Comparison of therapeutic efficacy and host toxicity of two different 131I-labelled antibodies and their fragments in the GW-39 colonic cancer xenograft model

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 presence of a concomitant bulky tumor can decrease the uptake and therapeutic efficacy of radiolabeled antibodies in small tumors.

We describe the effects that a concomitant large tumor mass can exert on the therapeutic efficacy of radioimmunotherapy against small tumors, using the nude mouse/GW-39 human colonic cancer model. The tumor uptake 7 days p.i. of i.v.-injected 131I-labeled anti-CEA MAb (NP-4) and anti-CSAp MAb (Mu-9) in small (less than 0.2 g) s.c. GW-39 tumors was approximately 3-fold lower in animals with a concomitant large (greater than 1.0 g) GW-39 tumor than in the absence of a large tumor. An inverse correlation between the mass of the tumor burden and the 131I levels in the blood was observed, indicating that a large tumor mass may act as a sink for the injected radiolabeled antibody. Increasing antibody protein dose did not reverse the reduced uptake in the small s.c. tumors. The therapeutic efficacy of a single 0.25-mCi injection of 131I-labeled anti-CSAp MAb Mu-9 towards intrapulmonary GW-39 metastases was tested in nude mice bearing either small or large GW-39 s.c. tumors. Over 80% of the animals with small s.c. GW-39 tumors survived 18 weeks after tumor transplantation, whereas less than 20% of the animals bearing large s.c. tumors survived past 13 weeks. Dosimetric calculations, based on biodistribution data over time, indicated that the presence of a large s.c. tumor mass may have decreased the radiation dose to the intrapulmonary tumors almost two-fold. However, the radiation dose to the blood was also decreased in the animals with the large tumor burden. Therefore, the animals with larger tumor burden may also have been able to sustain higher doses of the radioantibody. The presence of a large tumor mass can thus affect the biodistribution and therapeutic efficacy of radioiodinated antibodies. We suggest that bulky tumors can adsorb a considerable amount of the injected dose, thereby reducing the total amount of MAb available for binding to the smaller tumors.     

Successful radioimmunotherapy for lung metastasis of human colonic cancer in nude mice.

The potential for radioimmunotherapy as an adjuvant treatment for early disseminated colonic cancer was investigated in an experimental lung metastasis model. Nude mice receiving intravenous injection with a suspension of human colonic cancer cells (GW-39) developed multiple (10-100) tumor nodules throughout the lungs, and more than 50% of the animals died of extensive tumor involvement within 5-10 weeks. Groups of eight or nine animals bearing 7-day-old tumor transplants were treated with a single intravenous injection of radioiodinated agents: either 0.15 or 0.30 mCi of whole IgG of the NP-4 murine monoclonal antibody (MAb) against carcinoembryonic antigen (CEA) or 0.15 or 0.30 mCi of whole IgG of Immu-31, an anti-alpha-fetoprotein (anti-AFP) MAb. Treatment of animals with 0.15 or 0.30 mCi of 131I-labeled NP-4 IgG 7 days after injection of tumor cells resulted in survival for 23 weeks after tumor implantation in four of eight and seven of nine animals, respectively. Microscopic examination revealed that over 90% of the lung tumor colonies had no evidence of surviving cells. Animals treated with 0.30 mCi of anti-AFP, an irrelevant MAb, survived 4 weeks longer than controls. Toxicity was evident in four of the 17 animals given 0.30 mCi of NP-4 IgG (specific) or anti-AFP IgG (irrelevant) MAb. These animals died within 1-3 weeks after radioantibody injection, suggesting that death was related to the radiation dose. None of the animals given 0.15 mCi of 131I-MAb died within this period.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enhanced clearance of radiolabeled murine monoclonal antibody by a syngeneic anti-idiotype antibody in tumor-bearing nude mice.

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.     

Comparison of tumor targeting of mouse monoclonal and goat polyclonal antibodies to carcinoembryonic antigen in the GW-39 human tumor-hamster host model

We have evaluated 4 radioiodinated mouse monoclonal anticarcinoembryonic antigen antibodies (MAbs) by using the GW-39 human colorectal tumor xenograft transplanted i.m. in immunocompetent hamsters to determine whether there were any differences in their tumor localization properties. Additional comparisons were made to affinity-purified goat anticarcinoembryonic antigen antibody. Statistically significant differences were found in the percentage/g of tumor uptake and tumor/nontumor ratios among the antibodies, so that the antibodies could be ranked according to their tumor localization properties (NP-2 greater than NP-4 = goat antibody greater than NP-1 greater than NP-3). Although statistical differences were found, tumor/nontumor values generally were not distinguished by a factor of more than 1.5, suggesting that these differences may not be biologically significant. F(ab')2 fragments of NP-2 were found to be superior to NP-4 F(ab')2 fragments, giving tumor/liver and tumor/blood ratios of 16 and 11.5, respectively, within 3 days, in comparison to 5.4 and 3.8 for NP-4 F(ab')2 fragments. Mixtures of all of the MAbs or a mixture of NP-2 and NP-4 did not improve tumor localization, in comparison to NP-2 alone. These studies suggest that mixtures of these anticarcinoembryonic antigen MAbs may not afford better tumor imaging than the use of a certain single antitumor MAb.     

Radiolabeled monoclonal antibody 15 and its fragments for localization and imaging of xenografts of human lung cancer

Monoclonal antibody (MAb) 15 and its F(ab')2 and Fab fragments were radioiodinated, and their biodistribution and imaging were compared in BALB/c nude mice bearing a xenograft of a human lung cancer (TKB-2). Association constants for 125I-labeled MAb 15 IgG, F(ab')2, and Fab were 1.9 X 10(9), 1.8 X 10(9), and 3.7 X 10(8) M-1, respectively. Immunoreactive fractions ranged from 0.59 to 0.50. Cultured TKB-2 cells expressed 1.1 X 10(4) binding sites/cell for MAb 15 IgG in vitro. The binding of a control antibody and the binding of its fragments to TKB-2 cells were less than 3% of the input doses. The mice with the TKB-2 tumors were given simultaneous injections of 10 microCi of 131I-labeled MAb 15 or its fragments and 10 microCi of 125I-labeled control IgG or its fragments. With MAb 15 IgG, the percentage of the injected dose bound per gram of tissue (ID/g) of the tumor was 3.68% at day 7, when the localization index (LI) was 4.38. At day 2 after MAb 15 F(ab')2 injection, 1.12% of the ID/g was localized in the tumor and the LI was 3.04. After MAb 15 Fab injection, the percentage of the ID/g of the tumor was 0.31% and the LI was 2.58 at day 1. MAb 15 IgG, F(ab')2, and Fab cleared from the blood early, with a half-life of 33, 16, and 9 hours, respectively. The distributions of MAb 15 and its fragments in the normal organs did not differ from those of the control. Radioimaging with 100 microCi of 131I-labeled MAb 15 and its fragments showed that 42%, 44%, and 32% of the total-body count were localized in the tumor with IgG at day 7, F(ab')2 at day 2, or Fab at day 1, respectively. Because the radioactivity remaining in the tumor with Fab was low, the image was insufficient. Throughout the period, less than 10% of the control IgG and its fragments remained in the tumor. Microautoradiography confirmed the binding of MAb 15 and its fragments to the tumor cells. In this study the F(ab')2 was the best compromise between the slowly cleared IgG and the poorly localized Fab in tumor imaging.     

An idiotypic replica of carcinoembryonic antigen inducing cellular and humoral responses directed against human colorectal tumours.

A monoclonal anti-idiotypic antibody (anti-Id MAb) 708 (IgG2b), which inhibited the binding of NCRC23 (IgG1) MAb to CEA and prevented radiolabelled CEA from binding to MAb NCRC23, was produced. No recognition of 3 other anti-CEA antibodies, 3 other IgG1 or 2 IgM MAbs was observed with this anti-idiotypic antibody. When an immunoblotting technique was used, 708 anti-Id MAb failed to bind to isolated heavy or light chains of MAb NCRC23, whereas binding was observed with intact antibody. Mouse, rat and human lymphocytes (in vitro) were immunized with 708 anti-Id MAb and the resultant Ab3 antibodies all inhibited binding of labelled 708 anti-Id MAb to MAb NCRC23 and also reacted with CEA, showing that 708 anti-Id MAb induced anti-CEA antibody responses. Similarly, mice immunized with 708 anti-Id MAb could be restimulated in vitro with either CEA or tumour cells expressing CEA which induced specific T-cell proliferative responses. Human tumour-infiltrating lymphocytes isolated from colorectal tumours or peripheral blood T cells from cancer patients were stimulated in vitro with 708 anti-Id MAb or an irrelevant IgG2b antibody. Six days later both sets of lymphocytes were restimulated with CEA, and lymphocytes primed to 708 anti-Id MAb proliferated in response to CEA. These results suggest that 708 anti-Id MAb can act as an idiotypic replica of CEA and stimulate cellular and humoral anti-CEA immune responses. It is therefore of great interest as an idiotypic vaccine against colorectal cancer.     

Comparative localization of murine monoclonal antibody Me1-14 F(ab')2 fragment and whole IgG2a in human glioma xenografts

Monoclonal antibodies (MAbs) targeted to glioma-associated antigens may allow the selective delivery of imaging and therapeutic agents to brain tumors; the use of MAb fragments may be a strategy to further improve tumor uptake of such agents relative to normal tissues. In this study, we have examined the in vivo localization of radioiodinated MAb Me1-14, a murine immunoglobulin G2a (IgG2a) reactive with gliomas, and its F(ab')2 fragment in s.c. and intracranial xenografts of human glioma cell line D-54 MG in athymic mice. The radiolabeled F(ab')2 fragment of Me1-14 was demonstrated to possess in vitro binding affinity and immunoreactivity comparable to that of whole IgG. Direct comparison of IgG and F(ab')2 biodistribution in s.c. xenograft-bearing mice showed higher tumor: normal tissue ratios of the F(ab')2 fragment compared to IgG. In intracranial tumor-bearing mice paired-label analysis using a nonspecific protein control showed earlier specific tumor localization by the F(ab')2 fragment of Me1-14 compared to IgG. Blood-to-tumor transfer constants (K1) derived for Me1-14 F(ab')2 were significantly greater than those for whole Me1-14 (P = 0.01). Estimated radiation dosimetry revealed that 131I-labeled Me1-14 F(ab')2 would deliver higher radiation doses to tumor than to normal tissues. These studies demonstrate that the F(ab')2 fragment of Me1-14 may be a potential agent for immune-directed brain tumor diagnosis and therapy.     

Biodistribution of indium-111-labeled OC 125 monoclonal antibody intraperitoneally injected into patients operated on for ovarian carcinomas

The biodistribution of 111In-labeled monoclonal antibody (MAb) OC 125 was studied after i.p. injection in 28 patients who underwent surgery for ovarian carcinoma. Group I (eight patients) received intact 111In-labeled OC 125 MAb, Group II (three patients) intact 111In-labeled irrelevant NS, Group III (five patients) intact 111In-labeled OC 125 MAb associated with 20 mg of the same unlabeled MAb and Group IV (12 patients) F(ab')2 fragments of 111In-labeled OC 125 MAb. The patients were operated on 1 to 3 days after i.p. injection, and the surgeon removed large tumor fragments and/or small tumor nodules and, in some patients, collected the residual perfusion fluid from which malignant cell clusters were isolated. Uptake by large tumor fragments at 24 h was low: 0.0031 +/- 0.0032% injected dose per gram (%ID/g) for Group I and 0.0024 +/- 0.0022%ID/g for Group IV. It was moderately higher than that of Group II (0.0014 +/- 0.0006%ID/g) and Group III (0.0015 +/- 0.0007%ID/g). Uptake by small tumor nodules (0.1302 +/- 0.0802%ID/g at 72 h for Group I) and malignant cell clusters (median: 0.3322, with a maximum value of 4.1614%ID/g at 24 h for Group IV) was markedly higher. Tumor-to-normal tissue ratios with OC 125 MAb [intact or F(ab')2 fragments] ranged between 0.1 and 8.5 for large tumor fragments and 2 and 8,700 for small tumor nodules and malignant cell clusters. It would thus appear that RIT is feasible if an appropriate radionuclide can be selected for antibody labeling.     

Antiidiotype antibodies in cancer patients receiving monoclonal antibody to carcinoembryonic antigen

The initial 10 patients of a Phase I clinical trial involving multiple injections of murine monoclonal anti-carcinoembryonic antigen (CEA) antibody, NP-2, were studied for the presence in their sera of antiidiotypic antibody. Most patients had advanced gastrointestinal adenocarcinoma and received 1 mg/m2 monoclonal antibody three times weekly, or once a week, resulting in five to 13 injections over 12 to 240 days. Antiidiotype antibody was detected with a blocking radioimmunoassay using [125I]NP-2-F(ab')2 binding to CEA-coated microwells and [125I]NP-4-F(ab')2 as a control antibody. Five out of 10 patients demonstrated 65-96% inhibition of NP-2 binding at 1/20 dilution of serum compared to NP-2 binding in the presence of pretreatment sera. The inhibitory activity was preserved after adsorption over a polyclonal mouse IgG immunoadsorbent whereas exposure to a NP-2 affinity column completely depleted the activity. Specificity testing, including the blocking effect of patient sera on the control antibody NP-4, and interference by the possible presence of circulating NP-2, circulating CEA, and human anti-CEA activity, confirmed that the inhibition observed was specific for NP-2 and was caused by an agent with CEA-like characteristics. Longitudinal studies demonstrated that elevated titers of antiidiotypic antibody appeared later in the course of immunization than did antibody against mouse immunoglobulin. These studies indicate that patients can be sensitized to the idiotype (anti-combining site and/or combining site-related) of monoclonal antibodies to CEA following multiple infusions.     

Selective tumor localization of radiolabeled anti-human melanoma monoclonal antibody fragment demonstrated in the nude mouse model

Monoclonal antibodies (Mab) directed against distinct epitopes of the human 240 kD melanoma-associated antigen have been evaluated for their capacity to localize in human melanoma grafted into nude mice. A favorable tumor to normal tissue ratio of 13 was obtained with intact 131I-labeled MAb Me1-14. This ratio was further increased to 43 and 23 by the use of F(ab')2 and Fab fragments, respectively. The specificity of tumor localization was demonstrated by the simultaneous injection of F(ab')2 fragments from MAb Me1-14 and anti-CEA MAb 35, each labeled with a different iodine isotope, into nude mice grafted with a melanoma and colon carcinoma. The fragments from both MAb localized with perfect selectivity in their relevant tumor as shown by differential whole body scanning and by direct measurement of the two isotopes in tumors and normal tissues. These in vivo experimental results suggest that the F(ab')2 fragment from MAb Me1-14 is suitable for melanoma detection by immunoscintigraphy in patients.     

Radioimmunotherapy of human colon carcinoma by 131I-labelled monoclonal anti-CEA antibodies in a nude mouse model

A mixture of 3 MAbs directed against 3 different CEA epitopes was radiolabelled with 131I and used for the treatment of a human colon carcinoma transplanted s.c. into nude mice. Intact MAbs and F(ab')2 fragments were mixed because it had been shown by autoradiography that these 2 antibody forms can penetrate into different areas of the tumor nodule. Ten days after transplantation of colon tumor T380 a single dose of 600 microCi of 131I MAbs was injected i.v. The tumor grafts were well established (as evidenced by exponential growth in untreated mice) and their size continued to increase up to 6 days after radiolabelled antibody injection. Tumor shrinking was then observed lasting for 4-12 weeks. In a control group injected with 600 microCi of 131I coupled to irrelevant monoclonal IgG, tumor growth was delayed, but no regression was observed. Tumors of mice injected with the corresponding amount of unlabelled antibodies grew like those of untreated mice. Based on measurements of the effective whole-body half-life of injected 131I, the mean radiation dose received by the animals was calculated to be 382 rads for the antibody group and 478 rads for the normal IgG controls. The genetically immunodeficient animals exhibited no increase in mortality, and only limited bone-marrow toxicity was observed. Direct measurement of radioactivity in mice dissected 1, 3 and 7 days after 131I-MAb injection showed that 25, 7.2 and 2.2% of injected dose were recovered per gram of tumor, the mean radiation dose delivered to the tumor being thus more than 5,000 rads. These experiments show that therapeutic doses of radioactivity can be selectively directed to human colon carcinoma by i.v. injection of 131I-labelled anti-CEA MAbs.     

Polyclonal and monoclonal anti-CEA antibodies in immunolocalization of colorectal cancer

Antibodies to carcinoembryonic antigen (CEA) from sheep and monkey were immunoadsorbent purified. Mouse monoclonal antibody (MAb) anti-CEA I-38S1 and Fab fragments of this antibody were prepared from mouse ascitic fluid. The IgG preparations were labelled with 123I or 131I, the Fab fragments with 131I. The antibody reactivity was unchanged after labelling. Patients with advanced colorectal carcinomas received an intravenous injection of 50-200 MBq 123I or 30-160 MBq 131I coupled to 250-500 micrograms antibody or antibody fragment. Patient examinations were performed using emission tomography (SPECT) and/or conventional gamma camera scintigraphy. The specific localization of labelled anti-CEA to tumor was compared to known tumor localized by CAT-scan, other x-ray methods or laparotomy, 50% of known tumors were accurately localized with sheep anti-CEA. In contrast, 70-80% of known tumor sites were correctly localized with polyclonal monkey anti-CEA antibodies, with monoclonal anti-CEA antibodies or with Fab fragments of the latter. A few previously unknown tumors were detected.     

Selective radiation of hematolymphoid tissue delivered by anti-CD45 antibody.

The ability to deliver radiation selectively to lymphohematopoietic tissues may have utility in conditions treated by myeloablative regimens followed by bone marrow transplantation. Since the CD45 antigen is the most broadly expressed of hematopoietic antigens, we examined the biodistribution of radiolabeled anti-CD45 monoclonal antibodies in normal mice. Trace 125I or 131I-labeled monoclonal antibodies 30G12 (rat IgG2a), 30F11 (rat IgG2b), and F(ab')2 fragments of 30F11 were injected i.v. at doses of 5 to 1000 micrograms. For both intact antibodies, a higher percentage of injected dose/g (% ID/g tissue) in blood was achieved with higher antibody doses. However, as the dose of antibody was increased, the % ID/g in the target organs of spleen, marrow, and lymph nodes decreased. At doses between 5 and 10-micrograms, % ID/g in these tissues exceeded that in lung, the normal organ with the highest concentration of radiolabel. In contrast, thymus was the only hematopoietic organ in which the % ID/g increased with increasing antibody dose, although at high dose the % ID/g was still far below that achieved in the other hematopoietic organs. Antibody 30F11 F(ab')2 fragments were cleared more quickly than intact antibody from blood and from both target and nontarget organs, although the relationship between increasing antibody dose and decreasing % ID/g in spleen, marrow, and lymph nodes was observed. The time-activity curves for each dose of antibody were used to calculate estimates of radiation absorbed dose to each organ. At the 10-micrograms dose of 30G12, the spleen was estimated to receive a radiation dose that was 13 times more than lung, the lymph nodes 3 to 4 times more, and the bone marrow 3 times more than lung. For each antibody fragment dose, the radiation absorbed dose per MBq 131I administered was lower because the residence times of the fragments were shorter than those of the intact antibody. Thus these estimates suggested that the best "therapeutic ratio" of radiation delivered to target organ as compared to lung was achieved with lower doses of intact antibody. We have demonstrated that radiolabeled anti-CD45 monoclonal antibodies can deliver radiation to lymphohematopoietic tissues with relative selectivity and that the relative uptake and retention in different hematolymphoid tissues change with increasing antibody dose.     

Radioimmunotherapy of human colonic cancer xenografts with 90Y labeled monoclonal antibodies to carcinoembryonic antigen

Monoclonal antibodies (MAbs) to carcinoembryonic antigen (CEA) or alpha-fetoprotein (AFP) were conjugated with diethylenetriaminepentaacetic acid and radiolabeled with 90Y at a specific activity of 4.0-6.0 mCi/mg. Approximately 50% of the radiolabeled anti-CEA antibody (90Y-labeled NP-2) bound to an immunoadsorbent containing CEA while analysis by high performance liquid chromatography revealed that 95-98% of the 90Y was associated with immunoglobulin. Less than 5% of the 90Y dissociated from either MAb after incubation in plasma for 48 h at 37 degrees C. After injection into nude mice, 98% of the circulating radioactivity remained associated with antibody and no loss of immunoreactivity was observed at 3 days. To evaluate 90Y-labeled NP-2 as a therapeutic agent, varied doses (10-100 microCi) were administered as a single i.v. injection into groups of nude mice bearing s.c. implants (0.3-0.4 g) of a CEA-producing human colonic cancer xenograft, GW-39. At the 10-microCi dose, no inhibition of tumor growth was observed. After 28 days, tumor growth was inhibited by as much as 77% in mice treated with 50 microCi of 90Y-labeled NP-2 as compared to tumor growth in control animals given 90Y-labeled anti-AFP. Doses higher than 50 microCi (75 and 100 microCi) were toxic to most of the animals, killing them within 2-3 weeks after administration. Marked suppression of circulating leukocytes was observed with 20 and 50 microCi by 1-2 weeks postinjection, but they returned to normal levels 3-4 weeks later. These studies show that treatment with 90Y-labeled MAbs against CEA can produce significant antitumor effects. However, toxicity to the bone marrow may limit the therapeutic efficacy of systemically administered 90Y-labeled MAbs.     

Tumor targeting with radiolabeled antibodies in a human carcinoembryonic antigen transgenic mouse model

Mice transgenic for the carcinoembryonic (CEA) gene were used to study the biodistribution and tumor targeting of a radioiodinated monoclonal antibody (MAb), T84.66. The specificity of antibody uptake in tumors was assessed in mice bearing a CEA-transfected syngeneic tumor as well as the antigen-negative parental tumor. With high CEA-expressing tumors, the percent injected dose per gram (%ID/g) approached 30% at 48 hr. Tumor uptake in antigen-positive tumors was 5-8-fold higher than that observed in the antigen-negative parental tumors. Only antigen-positive tumors were visualized by immunoscintigraphy. The tumor targeting obtained in athymic nude mice bearing human tumor xenografts was similar to that observed with CEA-expressing murine tumors implanted in either athymic nude or transgenic mice. The degree of localization of CEA-transfected murine tumors was related with the level of antigen expression. Circulating antigen-radio-antibody complexes were not detected while blood clearance of radio-antibody was similar between transgenic and non-transgenic mice. With the exception of the large bowel, the distribution of radioiodinated MAb in normal tissues was similar in both CEA transgenic and non-transgenic mice. Increased localization of intact antibody was observed in the large bowel from transgenic mice, suggesting specific targeting to antigen-positive normal tissues. These results suggest that the CEA transgenic mouse model will be useful in the development of antibodies for radio-immunodetection and treatment of carcinomas expressing CEA.     

Antibody protein dose and radioimmunodetection of GW-39 human colon tumor xenografts

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.     

Distinguishing malignant mesothelioma from pulmonary adenocarcinoma: an immuno-histochemical approach using a panel of monoclonal antibodies

A panel of six monoclonal antibodies (MAbs) was employed to evaluate antigen expression in pulmonary adenocarcinomas and mesotheliomas. Monoclonal anti-human milk fat globulin (HMFG-2), anti-carcinoembryonic antigen (NP-2), anti-epithelial membrane antigen (EMA), anti-cytokeratin (PKK-1), anti-tumor-associated antigen 72 (B72.3), and anti-human myelomonocytic antigen (Leu M-1) antibodies were used to localize their respective antigens in formalin-fixed, paraffin-embedded tumors by using the avidin-biotin-complex immunoperoxidase technique. In all, 28 mesotheliomas obtained from Ohio State University Anatomic Pathology files and from a Southwest Oncology Group (SWOG) protocol were compared to 22 pulmonary adenocarcinomas by using this MAb panel. None of the mesotheliomas demonstrated positive staining with MAbs NP-2 (anti-CEA) or Leu M-1. However, 95% (21/22) of adenocarcinomas stained with one of these two antibodies. Although neither of these two MAbs stained all adenocarcinomas, each antibody demonstrated positive immunostaining in more than 90% of the adenocarcinomas studied. Therefore, MABs NP-2 and Leu M-1 are, individually, quite useful for distinguishing mesothelioma from adenocarcinoma. However, in our study, no single MAb could be used to distinguish these two tumor types in every case. MAb B72.3 stained 91% (20/21) adenocarcinomas but also stained 7% (2/28) of mesotheliomas. MAb HMFG-2 reacted positively with 95% of adenocarcinomas, but also stained 39% of the mesotheliomas, usually in a membranous pattern. MAbs EMA and PKK-1 were not found useful in distinguishing mesothelioma from adenocarcinoma. We conclude that MAbs Leu M-1 and NP-2 were both useful in distinguishing mesothelioma from pulmonary adenocarcinoma in that positive staining was demonstrated in adenocarcinomas and not mesotheliomas.     

Selection of radioimmunoconjugates for the therapy of well-established or micrometastatic colon carcinoma

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. Int. J. Cancer 72:477–485, 1997. © 1997 Wiley-Liss, Inc.     

Murine monoclonal antibodies raised against human non-small cell carcinoma of the lung: specificity and tumor targeting

The tumor targeting properties of murine monoclonal antibodies (MAbs) generated in our laboratory against non-small cell carcinoma of the lung have been investigated in nude mouse xenograft models. The MAbs selected for evaluation, RS5-4H6, RS7-3G11, and R511-51, have pancarcinoma reactivity, as shown by immunoperoxidase staining of the majority of tumors from the lung as well as breast, colon, kidney, and ovary. The localization of the three MAbs which bind to distinct antigens, and exhibit different levels of cross-reactivity with normal human epithelial tissues, are compared. The MAbs are of the IgG1 isotype. Since these MAbs were reactive with Calu-3, a human adenocarcinoma of the lung cell line grown as xenografts in nude mice, this system was selected as our initial tumor target. The MAbs were found to localize preferentially to the heterotransplanted tumors, with from 6.6 to 8.6% of the injected dose per gram accreting in the tumor at 7 days. Tumor/nontumor ratios of up to 9.7 were seen with one MAb at day 14. The targeting of MAb RS11-51 and F(ab')2 fragments of RS11-51 in GW-39, a human colon cancer grown in nude mice, was also studied. Accretion of intact RS11-51 and F(ab')2 fragments into GW-39 was greatly increased compared to Calu-3. In view of the high frequency of antigen expression on a wide variety of tumors, and the ability to target in vivo, these new MAbs may have potential use in the imaging and therapy of cancer.