Development and Characterization of Chimeric Anti-carcinoembryonic Antigen Monoclonal Antibodies and Their Fab Fragments

In an attempt to reduce the immunogenicity of two different murine anti-carcinoembryonic antigen (CEA) monoclonal antibodies (MAbs), KM10 and A10, we produced recombinant mouse/human chimeric MAbs and the respective Fab fragments carrying the variable regions of the murine MAbs. Chimeric A10 Fab fragment was expressed in Escherichia coli , and produced in large quantities in a mini-jar fermentation system. In competitive binding assays, chimeric MAbs and their Fab fragments showed identical specificity to human CEA epitopes, as compared to the parental MAbs or Fab fragments. Both chimeric Fab fragments exhibited strong immunohistochemical reactivity with various gastrointestinal carcinomas and no reactivity with CEA-related antigens, such as NCA (nonspecific cross-reacting antigen) or BGPI (biliary glycoprotein I). Furthermore, chimeric KM10 MAb elicited substantially higher antibody-dependent cellular cytotoxicity than the murine MAb. Complement-dependent cytotoxicity in vitro was much weaker with chimeric KM10 MAb. These results indicate that chimeric MAbs or Fab fragments could potentially replace the parental murine antibodies or their Fab fragments in therapy or diagnosis of human gastrointestinal carcinomas.     

Comparison of the pharmacokinetics, biodistribution and dosimetry of monoclonal antibodies OC125, OV-TL 3, and 139H2 as IgG and F(ab')2 fragments in experimental ovarian cancer.

Monoclonal antibody (MAb) 139H2 was previously shown to localise specifically into ovarian cancer xenografts in nude mice. MAb 139H2 was compared with MAbs OC125 and OV-TL 3, all reactive with ovarian carcinomas, for the binding characteristics as IgG and F(ab')2 fragments with the use of the OVCAR-3 cell line grown in vitro and as s.c. xenografts. Immunoperoxidase staining of OVCAR-3 tissue sections with MAbs OC125 and 139H2 was heterogeneous, whereas MAb OV-TL 3 showed homogeneity. No differences in binding were observed between IgG and F(ab')2. The avidity expressed as apparent affinity constants of MAbs OC125, OV-TL 3 and 139H2 for OVAR-3 cells were 1 x 10(9) M-1, 1 x 10(9) M-1, and 1 x 10(8) M-1, while the number of antigenic determinants were 5 x 10(6), 1 x 10(6) and 7 x 10(6), respectively. In OVCAR-3 bearing nude mice the blood half-lives of the MAbs as IgG and F(ab')2 were approximately 50 h and 6 h, respectively. Maximum tumour uptake for the whole MAbs OC125, OV-TL 3, 139H2 and a control MAb 2C7 was 8.5%, 17.7%, 11.1% and 2.5% of the injected dose g-1, reached at 72 h after injection. For the respective F(ab')2 fragments, the maximum values were 5.2%, 10.0%, 5.5% and 1.9% of the injected dose g-1, reached between 6 h and 15 h. Tumour to non-tumour ratios were more favourable for the F(ab')2 fragments as compared to those for MAbs as IgG. Biodistribution in mice bearing a control tumour confirmed the specificity of tumour localisation of MAbs OC125, OV-TL 3 and 139H2. After injection of a tracer dose of 10 microCi of radiolabelled MAbs OC125, OV-TL 3 and 139H2 as IgG, tumours received 38 cGy, and 9 cGy. In our OVCAR-3 model, a ranking in efficiency in tumour localisation would indicate MAb OV-TL 3 as most favourable MAb, but cross-reactivity with subpopulations of human white blood cells might hamper its clinical use. Dosimetric data indicate a 4-fold higher radiation absorbed dose to tumours for IgG compared with F(ab')2 fragments.     

Tumor targeting with newly designed biparatopic antibodies directed against two different epitopes of the carcinoembryonic antigen (CEA)

In an attempt to improve tumor targeting and tumor retention time of monoclonal antibodies (MAbs), we prepared biparatopic antibodies (BpAbs) having the capability of binding 2 different non-overlapping epitopes on the same target antigen molecule, namely, the carcinoembryonic antigen (CEA). Six BpAbs were constructed by coupling 2 different Fab' fragments from 4 different specific anti-CEA MAbs recognizing 4 CEA epitopes (Gold 1-4). Demonstration of the double paratopic binding of these antibodies for CEA was confirmed in vitro by inhibition radioimmunoassay and cross-inhibition analysis by surface plasmon resonance (SPR; BIACORE) technology. Using the latter technique, the affinity constants for CEA immobilized onto the sensor chip were found to range from 0.37 to 1.54 x 10(9) M(-1) for the 4 parental F(ab')2 fragments and from 1.88 to 10.14 x 10(9) M(-1) for the BpAbs, demonstrating the advantage of biparatopic binding over conventional F(ab')2 binding. The Ka improvement was particularly high for BpAb F6/35A7 and BpAb F6/B17 with a 9.5- and 8.1-fold increase, respectively, as compared with the parental F(ab')2. In vivo, the 6 BpAbs were compared with their 2 respective parental F(ab')2 by injection of 131I-BpAb/125I-F(ab')2 parental fragments into nude mice xenografted with the human colon carcinoma T380. Dissection 72 hr post-injection demonstrated that BpAb B17/CE25 and BpAb F6/B17 gave higher tumor uptake than that of their parental F(ab')2. This finding is particularly interesting for BpAb F6/B17, which compared favorably with the F6 F(ab')2, one of the best parental F(ab')2 fragments used in our study.     

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.     

Penetration and binding of radiolabeled anti-carcinoembryonic antigen monoclonal antibodies and their antigen binding fragments in human colon multicellular tumor spheroids

The binding and penetration of two 125I-labeled anti-carcinoembryonic antigen (CEA) monoclonal antibodies (MAb) and their F(ab')2 and Fab fragments were measured in multicellular spheroids of poorly (HT29) and moderately well differentiated (Co112) human colon adenocarcinomas which express different amounts of CEA. Spheroids cultured in vitro model tumor microenvironments where poor vascular supply may modulate antigen expression and accessibility. The two MAb studied, 202 and 35, were shown previously to react with different CEA epitopes and to have high affinities of 1.2 and 5.8 X 10(9) M-1, respectively. MAb 202 has also been shown to cross-react with antigens present on human granulocytes and normal epithelial cells from human lung and pancreas. Specific binding of intact MAb and fragments of both antibodies was demonstrated for both types of human colon carcinoma spheroids compared to mouse colon carcinoma (CL26) and mammary tumor (EMT6/Ro) spheroids. Total binding of MAb and fragments was greater (1.5- to 2.5-fold) after 4 h compared to 1 h of exposure; the amount of binding compared to control IgG1 was 5- to 30-fold greater after 1-h incubation and 15 to 200 times greater after 4 h. This binding was stable as demonstrated by short and long wash experiments at 37 degrees and 4 degrees C. The binding of F(ab')2 and Fab fragments of the anti-CEA MAb 35 to spheroids of human colon Co112 was almost 2-fold greater than that of the intact MAb. However, for MAb 202, the binding of intact MAb and F(ab')2 was greater than that of Fab fragments. In addition the binding of both intact and F(ab')2 fragments of MAb 202 was greater than that obtained with MAb 35. Specific binding of both antibodies to HT29 spheroids, which express less CEA, was decreased for MAb and fragments of both 202 and 35. Autoradiography and immunoperoxidase experiments were performed to determine the penetration of MAb and fragments after incubation with intact spheroids. Comparisons were made with labeled MAb directly applied to frozen sections of spheroids. F(ab')2 and Fab fragments of both antibodies were bound at the surface of intact spheroids and penetrated to eight to ten cells, but the intact MAb were localized mainly at the spheroid surface and the outer one to three cell layers. There was much less binding at the surfaces of HT29 compared to Co112 spheroids. An enzyme immunoassay using MAb 35 and 202 demonstrated that Co112 spheroids produced about 8-fold more CEA/mg of cell protein than did monolayer cultures.(ABSTRACT TRUNCATED AT 400 WORDS)     

Swine monoclonal antibodies of high affinity and specificity to carcinoembryonic antigen

To avoid the exclusive use of rodent monoclonal antibodies (MAbs) in patients for the detection of tumors by immunoscintigraphy and for radioimmunotherapy, swine MAbs were produced that are directed against carcinoembryonic antigen (CEA). Spleen cells from 2 pigs immunized with purified colon carcinoma CEA were fused with a nonsecreting mouse myeloma cell line by conventional methods, except that a particularly long immunization protocol and large amounts of spleen and myeloma cells were used. Of 1,200 growing hybrids tested, 20 were found initially to produce antibodies binding to radiolabeled CEA. Seven stable clones producing anti-CEA MAbs for more than 6 months were derived from these hybrids by repeated subcloning. The pig origin of the seven MAbs was demonstrated in a solid-phase CEA enzyme immunoassay where anti-pig immunoglobin (Ig) antibodies coupled to peroxidase gave a positive reaction while anti-mouse Ig antibodies were entirely negative. All swine MAbs were of the IgG isotype. Three anti-CEA MAbs showed no cross-reactivity with granulocytes, while four others gave various degrees of reactivity with different granulocyte glycoproteins. Competitive binding to CEA performed for two purified swine MAbs showed that they recognized two different epitopes. The affinity constants measured for these two MAbs by Scatchard plot on purified CEA were high (1.2 X 10(9) and 1.2 X 10(10) liter/mol). One of the MAbs was tested in vivo for tumor localization by injection, after radiolabeling, in nude mice bearing human colon carcinoma xenograft. High ratios of tumor to normal tissue were obtained with mean values of 10.5 for intact MAbs and of 26.8 for F(ab')2 fragments of the porcine MAb. The results showed that heterofusion with this particular protocol can be used to produce swine MAbs of high affinity and specificity for a well-defined tumor marker. These reagents may have an important clinical utility, particularly in patients who became sensitized to mouse immunoglobulins.     

A comparison of the in vitro and in vivo activities of IgG and F(ab')2 fragments of a mixture of three monoclonal anti-Her-2 antibodies.

PURPOSE: We have demonstrated previously that a mixture of three anti-Her-2 monoclonal antibodies (MAbs) that bind to different epitopes on the extracellular domain of Her-2 expressed on a human breast cancer cell line has more potent antitumor activity than the individual MAbs both in vitro and in xenografted severe combined immunodeficient mice. Because the activity of Herceptin is Fc dependent, we determined whether this would also be the case when a mixture of these three anti-Her-2 MAbs was used. EXPERIMENTAL DESIGN: IgG and highly purified F(ab')(2) fragments of the anti-Her-2 MAbs and Herceptin were prepared and evaluated for their ability to induce cell death, inhibit vascular endothelial growth factor secretion, and mediate antibody-dependent cellular cytotoxicity and complement-mediated cytotoxicity in vitro. They were also compared for their abilities to induce regression of large BT474 tumors in severe combined immunodeficient mice. RESULTS: All of the F(ab')(2) fragments were >95% pure and, as expected, did not mediate antibody-dependent cellular cytotoxicity or complement-dependent cytotoxicity in vitro. The in vitro antiproliferative and proapoptotic effects of the IgGs and F(ab')(2) fragments were similar. In contrast, the IgGs had significant antitumor activity in vivo, whereas their F(ab')(2) fragments were only marginally effective even at 5-fold higher doses to offset their shorter half-lives. CONCLUSIONS: These results confirm the importance of the Fc portion of Herceptin for optimal in vivo activity and demonstrate that even a mixture of three anti-Her-2 MAbs that are highly effective at inducing cell death in vitro requires Fc-mediated effector function for optimal in vivo activity.     

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.     

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.     

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.     

Radioimmunodetection of human glioma xenografts by monoclonal antibody to epidermal growth factor receptor

Murine IgG2a monoclonal antibody (MAb) 425 specifically detects epidermal growth factor receptor, which is expressed on human gliomas and tumors of other tissue origin but rarely on normal brain tissues, and not at all on bone marrow and peripheral blood cells. 131I-labeled F(ab')2 fragments of this MAb injected into nude mice grafted with U-87 MG glioma cells preferentially localized in tumor tissue compared to normal mouse tissues, as determined by differential tissue counting of radioactivity. The mean tumor-to-tissue ratios of radioactivity ranged between 8.2 (blood) and 55.8 (muscle) at 2 days after the injection of 15 muCi of 131I-425 F(ab')2/mouse. Radiolabeled fragments of an anti-hepatitis virus IgG2a MAb did not localize in tumors. The localization index derived from the ratios of specific antibody to indifferent antibody in tumor tissue relative to blood was 9.94 at 2 days following the MAb injection. The labeled MAb did not localize in a xenograft of colorectal cancer tumor, which does not express the epidermal growth factor receptor. Tumors could be located by whole-body gamma-scintigraphy without background subtraction following the injection of 100 muCi of radiolabeled MAb 425 F(ab')2 fragments. The data suggest that MAb 425 is a likely candidate for clinical diagnostic and radioimmunotherapy trials.     

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.     

Enhancement of colorectal tumor targeting using a novel biparatopic monoclonal antibody against carcinoembryonic antigen in experimental radioimmunoguided surgery.

Biparatopic CEA, carcinoembryonic antigen (MAb) was newly designed and tested as to whether it enhanced the accuracy of tumor detection by reducing non-specific binding in experimental radioimmunoguided surgery. Biparatopic MAb was prepared by using cross-linking of reduced Fab' fragments from PR1A3 and T84.66. Fifty-nine tumors from 2 human colorectal carcinoma cell lines with high (KM-12c) and low (Clone A) carcinoembryonic antigen (CEA) expression were successfully implanted subcutaneously on the backs of 42 nude mice. Tumors were localized using 125I-labeled MAbs: IgG, F(ab')(2) and Fab' of PR1A3, and biparatopic MAb of PR1A3 and T84.66. Radioactivity counted on a portable radioisotope detector correlated well with that counted on a gamma counter (p < 0.001). Accumulations of radioactivity in control mice without tumorigenesis were the greatest in PR1A3 IgG-pretreated mice and the least in biparatopic MAb-pretreated mice. Tumors of 2 cell lines did not differ in the distribution of radiolabeled MAbs. Localization indices of the tumor in various organs revealed 1.3 to 4.1 in PR1A3 IgG-pretreated mice, 2.4 to 6.6 in fragment MAbs of PR1A3-pretreated mice and 2 to 4.6 in biparatopic MAb-pretreated mice. Silver grains and immune staining were predominantly distributed in tumor cells of all types of MAb-pretreated mice. Sensitivity and specificity of tumor localization by radioimmunoguided surgery (RIGS) were the highest in the biparatopic MAb-pretreated mice (90.9% and 94.5%, respectively) and the least in the PR1A3 IgG-pretreated mice (50% and 72%). The biparatopic MAb using 2 anti-CEA MAbs against different epitopes achieved a great affinity and avidity with accurate localization of colorectal carcinoma in experimental radioimmunoguided surgery.     

Stable, genetically engineered F(ab')(2) fragments of chimeric TNT-3 expressed in mammalian cells

F(ab')(2) fragments are desirable structural derivatives of monoclonal antibodies (MAbs) because of their pharmacokinetic properties and bivalent binding to antigen. Production of these fragments, however, has proven difficult because of the variable sensitivity of intact antibodies to proteolytic enzymes, which can result in very low yields and unstable product. To circumvent these problems, we attempted to apply genetic engineering methods to generate stable F(ab')(2) fragments in NSO murine myeloma cells using the glutamine synthase expression system. For these studies, the chimeric MAb, chTNT-3, directed against necrotic regions of solid tumors, was used to generate several F(ab')(2) variants, which contained between one and three cysteine residues at the end of the hinge region. In addition, two different affinity tags (his tag, streptactin tag) were used with each variant to determine the best tag for purification procedures. Stability was measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by antigen binding studies and the constructs were tested in vivo to measure their pharmacokinetic properties and biodistribution in normal organs and tumor. The results of these studies show that 3 cysteine residues are required to produce stable F(ab')(2) fragments and that either purification tag can be used with this variant to produce suitable reagents for in vivo studies. Those constructs containing one or two cysteines were found to be unstable and broke down to Fab fragments regardless of the purification tag used. These studies demonstrate that stable, clinically useful F(ab')(2) fragments of chTNT-3 can be produced in mammalian cells by genetic engineering methods.     

Targeted therapy of athymic mice bearing GW-39 human colonic cancer micrometastases with 131I-labeled monoclonal antibodies.

The therapeutic potential of radiolabeled antibodies is usually evaluated in experimental animal models bearing s.c. xenografts. We have established a micrometastatic model of the GW-39 human colonic carcinoma in the nude mouse lung (J. Natl. Cancer Inst., 83: 627-632, 1991) and presented preliminary findings on the efficacy of a 131I-anticarcinoembryonic antigen (CEA) antibody in this model. We now extend our observations on the use of radioiodinated labeled monoclonal antibodies (MAbs) to treat multiple small tumor nodules. Biodistribution and dosimetry analysis was performed for intact and F(ab')2 of NP-4 anti-CEA IgG, Mu-9 anti-colon-specific antigen IgG, isotype-matched irrelevant anti-AFP IgG, and intact MAb 34A anti-lung endothelial IgG antibody. Comparisons were made for rad dose delivered to small s.c. tumors, normal lung, lung with tumor nodules, and isolated tumor nodules. Survival curves were generated for tumor-bearing animals treated 1, 7, or 14 days after tumor cell implantation with these antibodies using the maximal tolerated dose for intact antibodies (275 microCi) and for F(ab')2 fragments (1.2 mCi). The studies established the following observations: (a) in contrast to previous results in a bulky tumor model in hamsters, intact antibodies are more therapeutic than MAb fragments for both NP-4 and Mu-9; (b) tumor nodule size, even on the microscopic level, affects therapeutic outcome; antibodies were more effective when administered 7 days postimplantation (mean nodule diameter, 150 microns) compared with treatment 14 days postimplantation (mean nodule diameter, 750 microns); (c) administration of radioiodinated Mu-9 was exquisitely effective on single avascular tumor cells that had seeded in lung; irrelevant antibody was minimally radiotoxic; (d) as in the bulky disease model, the anti-colon-specific antigen p antibody delivers a higher rad dose than the anti-CEA antibody and is significantly more therapeutic in the micrometastasis model; (e) a higher affinity anti-CEA antibody (MN-14) recognizing the same epitope on CEA as NP-4 was equally therapeutic; (f) the use of MAb directed against the lung endothelium was not as therapeutic as a tumor-associated antibody; and (g) all tumor-associated antibodies were more efficacious than administration of the maximal tolerated dose of 5-fluorouracil and leucovorin in this human tumor-xenograft model. These results provide further support for the use of radioimmunotherapy in the handling of minimal disease, probably as part of an adjuvant treatment regimen.     

Tumor-cell killing by MAbs against fucosyl GM1, a ganglioside antigen associated with small-cell lung carcinoma.

Monoclonal antibodies (MAbs) reactive with the ganglioside fucosyl GM1 (Fuc-GM1), an antigen associated with small-cell carcinoma of the lung (SCLC), were tested for their ability to mediate tumor-cell killing in vitro in conjunction with humoral and cellular effectors and to inhibit tumor engraftment in nude mice in vivo. MAbs F12 and F15, both IgG3k, induced human complement-mediated cytolysis of 3 Fuc-GM1-expressing tumor cell lines: one rat hepatoma cell line, H4-II-E, and 2 human SCLC cell lines, NC1 H69 and NC1 H128. F12 and F15 also induced ADCC of these cell lines in the presence of either murine or human effector cells. Addition of sub-cytolytic amounts of fresh human serum as complement source resulted in enhanced ADCC induced by MAb F12 (IgG3). Also a Fuc-GM1-reactive MAb of IgM isotype, F9, was able to induce such complement-aided ADCC (CADCC). F12 and F15 both proved to effectively inhibit engraftment of H4-II-E tumors in nude mice. A single dose of a modest amount (40 micrograms) of MAb conferred 65 to 100% protection against development of tumors. Our results demonstrate that Fuc-GM1 can act as a target antigen on tumor cells for specific immunotherapy in vitro and in a mouse model in vivo. Complement and murine and human mononuclear effector cells were effective mediators of tumor cytolysis in vitro in the presence of murine Fuc-GM1-reactive MAbs. Our results also suggest that humoral and cellular effectors may co-operate in specific tumoricidal reactions and that these may be induced by antibodies of both IgG and IgM isotypes.     

In vivo targeting of malignant melanoma by 125Iodine- and 99mTechnetium-labeled single-chain Fv fragments against high molecular weight melanoma-associated antigen.

Monoclonal antibodies (MAbs) against high-molecular-weight melanoma-associated antigen (HMW-MAA) have been used in vivo to target melanoma. More recently, single chain Fv (scFv) antibody fragments against HMW-MAA have been described that may improve melanoma targeting. However, there have been few in vivo studies with antimelanoma scFvs because these have proved difficult to label with isotopes (e.g., 99mTc) suitable for imaging. We have generated a series of scFvs against HMW-MAA by chain shuffling and antibody phage selection on melanoma cells. In preliminary experiments we identified one scFv (RAFT3) as suitable for in vivo melanoma targeting. Direct radiolabeling of RAFT3 scFv with 99mTc was simple, yielding a radiochemical purity of >90%. The label remained stable for 24 h in vitro. 125I- and 99mTc-labeled RAFT3 scFv were tested in a nude mouse xenograft model for human melanoma and were compared with the parent MAb LHM2 and its F(ab')2 fragment versus nonmelanoma-specific MAb and scFv. RAFT3 scFv accumulated specifically in the tumor and showed greater tumor specificity compared with LHM2 with faster pharmacokinetics (t(1/2)alpha, 8 min; t(1/2)beta, 189 min; and t(1/2)alpha, 37 min; t(1/2)beta, 384 min, respectively) and reduced background in liver, lung, and spleen. Nonspecific accumulation of 99mTc-labeled RAFT3 scFv in the kidney was high but tumor:normal tissue ratios were better compared with 125I-labeled RAFT3 scFv and LHM2 F(ab')2. Overall, tumor-targeting efficiency at equivalent time points was scFv > IgG > F(ab')2 in good agreement with previously described scFvs engineered for 99mTc labeling. We discuss the potential use of RAFT3 scFv for imaging and therapy of metastatic melanoma.     

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.     

Monoclonal antibody and F(ab')2 fragment delivery to tumor in patients with glioma: comparison of intracarotid and intravenous administration

Non-i.v. delivery of radiolabeled monoclonal antibodies (MAbs) has been shown to increase tumor uptake and decrease dose to normal tissues. In this study, we have examined the potential advantage of intracarotid (i.c.) versus i.v. administration for the delivery of an intact MAb and a F(ab')2 fragment to tumor in patients with gliomas. Three patients received 10-50 mg of 81C6 IgG2b, a MAb reactive with the glioma-associated extracellular matrix antigen tenascin, and three received 5-20 mg of the F(ab')2 fragment of Mel-14, which is reactive with gliomas and melanomas. Paired-injection protocols, in which one-half of the MAb was labeled with 131I and administered by i.c. injection, and one-half was labeled with 125I and simultaneously administered by i.v. injection, were used. For both 81C6 IgG2b and Mel-14 (Fab')2, no differences in blood clearance half-times or urinary excretion rates of radioiodine were observed between i.c.- and i.v.-administered activity. Analysis of biopsy samples revealed i.c.:i.v. uptake ratios of 1.02 +/- 0.04, 0.95 +/- 0.03, and 1.03 +/- 0.05 for the accumulation of 81C6 IgG2b in temporalis muscle, normal brain, and glioma, respectively. Similarly, the i.c.:i.v. uptake ratios for Mel-14 F(ab')2 in these tissues were 0.98 +/- 0.04 (SD), 1.00 +/- 0.05, and 1.04 +/- 0.05. When the differences in percentage of injected dose/g uptake after i.c. and i.v. administration were compared, no statistically significant advantage for i.c. delivery was seen (P = 0.22-0.61). These data indicate that i.c. administration of MAb 81C6 IgG2b and Mel-14 F(ab')2 fragments offers no delivery advantage to offset the small but finite risk involved in cannulation and injection of the internal carotid artery.     

Evaluation of L6, an anti-carcinoma murine monoclonal antibody, in tumor-bearing nude mice

L6 is a murine monoclonal antibody (MAb) binding to cells of most human carcinomas, mediating antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity, and inhibiting tumor growth in nude mice [10]. Fab and F(ab')2 fragments of L6, as well as intact MAb, have been evaluated for immunospecific localization in nude mice xenografted with a human lung carcinoma. They were compared with preparations of an isotype-matched control immunoglobulin, P1.17, after labelling with 125I or 131I. L6 Fab fragments prepared from MAb L6 and labelled with 67Ga via desferrioxamine were also tested. The data suggest that MAb L6 may be useful for in vivo detection of human carcinomas.