Antibody In Nude Mice Research Articles

Abstract A032: Targeting pancreatic cancer senescence with ImmunoPET

Abstract Pancreatic cancer is the fourth leading cause of cancer-related deaths for men and women, with five-year overall survival rates around 10 percent. Despite years of cancer research and new immunotherapy solutions, pancreatic cancer is still mainly treated by chemotherapies, starting with gemcitabine. Many cytostatic drug combinations have been shown, in a wide range of cancers, to induce senescence, a persistent, anti-apoptotic process characterized almost exclusively by beta galactosidase activity. Studies inducing senescence with the combination of trametinib and palbociclib (TP) have linked several tumorigenic and pro-metastatic factors in murine pancreatic cancer models to the senescence-associated secretory profile (SASP), giving rise to new antigen targets during the induction of senescence. Accordingly, senescence has recently been elevated as a new hallmark of cancer. There is thus a critical need for antigen-based tools to noninvasively identify markers of senescence in vivo. Senescence targets comprise of shed antigens (VEGF and IL-6) in the context of senescence-inducing therapy with trametinib and palbociclib (TP), when membrane bounds targets such as urokinase plasminogen activated receptor (uPAR) are potentially enriched. We show that VEGF and IL-6 targeting antibodies labeled with zirconium-89 deferoxamine (89Zr-DFO) bind cells treated with TP though in vivo, marked reduction in tumor targeting is seen. Preloading and targeting antigen shed are being actively considered as alternative imaging approaches. For membrane bound antigens associated with senescence, here we report the latest work developing uPAR immunoPET agents. First, we have shown that attachment of 89Zr-DFO preserves the binding function of both murine (muPAR) and human uPAR (huPAR) antibodies with high cross-reactivity of the murine uPAR (muPAR) antibody with huPAR. Cell uptake was increased two-fold in murine KPC cells with senescence-inducing TP treatment. In vivo, accumulation in subcutaneously implanted murine KPC pancreatic tumors was observed with TP-treated mice; however lower liver and blood activity was imaged by PET/CT and confirmed by terminal biodistribution at 144h. Human uPAR-targeting antibody in nude mice bearing flank MiaPaCa2 tumors showed strong antibody uptake by 144 h, with increased tumor uptake for mice undergoing TP therapy. Preloading studies at 10 µg, 40 µg, and 100µg at 4h prior to injection of 40µg 89Zr-DFO-huPAR showed improved tumor uptake kinetics with the highest preloading level, though overall tumor uptake was decreased at the higher radiolabeled antibody dose used, thus more optimization is needed. The development and optimization of 89Zr-DFO-uPAR antibodies for human and murine targets provides a superior means of identifying pancreatic cancer and pancreatic cancer senescence than that afforded by shed antigens. Future directions include further dose optimization and testing uptake in other pancreatic models, senescence-inducing drug combinations, and possible antibody-conjugated senolytic or endoradiotherapy strategies. Citation Format: Edwin C. Pratt, Riccardo Mezzadra, Tara Viray, Spencer Kaminsky, Scott W. Lowe, Jason S. Lewis. Targeting pancreatic cancer senescence with ImmunoPET [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr A032.

Abstract 3551: Oncolytic adenovirus serotype 35-mediated activation of NK cells leads to efficient anti-tumor effects

Abstract Oncolytic adenoviruses (OAds) are getting much attention as cancer immunotherapy agents. Almost all OAds are based on human adenovirus serotype 5 (Ad5) (OAd5). However, OAd5 has several drawbacks which possibly attenuate the anti-tumor effects of OAd5. More than 80% of adults have neutralizing antibodies against Ad5. The expression of coxsackievirus-adenovirus receptor (CAR), which is the primary infection receptor of Ad5, often declines on malignant tumor cells. Previously, we have developed a novel OAd fully composed of human adenovirus serotype 35 (Ad35) (OAd35) (Ono et al., Mol.Ther.Oncolytics, 2021). Only 20% or fewer adults have neutralizing antibodies against Ad35. Ad35 recognizes CD46, which is highly expressed on malignant tumor cells, as an infection receptor. OAd35 showed efficient tumor cell lysis activities in many types of tumor cells; however, the mechanism of anti-tumor effects of OAd35 remains to be fully evaluated. In this study, we examined the mechanism of anti-tumor effects of OAd35. Although OAd35 showed efficient tumor growth suppression at levels comparable to OAd5 following intratumoral administration in nude mice bearing H1299 human lung cancer xenografts, the genome copy numbers of OAd35 in the tumor were about 500-folds lower than those of OAd5 at 18 days after administration and were almost background levels. These data suggest that other mechanisms different from infection of tumor cells with OAd35 were involved in the anti-tumor effects of OAd35. Since previous studies reported that Ad35 efficiently activated innate immunity in natural killer (NK) cells, we examined whether OAd35-mediated activation of NK cells contributed to the anti-tumor effects of OAd35. Intratumoral administration of OAd35 resulted in efficient infiltration and activation of NK cells in H1299 tumors. In contrast, OAd5 did not apparently induce the infiltration and activation of NK cells in the tumors. OAd35-mediated activation and infiltration of NK cells were also found in B16 tumor-bearing immunocompetent mice. Next, in order to examine the roles of NK cells in the anti-tumor effects of OAd35, NK cells were depleted by intravenous injection of anti-GM1 antibody in nude mice, followed by intratumoral administration of OAd35. The depletion of NK cells significantly attenuated the anti-tumor effects of OAd35 on H1299 tumors. Furthermore, pretreatment with anti-GM1 antibody significantly increased the genome copy numbers of OAd35 in the tumor, suggesting that activated NK cells removed the OAd35-infected tumor cells. This study demonstrated that OAd35 significantly activated NK cells following intratumoral administration and that activated NK cells efficiently removed the tumor cells, leading to efficient tumor growth suppression. Citation Format: Ryosuke Ono, Kosuke Takayama, Fuminori Sakurai, Hiroyuki Mizuguchi. Oncolytic adenovirus serotype 35-mediated activation of NK cells leads to efficient anti-tumor effects [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3551.

T86: New approaches to the rational design of anticancer drugs

Discovery of new pharmacologically active small molecules is an important and rapidly expanding area of modern molecular pharmacology. Given a limited number of proteins that are druggable, it is important to identify as many chemical effectors as possible to define the best regimen of anti-cancer therapy in each particular case. An E3 ubiquitin ligase, Mdm2, which mediates ubiquitin-dependent degradation of the critical tumor suppressor p53, is a promising target for small molecule inhibitors. Using a hybrid approach which combines the rational design of small molecules selected from the virtual library and the high-content screening using cancer cell lines we discovered several new inhibitors of the p53-Mdm2 interaction. These compounds were able to activate and stabilize the p53 protein causing massive apoptosis preferably in p53-positive cells at rates higher than the well-known inhibitor of Mdm2, Nutlin-3. The molecular mechanisms of their action will be discussed. As another example of rational design of potential anti-cancer drugs, we will talk about artificial nano-Matrix-Imprinted -Polymers (MIPs) that recognize the structure of peptides and other biological molecules and thus dubbed as “plastic antibodies”. We have generated such nanoparticles against the surface region of the oncogenic receptor, EGFR, which is overexpressed in many forms of solid tumors. Selection of the linear epitope for creating “plastic antibodies” against the receptor was performed by analysis of a three-dimensional structure of the protein. The obtained “plastic antibodies” were specific against the epitope of EGFR. These plastic antibodies when loaded with a genotoxic drug, doxorubicin, were able to specifically induce cell death of breast cancer cell lines that overexpress the EGFR receptor. Experiments in vivo using xenografts of breast cancer cell lines pre-incubated with these plastic antibodies in nude mice showed that they have a pronounced therapeutic effect. Furthermore, since the commercial drug, Cetuximab, recognizes an epitope of EGFR, different from the one recognized by our plastic antibodies, it is likely that the latter may increase the efficacy of the commercial monoclonal antibody. Collectively, we demonstrate that the rationally designed small molecules can be potent and specific drugs for anti-cancer therapy.

Radioimmunoimaging with Mixed Monoclonal Antibodies of Nude Mice Bearing Human Lung Adenocarcinoma Xenografts

The present study was conducted to evaluate radioimmunoimaging (RII) and in vivo distribution of mixed antibodies 99mTc-EGFR-mAb and 99mTc-CD44- mAb in nude mice bearing human lung adenocarcinoma xenografts. Single and mixed applications of the two radiolabeled monoclonal antibodies (mAbs) were compared. Direct labeling of 99mTc was applied to radiolabel the EGFR and CD44 mAbs. The properties of the radiolabeled antibodies were then characterized. RII and assessment of the distribution of the antibodies in nude mice bearing lung adenocarcinoma xenografts were achieved by applying separate and combined doses of 99mTc-EGFR-mAb and 99mTc-CD44-mAb. The labeling rates of 99mTc for EGFR-mAb and CD44-mAb were 91.5% ±3.8% and 92.3% ± 4.1% respectively, with specific activities of 2.8 and 2.9 MBq/μg, respectively, and radiochemical purities (RCP) of 96.5% and 96.2%. The radioactivity uptake of the combined application of both radiolabeled antibodies was clearly higher than with a single application of either alone. The relative values of target-to-nontarget (T/ NT) measured through the regional interest (ROI) technique were 5.59 ± 0.42 (mixed antibodies), 2.78 ±0.20 (99mTc-EGFR-mAb), and 2.28 ± 0.16 (99mTc-CD44-mAb) in the RII. The body distribution of the radiolabeled antibodies and their imaging results were basically identical. Application of the mixed antibodies with 99mTc- EGFR-mAb and 99mTc-CD44-mAb can increase the radioactivity uptake of tumor tissue, leading to more ideal target-to-nontarget ratios, and therefore superior results.

Production of anti-double-stranded DNA antibodies in activated lymphocyte derived DNA induced lupus model was dependent on CD4+T cells

Our previous study demonstrated that activated lymphocyte derived DNA (ALD-DNA) could function as an autoantigen to induce production of anti-double-stranded DNA (anti-dsDNA) antibodies in syngeneic BALB/c mice. Here we carefully evaluated the potential role of T cells in the induction of anti-dsDNA antibody. We demonstrated that ALD-DNA could effectively induce production of anti-dsDNA antibodies in vivo and in vitro. In contrast, ALD-DNA could not induce the generation of anti-dsDNA antibodies in nude mice. We further showed that in vivo depletion of CD3(+) T cells blocked the induction of anti-dsDNA antibodies in BALB/c mice. Notably, we demonstrated that CD4(+) but not CD8(+) T cells conferred ALD-DNA to induce anti-dsDNA antibodies. Finally, we demonstrated that adoptive transfer of CD4(+) T cells could rescue ALD-DNA induced anti-dsDNA antibodies in nude mice. Our results suggested that T helper cells were required for ALD-DNA to induce anti-dsDNA antibodies. These findings could further our understanding about the immunogenic properties of DNA and throw new light on SLE pathogenesis.

Animal biodistribution and pharmacokinetics study of ~(131)I-labelled rch24

Objective To evaluate biodistribution and pharmacokinetics pattern of ~(131)I-labeled rch24which is the region-grafted (humanized) anti-carcinoembryonic antigen (CEA) monoclonal antibody in nude mice. Methods Nude mice bearing cancer xenografts received intravenous injections of ~(131)I- rch24, then blood, plasma, heart, liver, spleen, lung, kidney, tumor and other tissues were taken at different time point for determination the concentration of radioactivity and calculate the T/NT value. Nude mice were packeted randomly to four group of high, medium, low dose and continuous administration, blood drug concentration was detected by ELISA method at the different intervals. Then, draw the concentration-time curve and calculate the pharmacokinetics paramete. Results After administration, radioactivity of the tumour was significantly enhanced whereas radioactivity of normal tissues decreased gradually. For single administration, at the dose of low to medium, pharmacokinetics pattern was linearity -kinetics whereas for high dose group,pharmacokinetics paramete shown some behavior of non-linearity-kinetics. Conclusion Our results suggest that the ~(131)I-labeled region-grafted (humanized) anti-CEA monoclonal antibody rch24 exhibit a considerable targeting activity so as to ~(131)I radioisotopes can be concentrated specifically in tumor. The pharmacokinetics pattern of this medicine was different at different dose. Key words: Carcinoembryonic antigen; Antibodies; monoclonal; ~(131)I; Tissue distribution; Pharmacokinetics

Inhibition of Breast Cancer Metastasis and Angiogenesis by Antiosteopontin Single-Chain Fv-Fc Fusion Protein

Osteopontin (OPN) is associated with many diseases, and its role in tumor growth and metastasis has been studied in breast cancers. Previous studies have described anti-OPN antibodies that could inhibit tumor cell adhesion and invasion in vitro, but until now, there are no systematic studies on antitumor effects of anti-OPN antibodies in vivo. In the present study, we have raised several anti-OPN single-chain variable fragments from phage antibody library and expressed them as single-chain variable fragment-constant region fragment fusion proteins in Chinese hamster ovary cells. Of them, two antibodies (1A12 and 2H8) were able to inhibit MDA-MB-435s breast cancer cell attachment, invasion, migration, and colony formation in soft agar. Furthermore, 1A12 and 2H8 inhibited the anti-apoptotic and prosurvival functions of OPN in human umbilical vein endothelial cell. In human umbilical vein endothelial cell capillary tube formation, chicken chorioallantoic membrane assay, and rabbit corneal micropocket assay, the two antibodies showed markedly inhibitory effects toward angiogenesis. We investigated antitumor effects of anti-OPN antibodies in nude mice by assessing xenograft tumor growth and lung metastasis potential. The results showed that the two antibodies were capable of delaying primary tumor growth and reducing spontaneous lung metastasis. Epitope mappings of these two anti-OPN antibodies were performed, and a new binding site of 1A12 was revealed. In summary, the present study has demonstrated the roles of anti-OPN antibodies in blocking the function of OPN, suggesting that they may have the potential to be developed for future clinical use.

TU‐C‐L100J‐04: Absorbed Dose Estimates Obtained Via PET Imaging for I‐124 Intact Antibody in Nude Mice

Purpose: To estimate tumor targeting and radiation dose in mice receiving I‐124‐labeled anti‐CEA (Carcinoembryonic Antigen) intact monoclonal antibody. Use of I‐124 as a PET emitter is complicated by high‐energy photons (603 keV and 723 keV at 63% and 10% respectively) emitted along with the positrons (23%). Method and Materials: Intact monoclonal antibody cT84.66 was labeled with I‐124 and injected via tail vein into nude mice bearing LS174T human colon tumors (0.04 to 0.19 g). PET imaging was performed using the Siemens MicroPET RF scanner at seven time points out to 8 days post‐injection. Standard energy window of 350–750 keV was employed. Average voxel values from organ volumes of interest were used to determine relative magnitudes of activity in tumor, liver, heart (blood) and whole body. Absolute activities were calculated by normalizing these PET data to dissection results at the last time point. Bi‐exponential functions were fitted to resultant activity vs time curves and integrated to establish à values and residence times. Organ‐to‐organ S factors were determined by Monte Carlo analysis of I‐124 in a 20g digital mouse phantom. Organ dose was then estimated as the matrix product of SÃ. Results: Tumors were visualized within one day of injection. Average residence times for blood, tumor, liver and whole body were 45, 6.5, 7.0 and 86 hours respectively. Mean dose for the tumors was 3.3 Gy/MBq; associated liver and whole body average values were only 0.60 and 0.31 Gy/MBq respectively. Conclusions: Small colon tumors can be PET‐imaged in nude mice receiving the I‐124 anti‐CEA antibody cT84.66. By normalizing voxel results to those obtained at sacrifice, quantitative I‐124 uptakes can be measured and used to generate residence times. Ratios of tumor/liver and tumor/whole body dose were approximately 5 and 10 respectively.

Biodistribution study of murine monoclonal anti-GD3 antibody in nude mice bearing human melanoma xenografts for development of immunoscintigraphy.

Reactivity of the monoclonal antibody with the tumor markers is known to be different between cultured cells in vitro and transplanted tumors in vivo. The monoclonal antibody should be investigated regarding its specific accumulation in tumor-bearing mice for immunodetection or immunotherapy. We studied the biodistribution of radiolabeled monoclonal anti-GD3 antibody (IgM) in normal mice and nude mice bearing human melanoma xenografts. Tissue-to-blood distribution ratios of the antibody in the liver, spleen and kidney increased with time in both normal and melanoma-transplanted mice, but no significant changes were observed in other normal tissues up to 5 days after injection. Specific accumulation of the monoclonal anti-GD3 antibody in the grafted human melanoma (HMV-II) was observed 4 and 5 days after injection. On the other hand, no specific accumulation of standard murine IgM in the tissue of HMV-II was observed in mice bearing the HMV-II xenograft 5 days after injection. Because the tissue-to-blood ratio of the distribution in the tissue of HMV-II became larger than that of other tissues 4 and 5 days after administration, 4 days after the administration of the monoclonal anti-GD3 antibody were required for immunoscintigraphy. Accumulation of the monoclonal anti-GD3 antibody in other human melanomas (HMV-I, HMY-1 and SK-MEL188) inoculated into mice was also observed 4 days after the antibody administration. The monoclonal anti-GD3 antibody used in this study would be useful in immunodetection or immunotherapy.

Tumor radioimmunoimaging of chimeric antibody in nude mice with hepatoma xenograft

AIM:To study the radioimmunoimaging (RAII) using the human/mouse chimeric Ab to evaluate its targeting activity in animal models.METHODS: To chimeric Ab was labeled with (131)I. RAII was performed at different intervals after injection of radio-labeled Abs in nude mice with human hepatoma xenograft, and tissue distribution of radioactivity was measured. Comparison was made in the chimeric Ab between the single segment Ab and previous murine mAb against HBxAg.RESULTS: The experimental objects developed tumor-positive image after 2 days of radio-labeled Abs injection, and the peak accumulation of radioactivity fell on the 7th day. The tumor/liver ratioactivity of the chimeric Ab, single segment Ab, anti-HBx mAb, and the control group was 2.81 ± 0.21, 2.44±0.16, 4.60± 0.19, and 0.96 ±0.14, respectively.CONCLUSION: The genetic engineering Abs have a considerable targeting activity which can be used as a novel humanized vector in the targeting treatment of liver cancer.

The effect of antibody protein dose of anti-renal cell carcinoma monoclonal antibodies in nude mice with renal cell carcinoma xenografts.

Antibodies preferentially can direct radionuclides to solid tumors. However, antibody uptake in tumors is often highly heterogeneous. This heterogeneity may be overcome by increasing antibody protein dose. The biodistribution of increasing protein doses of radioiodinated antirenal cell carcinoma (RCC) monoclonal antibodies (MoAbs) G250 and RC 38 was studied in mice with NU-12 or SK-RC-52 RCC xenografts. In addition, MoAb affinity constants and antigen densities (Scatchard analysis) and MoAb processing (internalization) were determined in vitro. The relative uptake of G250 in NU-12 tumors was very high at low protein doses (125% injected dose/g [%ID/g]), but decreased at higher doses, suggesting tumor saturation. Indeed, saturation of G250 antigen occurred at 3 microg protein. In this model, 9200 G250 determinants per NU-12 cell could be targeted, which is only 6.1% of the 150,000 G250 determinants per NU-12 cell as determined in vitro. The RC 38 uptake in NU-12 tumors remained constant up to the 10 microg dose level (40% ID/g) and decreased at higher doses. RC 38 antigens were saturated at 25 microg of RC 38. With RC 38, 15% of the available RC 38 antigens per NU-12 tumor cell were targeted. In contrast, G250 uptake in SK-RC-52 tumors was very low at low antibody dose (4% ID/g at 1 microg) and increased with increasing protein dose. These differences in G250 biodistribution might be related to differences in the processing of G250 by the tumor cells. Our studies show that some RCC tumors can be saturated with anti-RCC MoAbs at low (25 microg) to very low (3 microg) protein doses. At nonsaturated doses relatively high tumor uptake can be achieved. Surprisingly, in NU-12 tumors only 6.1% and 15% of the available antigenic sites were targeted at the saturating dose levels with G250 and RC 38, respectively.

The effect of antibody protein dose of anti-renal cell carcinoma monoclonal antibodies in nude mice with renal cell carcinoma xenografts

Antibodies preferentially can direct radionuclides to solid tumors. However, antibody uptake in tumors is often highly heterogeneous. This heterogeneity may be overcome by increasing antibody protein dose.The biodistribution of increasing protein doses of radioiodinated antirenal cell carcinoma (RCC) monoclonal antibodies (MoAbs) G250 and RC 38 was studied in mice with NU-12 or SK-RC-52 RCC xenografts. In addition, MoAb affinity constants and antigen densities (Scatchard analysis) and MoAb processing (internalization) were determined in vitro.The relative uptake of G250 in NU-12 tumors was very high at low protein doses (125% injected dose/g [%ID/g]), but decreased at higher doses, suggesting tumor saturation. Indeed, saturation of G250 antigen occurred at 3 microg protein. In this model, 9200 G250 determinants per NU-12 cell could be targeted, which is only 6.1% of the 150,000 G250 determinants per NU-12 cell as determined in vitro. The RC 38 uptake in NU-12 tumors remained constant up to the 10 microg dose level (40% ID/g) and decreased at higher doses. RC 38 antigens were saturated at 25 microg of RC 38. With RC 38, 15% of the available RC 38 antigens per NU-12 tumor cell were targeted. In contrast, G250 uptake in SK-RC-52 tumors was very low at low antibody dose (4% ID/g at 1 microg) and increased with increasing protein dose. These differences in G250 biodistribution might be related to differences in the processing of G250 by the tumor cells.Our studies show that some RCC tumors can be saturated with anti-RCC MoAbs at low (25 microg) to very low (3 microg) protein doses. At nonsaturated doses relatively high tumor uptake can be achieved. Surprisingly, in NU-12 tumors only 6.1% and 15% of the available antigenic sites were targeted at the saturating dose levels with G250 and RC 38, respectively.

Synthesis and biodistribution of peptide based 99mTc/186Re-MAGIPG-D612 monoclonal antibody in nude mice bearing colon cancer xenografts.

A new bifunctional chelating agent MAGIPG was synthesized in good yield using Boc-p-nitro-phenylalanine as the starting material. 99mTc-labeled-MAGIPG-D612 conjugates reactive with human colon cancer were prepared in 3% to 15% yield with a radiochemical purity of 63% to 97%. 186Re-labeled MAGIPG-D612 conjugates were prepared with a 7% to 30% yield and a radiochemical purity of 82% to 98%. The biodistribution results demonstrate that these radioimmuno-conjugates selectively localized in LS174T human colon cancer xenografts.

Transduction of the macrophage colony-stimulating factor gene into human multidrug resistant cancer cells: enhanced therapeutic efficacy of monoclonal anti-P-glycoprotein antibody in nude mice.

To develop a therapeutic modality for overcoming multidrug‐resistant (MDR) cancer with anti‐MDR1 antibody, we examined the effect of macrophage colony‐stimulating factor (M‐CSF) gene transfection into MDR AD10 cells on therapy of MDR cancer with anti‐MDR1 antihody (MRK17) in nude mice. MDR human ovarian cancer (AD10) cells were transduced with the human M‐CSF gene inserted into an expression vector to establish gene‐modified cells capable of producing low (ML‐AD10), intermediate (MM‐AD10) and high (MH‐AD10) amounts of M‐CSF. Systemic administration of MRK17 resulted in significant dose‐dependent inhibition of subcutaneous growth of ML‐AD10 tumors. In contrast, systemic administration of recombinant M‐CSF in combination with MRK17 did not augment the therapeutic efficacy of MRK17 alone, but rather promoted the growth of the parent AD10 cells. To test the efficacy of in vivo M‐CSF gene therapy combined with antibody, we mixed the parent AD10 cells with MH‐AD10 cells producing a large amount of M‐CSF, and inoculated the mixed cells subcutaneously. Treatment with MRK17 inhibited growth of the mixed cells more than that of the parent cells alone. Thus, combined therapy with anti‐MDR1 mAb and M‐CSF gene modification of MDR cancer cells may provide a new immunotherapeutic modality for overcoming MDR in humans.

Intraportal injection of monoclonal antibody in nude mice bearing hepatic metastases.

Using a model for hepatic human colorectal carcinoma metastases in athymic mice, we compared the selective [intraportal (ip)] and systemic [intravenous (iv)] injection of radiolabeled monoclonal antibody (mAb) strongly reactive against the cell line. Percent injected dose of radiolabeled antibody per gram (%id/g) of tumor or normal tissues was measured at selected time points (up to 5 days postinjection) within 3 dose levels: 0.1, 1.0, and 2.0 micrograms (micrograms). At each dose level, 3-9 animals were studied in each of 3 groups: animals receiving ip injection (group HT-29-15 ip), those receiving intravenous injection (group HT-29-15 iv), and those receiving isotype-matched control antibody via the intraportal route (group BL-3 ip). Significantly greater (P < 0.005) %id/g in tumor was seen in group HT-29-15 ip at all time points and dose levels compared to those in groups HT-29-15 iv or BL-3 ip. However, immediately after injection of mAb, there was no difference in tumor %id/g between groups HT-29-15 ip and HT-29-15 iv at the highest dose level. There was no increase in %id/g of mAb in normal liver and blood after ip injection compared to iv injection beyond day 1. Therefore ip injection resulted in higher tumor to liver and tumor to blood ratios compared to iv (P < 0.005). We conclude that delivery of mAb to hepatic metastases can be enhanced by selective injection; this has important implications in the design of future clinical trials utilizing radiolabeled mAb in the diagnosis and treatment of hepatic metastases.

Therapy with 125I-labelled internalized and non-internalized monoclonal antibodies in nude mice with human colon carcinoma xenografts

The therapeutic effects of 125I-labelled (18–97 MBq) monoclonal antibodies (MAb) C-242, C-215 and S-S.1 were studied in nude mice with human colorectal adenocarcinoma tumours. The antibodies were administered 2 or 10–16 days after implantation of the tumour cells. The monoclonal antibody C-242 was internalized into the tumour cells, C-215 was internalized to a lower degree while S-S.1 (unspecific MAb) was not internalized at all. No enhanced therapeutic effect of 125I-C-242 was observed, as a result of Auger electrons, compared with 125I-C-215 and 125I-S-S.1.

Comparison of the biodistribution of 75Se- and 131I-labelled monoclonal antibodies in nude mice

A monoclonal antibody, C-215, against colon cancer, was internally labelled with [ 75Se]methionine. The biodistribution was studied in tumour-bearing nude mice and compared with the biodistribution of [ 131I]C-215. The tissue uptake was divided into three parts: antibody bound to the antigen, antibody in the extracellular space and uptake of the released radionuclide. [ 75Se]C-215 showed a greater amount of antigen-bound antibody in the tumour, but also a greater unspecific uptake both in tumour and normal tissue.

Influence of antibody protein dose on therapeutic efficacy of radioiodinated antibodies in nude mice bearing GW-39 human tumor.

The biodistributions of three 131I-labeled murine monoclonal antibodies, NP-4 and Immu-14 anti(carcinoembryonic antigen), and Mu-9 anti-(colon-specific antigen p), were determined at antibody protein doses varying from 1 microgram to 1000 micrograms in nude mice with small (0.1-0.4 g) GW-39 human colonic cancer xenografts. For each antibody, the percentage of the injected dose per gram of tumor and tumor/nontumor ratios were constant over a wide protein dose range. However, at high protein doses (above 100 micrograms for NP-4 and Immu-14) the percentage of the injected dose per gram of tumor and tumor/nontumor ratios decreased. Assuming that the uptake of a control anti-(alpha-fetoprotein) antibody represents the amount of antibody that accumulates in the tumor nonspecifically (i.e., antigen-independently), it could be shown that for each antibody the amount of antibody protein that accumulates in the tumor specifically, increases linearly with the protein dose, reaching a plateau level at the highest doses tested. The growth inhibition of GW-39 tumor transplants in nude mice treated with 131I-labeled antibody at either low or high antibody protein dose was compared. These experiments indicated that, in this experimental model, enhanced antibody protein dose may decrease the therapeutic efficacy of radioiodinated antibodies. It is suggested that heterogeneous distribution at low protein dose, with intense localization around the blood vessels, may enhance the tumoricidal effect of radioantibodies.

Effects of tumour mass and circulating antigen on the biodistribution of111In-labelled F(ab′)2 fragments of human prostatic acid phosphatase monoclonal antibody in nude mice bearing PC-82 human prostatic tumour xenografts

We have evaluated the effects of tumour mass and circulating antigen (prostatic acid phosphatase, PAP) on the biodistribution and the incorporation of 111In-labelled F(ab')2 monoclonal antibody (MoAb) fragments directed against human PAP into human prostatic tumours (PC-82; 0.1-8.9 g) growing in nude mice. The radioactivities in the blood, liver, spleen, kidney and tumour were compared at 1, 3, 4 and 6 days after the intravenous administration of the antibody fragments. There was a significant correlation between the tumour size and the serum PAP concentration in the model employed. Even tissue of a small tumour (less than 0.1 g) had a high concentration of PAP, but it was not secreted into the circulation in detectable amounts when measured by radioimmunoassay (the lowest standard was 0.5 micrograms/l). The percentage uptake by tumours of the injected dose per gram of tissue (%ID/g) was inversely proportional to the tumour size at 24 h after the administration of 111In-labelled F(ab')2 fragments. This relationship had levelled off by 72 h and most likely reflected a better vascularisation of the smaller tumours. Our results show that the increase in tumour size and in the concentration of circulating antigen in the blood led to decreased tumour-to-blood ratios, since there was a tendency for higher blood activities in mice with larger tumours and higher serum PAP concentrations. There was no correlation between tumour size and label uptake by the liver during the follow-up over 144 h, although serum PAP concentrations ranged from 3.1 micrograms/l to 352 micrograms/l.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of Seven Iodine-Labelled Monoclonal Antibodies in Nude Mice with Human Colon Carcinoma Xenografts

The biokinetics of seven 131I-labelled monoclonal antibodies (MAbs), directed against human colon carcinoma and one 125I-labelled unspecific MAb have been examined. The study in nude mice, carrying human colon carcinoma, was intended to be a step in the selection of the most suitable antibody for clinical scintigraphy. The biological half-life in blood was found to be between 1.3 and 7.4 days for the different MAbs. Chromatography of plasma samples showed that the radioiodine was mainly bound to IgG-sized molecules. The (normal tissue)/blood ratios were similar for all the MAbs. The tumour/blood ratio was 0.41 for the unspecific MAb and 0.49-1.1 for the specific MAbs, and the tumour/muscle ratio was between 3.2 and 6.8 for the specific MAbs 6 days after injection. For one MAb tumour/blood and tumour/muscle ratios were 3.9 and 9.8 respectively 9 days after injection. Localization indices were at their highest 2.6 6 days after injection. For at least two of the monoclonal antibodies the tumour/blood and tumour/muscle ratios found are high enough to justify clinical trials regarding their usefulness for scintigraphy of colon cancer in man.