed maximum tumor accretion. At a 200:1 CM-11:NP-4 ratio, tumor uptake was reduced, suggesting inhibition of NP-4 binding to CEA within the tumor. Despite optimizing tumor uptake by delaying SA injection and adjusting its dose, the percentage of 131I-NP-4 in the tumor decreased 2- to 3-fold within 2 days after CM-11 injection. A similar effect was seen for 111In-labeled NP-4 IgG with CM-11. Injection of excess unlabeled NP-4 given to block CM-11 shortly after its injection failed to curtail the loss of NP-4 from the tumor. Our results suggest that high blood levels of MAb are important for sustaining NP-4 in the tumor. Radiation-dose predictions derived from biodistribution studies indicate that a higher tumor dose may be delivered using the SA method than with either 131I-NP-4 IgG or F(ab')2 alone. Use of the SA method with 90Y-labeled NP-4 IgG, as modeled from biodistribution studies with 111In-NP-4 IgG, would likely be limited by liver toxicity.

5-fluorouridine (FUR), an antineoplastic agent, was site-specifically conjugated to the carbohydrate moiety of a anticarcinoembryonic antigen (CEA) monoclonal antibody (MAb) by using amino-dextran as the intermediate carrier. The final immunoconjugate contains approximately 30-35 molecules of FUR per molecule of immunoglobulin, has immunoreactivity retained as examined by flow cytometry, and is cytotoxic to the target cells as examined by 75selenomethionine incorporation studies. In the GW-39/nude mouse model, the conjugate remained efficient in targeting the human colonic tumor and possessed greater inhibitory growth effects on the subcutaneous tumor than free FUR or an irrelevant antibody conjugate. In addition, the reduced host toxicity of the conjugate may permit the use of this agent in a high-dose therapy of this tumor system.

Our previous studies with a 90Y-labelled antibody against carcinoembryonic antigen (CEA) conjugated to the cyclic anhydride-DTPA (CA-DTPA) indicated that the accretion of 90Y in the bone may limit the application of 90Y-labelled antibodies for therapy. In this report, we have compared the tumor targeting of CA-DTPA-conjugated antibody to antibody conjugated with 4 isothiocyanatobenzyl (ITC-Bz) derivatives of DTPA in nude mice bearing a human colonic tumor xenograft. In biodistribution studies using an 111In-labelled anti-CEA murine monoclonal antibody (MAb), the CA-DTPA-conjugated MAb showed lower tumor uptake, faster blood clearance, and higher accretion in the liver than any of the 4 ITC-Bz-DTPA-conjugated MAbs. There were smaller differences among the 4 ITC-Bz-DTPA conjugates. Whole-body autoradiography of animals given 90Y-MAb prepared with the CA-DTPA or the ITC-Bz-DTPA showed less radioactivity in the bone with the ITC-Bz-DTPA-MAb than the CA-DTPA-MAb. 90Y uptake in the bone corresponded with regions of low proliferative activity as defined by 3H-labelled thymidine, suggesting that the 90Y was in the cortex rather than the marrow. These studies clearly show an advantage of the ITC-Bz-DTPA derivatives for 90Y and 111In labelling of MAbs.

This study investigates the influence of antibody protein dose on the radioimmunodetection of a CEA-producing, human colonic tumor xenograft (GW-39) using affinity-purified goat anti-carcinoembryonic antigen (CEA) antibody and a murine monoclonal anti-CEA antibody (NP-2). Hamsters bearing GW-39 tumors were given an equal mixture of 131I-labelled antibody and 125I-labelled irrelevant IgG at doses varying from 0.01 to 1.0 mg (0.1 to 10 mg/kg body weight) for each antibody preparation. No differences were found in the percentage of antibody in the tumor or the clearance rate from the blood as the antibody dose was increased. The concentration of antibody in the tumor increased proportionally with the antibody dose and maintained a linear relationship with increasing dose, indicating that antigenic sites in the tumor were not saturated with antibody. The concentration of irrelevant IgG in the tumor also increased proportionally as the dose was increased, but the concentration of irrelevant IgG in the tumor was less than the antibody and was removed more rapidly from the tumor than the antibody. By considering the amount of irrelevant IgG in the tumor as a measure of the amount of antibody non-specifically bound, we determined that there was no change in the amount of antibody in the tumor between days 3 and 7, and the amount of antibody increased uniformly for both the tumor and non-tumor tissues, resulting in no improvement in the tumor/non-tumor ratios with increasing antibody dose. External scintigraphy verified that the dose of the antibody did not influence tumor imaging. Thus, tumor accretion of antibody in this model is not dependent principally on the antibody protein dose, and other factors such as accessibility to antigen and antibody metabolism may play important roles in regulating the uptake of antibody in tumor.

The in vivo uptake, therapeutic potential, and host toxicity were evaluated for both the intact IgG and F(ab')2 fragment of 2 murine monoclonal antibodies (MAbs) directed against either carcino-embryonic antigen (CEA), called NP-4, or colon-specific antigen-p (CSAp), called Mu-9, in the GW-39 human colorectal carcinoma grown in the hamster cheek pouch. Mu-9 IgG and F(ab')2 were retained longer by the tumor than was the NP-4 IgG or F(ab')2, respectively. Localization of the two antibodies by micro-autoradiography revealed two distinct patterns. Mu-9 was seen densely around whole acini of tumor cells, whereas NP-4 was found around each individual cell, albeit less densely. The anti-tumor effects of 131I-labelled Mu-9 and NP-4 IgG were equal, but the therapeutic effectiveness of Mu-9 F(ab')2 was significantly higher than NP-4 F(ab')2, as measured by change in tumor size. A dose-dependent increase in host toxicity, as measured by change in body weight and change in peripheral white blood cells (p-WBCs), was observed with 0.5 to 3.0 mCi doses of 131I-IgG regardless of the MAb used. A 10-22% loss in body weight lasting 3-7 weeks and a 50-80% loss in pWBCs lasting 6-8 weeks were observed in these animals. In contrast, 3 x 2 mCi doses of 131I-NP-4 or Mu-9 F(ab')2 given at 3-day intervals, a schedule which was equally therapeutic to a single 2-mCi dose of 131I-IgG, resulted in only a 9% loss in body weight and a 50% loss in pWBCs that lasted only 1 week. This was followed by a complete recovery over the next 2-3 weeks. These data suggest that multiple doses of F(ab')2 can be as tumoricidal as a single dose of an intact MAb IgG, but significantly less toxic to the host.

The site-specific conjugation of methotrexate, 4-amino-N10-methylpteroylglutamic acid, to a monoclonal anti-carcinoembryonic antigen (CEA) antibody, using an intermediate amino-dextran carrier system, resulted in a ratio of 30-50 molecules of MTX per molecule of immunoglobulin. The immunoreactivity of the conjugate was analyzed using flow cytometry or a competitive binding assay, which showed that the conjugate has significant retention of the antigen-binding activity. The pharmacokinetic behavior of the immunoconjugate in BALB/c mice and tumor localization in hamsters indicated that the conjugate remained in the circulation with higher concentration than free antibody, and could recognize the tumor as efficiently as the unconjugated antibody. The high degree of drug incorporation with retained immunoreactivity makes this method preferable to direct antibody conjugation.