Specific localization of In-111-labeled monoclonal antibody versus 67-Ga-labeled immunoglobulin in mice bearing human breast carcinoma xenografts

Summary A murine monoclonal antibody reacting with more than 95% of all breast cancers was radiolabeled with In-111 and injected IP into nude mice bearing human breast carcinoma xenografts, together with Ga-67-labeled normal mouse immunoglobulin. Images were produced with a gamma camera in dual isotope mode. Tumors could be localized clearly with In-111-labeled specific monoclonal antibody, but improved visualization was obtained after computer-assisted subtraction of the image with Ga-67-labeled nonspecific immunoglobulin. The tumor-to-tissue contrast was improved from 2.3 to 5.9 after subtraction. Imaging with In-111-radiolabeled monoclonal antibody was superior to imaging with iodinated antibody. For the first time it was shown that images of two chemically related isotopes, Ga-67 and In-111, coupled to nonspecific and specific antibody, respectively, and simultaneously injected, can be subtracted to show the preferential uptake of the specific antibody in the tumor. As these isotopes are routinely used in clinical practice this technique may prove to be more practical for immunodetection of tumors in patients than existing imaging techniques.     

Use of radiolabeled monoclonal antibody to enhance vaccine-mediated antitumor effects

Radiolabeled monoclonal antibodies (mAb) have demonstrated measurable antitumor effects in hematologic malignancies. This outcome has been more difficult to achieve for solid tumors due, for the most part, to difficulties in delivering sufficient quantities of mAb to the tumor mass. Previous studies have shown that nonlytic levels of external beam radiation can render tumor cells more susceptible to T cell-mediated killing. The goal of these studies was to determine if the selective delivery of a radiolabeled mAb to tumors would modulate tumor cell phenotype so as to enhance vaccine-mediated T-cell killing. Here, mice transgenic for human carcinoembryonic antigen (CEA) were transplanted with a CEA expressing murine carcinoma cell line. Radioimmunotherapy consisted of yttrium-90 (Y-90)-labeled anti-CEA mAb, used either alone or in combination with vaccine therapy. A single dose of Y-90-labeled anti-CEA mAb, in combination with vaccine therapy, resulted in a statistically significant increase in survival in tumor-bearing mice over vaccine or mAb alone; this was shown to be mediated by engagement of the Fas/Fas ligand pathway. Mice receiving the combination therapy also showed a significant increase in the percentage of viable tumor-infiltrating CEA-specific CD8(+) T cells compared to vaccine alone. Mice cured of tumors demonstrated an antigen cascade resulting in CD4(+) and CD8(+) T-cell responses not only for CEA, but for p53 and gp70. These results show that systemic radiotherapy in the form of radiolabeled mAb, in combination with vaccine, promotes effective antitumor response, which may have implications in the design of future clinical trials.     

The natural sulfoglycolipid derivative SQAP improves the therapeutic efficacy of tissue factor-targeted radioimmunotherapy in the stroma-rich pancreatic cancer model BxPC-3

α-Sulfoquinovosylacyl-1,3-propanediol (SQAP) is a semi-synthetic derivative of natural sulfoglycolipid that sensitizes tumors to external-beam radiotherapy. How SQAP affects internal radiotherapy, however, is not known. Here, we investigated the effects of SQAP for radioimmunotherapy (RIT) targeting tissue factor (TF) in a stroma-rich refractory pancreatic cancer mouse model, BxPC-3. A low dose of SQAP (2 mg/kg) increased tumor uptake of the ¹¹¹In-labeled anti-TF antibody 1849, indicating increased tumor perfusion. The addition of SQAP enhanced the growth-inhibitory effect of ⁹⁰Y-labeled 1849 without leading to severe body weight changes, allowing for the dose of ⁹⁰Y-labeled 1849 to be reduced to half that when used alone. Histologic analysis revealed few necrotic and apoptotic cells, but Ki-67–positive proliferating cells and increased vascular formation were detected. These results suggest that the addition of a low dose of SQAP may improve the therapeutic efficacy of TF-targeted RIT by increasing tumor perfusion, even for stroma-rich refractory pancreatic cancer.     

Therapeutic Efficacy of C-Kit-Targeted Radioimmunotherapy Using 90Y-Labeled Anti-C-Kit Antibodies in a Mouse Model of Small Cell Lung Cancer

Small cell lung cancer (SCLC) is an aggressive tumor and prognosis remains poor. Therefore, the development of more effective therapy is needed. We previously reported that high levels of an anti-c-kit antibody (12A8) accumulated in SCLC xenografts. In the present study, we evaluated the efficacy of two antibodies (12A8 and 67A2) for radioimmunotherapy (RIT) of an SCLC mouse model by labeling with the ⁹⁰Y isotope.

Methods

¹¹¹In- or ¹²⁵I-labeled antibodies were evaluated in vitro by cell binding, competitive inhibition and cellular internalization assays in c-kit-expressing SY cells and in vivo by biodistribution in SY-bearing mice. Therapeutic efficacy of ⁹⁰Y-labeled antibodies was evaluated in SY-bearing mice upto day 28 and histological analysis was conducted at day 7.

Results

[¹¹¹In]12A8 and [¹¹¹In]67A2 specifically bound to SY cells with high affinity (8.0 and 1.9 nM, respectively). 67A2 was internalized similar to 12A8. High levels of [¹¹¹In]12A8 and [¹¹¹In]67A2 accumulated in tumors, but not in major organs. [¹¹¹In]67A2 uptake by the tumor was 1.7 times higher than for [¹¹¹In]12A8. [⁹⁰Y]12A8, but not [⁹⁰Y]67A2, suppressed tumor growth in a dose-dependent manner. Tumors treated with 3.7 MBq of [⁹⁰Y]12A8, and 1.85 and 3.7 MBq of [⁹⁰Y]67A2 (absorbed doses were 21.0, 18.0 and 35.9 Gy, respectively) almost completely disappeared approximately 2 weeks after injection, and regrowth was not observed except for in one mouse treated with 1.85 MBq [⁹⁰Y]67A2. The area of necrosis and fibrosis increased depending on the RIT effect. Apoptotic cell numbers increased with increased doses of [⁹⁰Y]12A8, whereas no dose-dependent increase was observed following [⁹⁰Y]67A2 treatment. Body weight was temporarily reduced but all mice tolerated the RIT experiments well.

Conclusion

Treatment with [⁹⁰Y]12A8 and [⁹⁰Y]67A2 achieved a complete therapeutic response when SY tumors received an absorbed dose greater than 18 Gy and thus are promising RIT agents for metastatic SCLC cells at distant sites.     

A versatile bifunctional chelate for radiolabeling humanized anti-CEA antibody with In-111 and Cu-64 at either thiol or amino groups: PET imaging of CEA-positive tumors with whole antibodies

Radiolabeled anti-carcinoembryonic antigen (CEA) antibodies have the potential to give excellent images of a wide variety of human tumors, including tumors of the colon, breast, lung, and medullar thyroid. In order to realize the goals of routine and repetitive clinical imaging with anti-CEA antibodies, it is necessary that the antibodies have a high affinity for CEA, low cross reactivity and uptake in normal tissues, and low immunogenicity. The humanized anti-CEA antibody hT84.66-M5A (M5A) fulfills these criteria with an affinity constant of >10 (10) M (-1), no reactivity with CEA cross-reacting antigens found in normal tissues, and >90% human protein sequence. A further requirement for routine clinical use of radiolabeled antibodies is a versatile method of radiolabeling that allows the use of multiple radionuclides that differ in their radioemissions and half-lives. We describe a versatile bifunctional chelator, DO3A-VS (1,4,7-tris(carboxymethyl)-10-(vinylsulfone)-1,4,7,10-tetraazacyclododecane) that binds a range of radiometals including 111 In for gamma-ray imaging and 64Cu for positron emission tomography (PET), and which can be conjugated with negligible loss of immunoreactivity either to sulfhydryls (SH) in the hinge region of lightly reduced immunoglobulins or surface lysines (NH) of immunoglobulins. Based on our correlative studies comparing the kinetics of radiolabeled anti-CEA antibodies in murine models with those in man, we predict that 64Cu-labeled intact, humanized antibodies can be used to image CEA positive tumors in the clinic.     

Combination therapy using the cyclooxygenase-2 inhibitor Parecoxib and radioimmunotherapy in nude mice with small peritoneal metastases of colonic origin

Background: Inhibition of the COX-2 enzyme has been shown to have a radiosensitizing effect in epithelial cancers. The aim of this study was to investigate whether the efficacy of radioimmunotherapy (RIT) using ¹³¹I-labeled anti-CEA monoclonal antibody MN-14 could be enhanced by co-administration of the selective COX-2 inhibitor Parecoxib in mice with small volume (1–3 mm) peritoneal carcinomatosis of colonic origin. Methods: First, the efficacy of 14 daily injections of Parecoxib monotherapy (0 – 0.2 – 1.0 – 5.0 – 25.0 mg/kg) was determined in mice with intraperitoneal LS174T xenografts. Second, the influence of Parecoxib (1.0 or 5.0 mg/kg) on the biodistribution of ¹²⁵I-MN-14 was assessed. Finally, the efficacy of RIT alone [125 μCi ¹³¹I-MN-14/mouse ≈ 1/4 of the maximal tolerated dose (MTD)] was compared with that of Parecoxib monotherapy and RIT combined with daily injections of Parecoxib (1.0 or 5.0 mg/kg). Results: Parecoxib had no measurable antitumor effect up to the highest dose level (25 mg/kg). Parecoxib had no effect on the uptake of ¹²⁵I-MN-14 in the intraperitoneal tumor xenografts or on normal tissue distribution. Median survival of the control mice and the mice treated with Parecoxib monotherapy (1.0 or 5.0 mg/kg) was 48.5 days, 52 days and 52 days (P=0.47). RIT alone significantly delayed the growth of the intraperitoneal xenografts resulting in a median survival of 87 days (P<0.0001). Mice treated with RIT + Parecoxib at 1.0 or 5.0 mg/kg had a median survival of 73.5 days and 76 days, respectively, which was not statistically different from survival after RIT alone (P=0.15). Conclusion: The COX-2 inhibitor Parecoxib does not enhance the therapeutic efficacy of RIT of experimental small volume peritoneal carcinomatosis of colonic origin.     

Tumor targeting of radiometal labeled anti-CEA recombinant T84.66 diabody and t84.66 minibody: comparison to radioiodinated fragments

Recombinant antibody fragments offer potential advantages over intact monoclonal antibodies in the radioimmunoscintigraphy (RIS) of solid tumors. Due to their smaller molecular size, antibody fragments have shown rapid tumor targeting and blood clearance, a more uniform tumor distribution and a lower potential to elicit a human immune response. Previously, we have expressed two genetically engineered antibody fragments, the T84.66 diabody (scFv dimer) and the T84.66 minibody (scFv-CH3 dimer), specific to carcinoembryonic antigen (CEA). When radioiodinated, both antibody fragments exhibited rapid tumor targeting and rapid blood clearance in xenografted mice. To extend and optimize their future clinical RIS utility with radiometals, these antibody fragments were conjugated with the macrocycle 1,4,7,10-tetraazacyclododecane N,N',N' ',N' "-tetraacetic acid (DOTA) and labeled with 111In. Tumor targeting and biodistribution studies were carried out in athymic mice xenografted with a human colorectal tumor cell line, LS174T. The [111In]T84.66 diabody (55 kDa) exhibited very rapid tumor targeting with 12.5 +/- 0.4% injected dose per gram (% ID g(-1) +/- standard error) at 2 h and reached a maximum of 13.3 +/- 0.9% ID g(-1) at 6 h. However, kidney uptake was observed to reached a peak of 183.5 +/- 21.0% ID g(-1) at 6 h, a result similar to that reported by others for other low molecular weight fragments labeled with radiometals. Preadministration of an oral dose of D-lysine resulted in a 59% lowering of the renal accumulation at 6 h, but was accompanied by a 31% reduction of tumor uptake to 9.2 +/- 1.2% ID g(-1). The second recombinant antibody fragment, the [111In]T84.66 minibody (80 kDa), displayed rapid tumor targeting of 14.2 +/- 6.1% ID g(-1) at 2 h, and reached a maximum activity of 24.5 +/- 6.1% ID g(-1) by 12 h. Renal uptake achieved a plateau of 12-13% ID g(-1) which cleared to 7.2% ID g(-1) at 72 h. However, hepatic uptake was elevated and reached a maximum of 26.0 +/- 1.0% ID g(-1) at 12 h in these xenograft-bearing mice. Experiments in nontumor bearing mice showed a reduction of hepatic activity at 12 h to 16.6 +/- 1.5% ID g(-1), indicative of an intrinsic hepatic accumulation of the [111In]DOTA-T84.66 minibody or metabolites. While the anti-CEA [111In]DOTA-T84.66 diabody and T84.66 minibody retain the rapid tumor targeting properties of the radioiodinated form, the normal organ accumulation (kidneys and liver, respectively) of the [111In]DOTA forms appeared problematic for RIS and RIT applications. Development of alternative blocking strategies or new metabolizable chelates are under investigation to enhance the utility of the radiometal form of these and other promising recombinant antibody fragments.     

Biodistribution and radioimmunotherapy of human breast cancer xenografts with radiometal-labeled DOTA conjugated anti-HER2/neu antibody 4D5

HER2/neu oncogene encodes a 185 kDa trans-membrane protein which is overexpressed in 20-30% of breast and ovarian cancers and portends a poor prognosis. We have studied the targeting and therapy of this oncoprotein with 4D5, a murine monoclonal antibody which recognizes a distinct epitope on the extracelluar domain of HER2/neu. We conjugated the antibody with an active ester of the macrocyclic chelating agent DOTA, radiolabeled the conjugate with either (111)In or (90)Y, and studied the antibody distribution and therapy, respectively, in athymic mice bearing xenografts of MCF7/HER2/neu, a human breast cancer cell line transfected with the HER2/neu oncogene. For the biodistribution of (111)In-labeled DOTA-4D5, a high specificity of tumor localization (30% ID/g) was seen with a tumor-to-blood ratio of greater than 2 at 48 h postinjection. Compared to a previously published study with (125)I-labeled 4D5 in beige nude mice bearing NIH3T3/HER2/neu xenografts [De Santes et al. (1992) Cancer Res. 52, 1916-1923], (111)In-labeled 4D5 antibody gave superior antibody uptake in tumor (30% ID/g vs 17% ID/g at 48h). In the therapy study, treatment of the nude mice bearing MCF7/HER2/neu xenografts with 100 microCi (3 microg) of (90)Y-labeled DOTA-4D5 caused a 3-fold reduction of tumor growth compared to untreated controls (injected with human serum albumin) in 40 days. Treatment of animals with 100 microCi of nonspecific antibody (90)Y-labeled DOTA-Leu16 (3 microg) had no tumor growth inhibition. Treatment with unlabeled DOTA-4D5 (3 microg) had a slight effect on tumor growth compared to untreated controls. When analyzed at the level of single animals, no effect was seen in seven of nine animals; however, in two of the animals, tumor growth inhibition was observed. Although a cold antibody therapeutic effect was unexpected at this dose level (3 microg), it may be possible that in some animals that 3 microg of antibody of (90)Y-labeled DOTA-4D5 augmented tumor growth reduction. To further explore the effects of cold antibody treatment alone, animals were treated with 100 or 400 microg of unlabeled 4D5 administered in two doses. These animals showed a 1.7-1.8-fold reduction in tumor growth over 28 days, a result less than that obtained with RIT only.     

90Y-Labeled Anti-ROBO1 Monoclonal Antibody Exhibits Antitumor Activity against Small Cell Lung Cancer Xenografts

IntroductionROBO1 is a membrane protein that contributes to tumor metastasis and angiogenesis. We previously reported that ⁹⁰Y-labeled anti-ROBO1 monoclonal antibody (⁹⁰Y-anti-ROBO1 IgG) showed an antitumor effect against ROBO1-positive tumors. In this study, we performed a biodistribution study and radioimmunotherapy (RIT) against ROBO1-positive small cell lung cancer (SCLC) models.MethodsFor the biodistribution study, ¹¹¹In-labeled anti-ROBO1 monoclonal antibody (¹¹¹In-anti-ROBO1 IgG) was injected into ROBO1-positive SCLC xenograft mice via the tail vein. To evaluate antitumor effects, an RIT study was performed, and SCLC xenograft mice were treated with ⁹⁰Y-anti-ROBO1 IgG. Tumor volume and body weight were periodically measured throughout the experiments. The tumors and organs of mice were then collected, and a pathological analysis was carried out.ResultsAs a result of the biodistribution study, we observed tumor uptake of ¹¹¹In-anti-ROBO1 IgG. The liver, kidney, spleen, and lung showed comparably high accumulation of ¹¹¹In-labeled anti-ROBO1. In the RIT study, ⁹⁰Y-anti-ROBO1 IgG significantly reduced tumor volume compared with baseline. Pathological analyses of tumors revealed coagulation necrosis and fatal degeneration of tumor cells, significant reduction in the number of Ki-67-positive cells, and an increase in the number of apoptotic cells. A transient reduction of hematopoietic cells was observed in the spleen, sternum, and femur.ConclusionsThese results suggest that RIT with ⁹⁰Y-anti-ROBO1 IgG is a promising treatment for ROBO1-positive SCLC.     

Reduction of kidney uptake in radiometal labeled peptide linkers conjugated to recombinant antibody fragments. Site-specific conjugation of DOTA-peptides to a Cys-diabody

Arano and co-workers (Arano et al. (1999) Cancer Res. 59, 128-134) have synthesized peptides with an N-terminal radioiodinated hippuric acid and a C-terminal lysine linked to antibody fragments via the epsilon-amino group of lysine that show reduced kidney uptake compared to antibody fragments directly radioiodinated. This approach takes advantage of the lysine specific carboxypeptidase activity of the kidney brush border enzymes that cleave off the radiolabeled peptide linker from the antibody fragment prior to uptake by proximal tubule cells. On the basis of their approach, we have synthesized a tetrapeptide with an N-terminal DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) and a C-terminal (N(epsilon)-maleoyl)lysine that was site-specifically conjugated to an anti-CEA diabody (Yazaki et al. (2001) Bioconjugate Chem. 12, 220-228) that was engineered to contain a C-terminal cysteine (Cys-diabody). Biodistributions of the In-111-radiolabeled conjugate in nude mice show significantly reduced kidney uptake (a maximum of 82%ID/g at 6 h) compared to In-111 radiolabeled DOTA-diabody (184%ID/g at 6 h) in which DOTA was conjugated to endogenous lysine residues using DOTA-active ester chemistry. To further reduce kidney uptake, a homologous compound with a C-terminal (N(epsilon)-amino-1,6-hexane-bis-vinyl sulfone)lysine was synthesized and site-specifically conjugated to the Cys-diabody. Biodistributions of this In-111-labeled conjugate reduced kidney uptake to 54%ID/g at 6 h. To explore the effect of the relative positions of the chelate vs the cys-diabody on kidney uptake, we also synthesized a tetrapeptide with an N-terminal bromoacetate for conjugation to Cys-diabody and a C-terminal (N(epsilon)-amidino-propyl-3-thio-vinylsulfonyl-DO3A)lysine. This peptide essentially reverses the positions of the chelate and Cys-diabody attachment points on the peptide, while retaining the linker length on the epsilon-amino group of the lysine. In this case, biodistributions of the In-111-radiolabeled conjugate in nude mice showed high kidney uptake (189%ID/g at 6 h), comparable to that obtained with the In-111-radiolabeled active ester conjugated DOTA-diabody (184%ID/g at 6 h). We conclude that the peptide linker strategy of Arano and co-workers to reduce kidney uptake can be successfully applied to chelate/radiometal complexes and requires that the chelate/radiometal be located at the N-terminus of the peptide and the antibody fragment attachment site on the epsilon-amino group of the lysine. Furthermore, we demonstrated a role for the attachment chemistry to the epsilon-amino group of the lysine on the magnitude of kidney uptake.     

In vivo therapeutic effect of CDH3/P-cadherin-targeting radioimmunotherapy

Purpose

We examined the possible efficacy of the yttrium-90 (⁹⁰Y)-labeled anti-CDH3/P-cadherin mouse monoclonal antibody (MAb-6) in radioimmunotherapy (RIT) for lung and colorectal cancers that express CDH3/P-cadherin.

Experimental design

MAb-6 was established using genetic immunization. The biodistribution of MAb-6 in nude mice with lung and colorectal cancers was examined by administering indium-111(¹¹¹In)-labeled MAb-6 to mice. The mice were prepared by inoculation of CDH3/P-cadherin-positive (EBC1, H1373, and SW948) and CDH3/P-cadherin-negative (A549 and RKO) tumor cells. Therapeutic effects and toxicity were investigated by administration of ⁹⁰Y-labeled MAb-6 (⁹⁰Y-MAb-6) to EBC, H1373, and SW948-inoculated mice.

Results

Our in vivo results confirmed the specific binding of MAb-6 to tumor cells after intravenous injections of ¹¹¹In-labeled MAb-6 to mice with tumors expressing CDH3/P-cadherin. A single intravenous injection of ⁹⁰Y-MAb-6 (100?μCi) significantly suppressed tumor growth in mice with tumors expressing CDH3/P-cadherin. Furthermore, two injections of ⁹⁰Y-MAb-6 led to complete tumor regression in H1373-inoculated mice without any detectable toxicity.

Conclusions

Our findings demonstrate that CDH3/P-cadherin-targeting RIT with ⁹⁰Y-MAb-6 is a promising strategy for the treatment for cancers expressing CDH3/P-cadherin.     

Simultaneous determination of alpha-fetoprotein and carcinoembryonic antigen in human serum by time-resolved fluoroimmunoassay

A novel simultaneous measurement method for alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA) in human sera by time-resolved fluoroimmunoassay (TR-FIA) is described. The proposed approach combines the use of europium-labeled anti-AFP antibody for AFP TR-FIA and biotinylated anti-CEA antibody complexed to samarium-labeled streptavidin for CEA TR-FIA. A 96-well microtiter plate coated with a mixture of anti-AFP and anti-CEA monoclonal antibodies was used for the assay. After it was reacted with a solution containing AFP and CEA, a mixture of anti-AFP antibody labeled with BHHCT-Eu(3+) and biotinylated anti-CEA antibody was added. The AFP concentration was determined by measuring the solid-phase fluorescence of the europium-labeled anti-AFP antibody at 615 nm. Then a BHHCT-Sm(3+)-labeled streptavidin-bovine serum albumin conjugate (SA-BSA) was added to react with the biotinylated anti-CEA antibody. After the reaction, the unreacted SA-BSA was washed out, and a 0.1 M NaOH solution containing 1.0 x 10(-5) M TOPO and 0.05% SDS was added to dissociate the samarium-labeled SA-BSA in the immune complex on the surface of the well into the solution. The CEA concentration was determined by measuring the solution fluorescence of 643 nm from the samarium-labeled SA-BSA. The present method gives detection limits of 0.07 ng/ml for AFP and 0.3 ng/ml for CEA. The coefficient variations of the method are less than 7%, and the recoveries are in the range of 90-110% for serum samples. The AFP and CEA concentrations in 27 human serum samples were determined by the present method as well as by single assay for comparison. A good correlation was obtained with the correlation coefficients of 0.990 for AFP and 0.973 for CEA.     

Comparison of CEA distribution in lesions and tumors of salivary glands as determined with monoclonal and polyclonal antibodies

Immunohistochemical localization of carcinoembryonic antigen (CEA) with conventional antibody to CEA (anti-CEA), nonspecific crossreacting antigen (NCA)-absorbed polyclonal antibody to CEA (NCAa-CEA), and monoclonal antibody to CEA (Mono-CEA) have been compared in obstructive lesions and salivary gland tumors. Normal salivary glands gave strong staining of the luminal borders of acinar cells with anti-CEA, whereas no staining occurred with Mono-CEA. Obstructive lesions showed occasionally marked staining with anti-CEA in some acinar cells, but there was no reaction with Mono-CEA. Of 69 pleomorphic adenomas examined, 34 were positively stained with anti-CEA, 18 with NCAa-CEA and 8 with Mono-CEA along the luminal borders of the tumor cells. The frequency of positive staining of material within tubular lumina was similar with all three immunoreagents. Neoplastic cells were positive with Mono-CEA in only three cases, while eight cases were positive with NCAa-CEA and 11 cases with anti-CEA. In salivary gland tumors true CEA is be found mainly at the border of tumor cells, but the frequency of positive reactions is low.     

Anti-CEA and other antibodies in the study of gastrointestinal tumors.

Localization of gastrointestinal tumors by means of labeled monoclonal antibodies is a new, sensitive and suitable technique currently used in several centers. Encouraging results have been documented with several monoclonal antibodies by different authors. This article reviews our experience with radioimmunoscintigraphy in 59 patients with colorectal cancer in follow-up, using 131I and 111In labeled B72.3, and in 16 patients with primary gastrointestinal tumors using 99mTc anti-CEA monoclonal antibody (type F023C5). The sensitivity of both B72.3 and anti-CEA was greater than 70% either for primary tumors and abdominal recurrences or distant metastases except hepatic ones. A significant gradient in antibody uptake was measured on surgical biopsies between tumors and normal tissues allowing a good in vivo contrast for gamma detection. We have defined the impact of some factors affecting in vivo tumor targeting. In fact, pharmacodynamics of MAbs, percentage of injected dose bound to tissues were measured, and in particular antigenic content in tumor nodules was quantified. Furthermore, the results of RIS were compared to those obtained by CT and other imaging modalities.     

A Single Domain–Based Anti-Her2 Antibody Has Potent Antitumor Activities

Human epidermal growth factor receptor 2 (HER2) is overexpressed in approximately 20% to 30% of breast cancers and various other types of cancers, which plays a vital role in the cancer progression. Monoclonal antibodies targeting Her2 are now used in the clinic to treat Her2 overexpression cancer patients. However, relapse or resistance is frequent with the current therapies. To generate a new treatment avenue against Her2, we immunized and selected a specific anti-Her2 single domain antibody C3 for further studies. The C3-Fc antibody drove antibody-dependent cell-mediated cytotoxicity against Her2-positive tumor cells in vitro and resulted in potent antitumor growth in vivo. These data suggest that the C3-Fc antibody may provide an alternative avenue for Her2-positive cancer therapy.     

Trifunctional antibody ertumaxomab: Non-immunological effects on Her2 receptor activity and downstream signaling

Background: The trifunctional antibody ertumaxomab bivalently targets the human epidermal growth factor receptor 2 (Her2) on epithelial (tumor) cells and the T cell specific CD3 antigen, and its Fc region is selectively recognized by Fcγ type I/III receptor-positive immune cells. As a trifunctional immunoglobulin, ertumaxomab therefore not only targets Her2 on cancer cells, but also triggers immunological effector mechanisms mediated by T and accessory cells (e.g., macrophages, dendritic cells, natural killer cells). Whether molecular effects, however, might contribute to the cellular antitumor efficiency of ertumaxomab are largely unknown. Methods: Potential molecular effects of ertumaxomab on Her2-overexpressing BT474 and SK-BR-3 breast cancer cells were evaluated. The dissociation constant K_d of ertumaxomab was calculated from titration curves that were recorded by flow cytometry. Treatment-induced changes in Her2 homodimerization were determined by flow cytometric fluorescence resonance energy transfer measurements on a cell-by-cell basis. Potential activation / deactivation of Her2, ERK1/2, AKT and STAT3 were analyzed by western blotting, Immunochemistry and immunofluorescent cell staining.Results: The K_d of ertumaxomab for Her2-binding was determined at 265 nM and the ertumaxomab binding epitope was found to not overlap with that of the therapeutic anti-Her2 monoclonal antibodies trastuzumab and pertuzumab. Ertumaxomab caused an increase in Her2 phosphorylation at higher antibody concentrations, but changed neither the rate of Her2-homodimerization /-phosphorylation nor the activation state of key downstream signaling proteins analyzed.Conclusions: The unique mode of action of ertumaxomab, which relies more on activation of immune-mediated mechanisms against tumor cells compared with currently available therapeutic antibodies for breast cancer treatment, suggests that modular or sequential treatment with the trifunctional bivalent antibody might complement the therapeutic activity of other anti-Her2/anti-ErbB receptor reagents.     

Radioimmunotherapy for breast cancer: treatment of a patient with I-131 L6 chimeric monoclonal antibody.

We report the first treatment of metastatic breast cancer by systemic radioimmunotherapy. The serial therapy doses were chosen based on quantitative imaging data in a treatment planning approach. A terminally ill patient with aggressive, locally advanced breast cancer who had failed radiation treatment and chemotherapy was injected intravenously with radiolabeled I-131 chimeric L6, a human-mouse chimeric lgG1 monoclonal antibody to adenocarcinoma. Initially, an imaging 10 mCi dose of I-131 chimeric L6 (dose 1) deposited 8.8% of the injected dose in her chest wall tumor at 48 hours. Ten days later the patient was given a 150 mCi I-131 chimeric L6 dose (dose 2) followed three weeks later by a 100 mCi dose (dose 3). Tumor uptake and retention were comparable for doses 1 and 2, and decreased for dose 3. Following dose 3 the patient developed a manageable thrombocytopenia and transient Grade IV granulocytopenia. The tumor was observed to decrease in size with peak tumor regression occurring two weeks after dose 3. This partial response (PR) was achieved by radioimmunotherapy at a time when conventional therapy had been unable to impact the growth of the patient's massive and aggressive tumor.     

Trifunctional antibody ertumaxomab: Non-immunological effects on Her2 receptor activity and downstream signaling

Background: The trifunctional antibody ertumaxomab bivalently targets the human epidermal growth factor receptor 2 (Her2) on epithelial (tumor) cells and the T cell specific CD3 antigen, and its Fc region is selectively recognized by Fcγ type I/III receptor-positive immune cells. As a trifunctional immunoglobulin, ertumaxomab therefore not only targets Her2 on cancer cells, but also triggers immunological effector mechanisms mediated by T and accessory cells (e.g., macrophages, dendritic cells, natural killer cells). Whether molecular effects, however, might contribute to the cellular antitumor efficiency of ertumaxomab are largely unknown.

Methods: Potential molecular effects of ertumaxomab on Her2-overexpressing BT474 and SK-BR-3 breast cancer cells were evaluated. The dissociation constant K_d of ertumaxomab was calculated from titration curves that were recorded by flow cytometry. Treatment-induced changes in Her2 homodimerization were determined by flow cytometric fluorescence resonance energy transfer measurements on a cell-by-cell basis. Potential activation / deactivation of Her2, ERK1/2, AKT and STAT3 were analyzed by western blotting, Immunochemistry and immunofluorescent cell staining.

Results: The K_d of ertumaxomab for Her2-binding was determined at 265 nM and the ertumaxomab binding epitope was found to not overlap with that of the therapeutic anti-Her2 monoclonal antibodies trastuzumab and pertuzumab. Ertumaxomab caused an increase in Her2 phosphorylation at higher antibody concentrations, but changed neither the rate of Her2-homodimerization /-phosphorylation nor the activation state of key downstream signaling proteins analyzed.

Conclusions: The unique mode of action of ertumaxomab, which relies more on activation of immune-mediated mechanisms against tumor cells compared with currently available therapeutic antibodies for breast cancer treatment, suggests that modular or sequential treatment with the trifunctional bivalent antibody might complement the therapeutic activity of other anti-Her2/anti-ErbB receptor reagents.     

Optimal antibody-radionuclide combinations for clinical radioimmunotherapy: a predictive model based on mouse pharmacokinetics

A theoretical comparison was made of radioimmunotherapy (RIT) dosimetry estimates for eight radionuclides (⁹⁰Y,¹⁰⁵Rh, ¹³¹I, ¹⁵³Sm, ¹⁸⁶Re, ¹⁸⁸Re,¹⁹⁸Au, ²¹¹At) conjugated to IgG, F(ab′)₂, and Fab antibody forms. Antibody pharmacokinetics, derived from a nude mouse animal model were combined with appropriate physical data and S values to evaluate absorbed dose to a 0.5 kg centrally located tumor, total body and kidney. Radioimmunoconjugates of F(ab′)₂ with ⁹⁰Y, ¹⁵³Sm and ¹⁸⁶Re were predicted to be the most promising for RIT.     

In Vitro and In Vivo Evaluation of Cysteine and Site Specific Conjugated Herceptin Antibody-Drug Conjugates

Antibody drug conjugates (ADCs) are monoclonal antibodies designed to deliver a cytotoxic drug selectively to antigen expressing cells. Several components of an ADC including the selection of the antibody, the linker, the cytotoxic drug payload and the site of attachment used to attach the drug to the antibody are critical to the activity and development of the ADC.The cytotoxic drugs or payloads used to make ADCs are typically conjugated to the antibody through cysteine or lysine residues. This results in ADCs that have a heterogeneous number of drugs per antibody. The number of drugs per antibody commonly referred to as the drug to antibody ratio (DAR), can vary between 0 and 8 drugs for a IgG₁ antibody. Antibodies with 0 drugs are ineffective and compete with the ADC for binding to the antigen expressing cells. Antibodies with 8 drugs per antibody have reduced in vivo stability, which may contribute to non target related toxicities.In these studies we incorporated a non-natural amino acid, para acetyl phenylalanine, at two unique sites within an antibody against Her2/neu. We covalently attached a cytotoxic drug to these sites to form an ADC which contains two drugs per antibody.We report the results from the first direct preclinical comparison of a site specific non-natural amino acid anti-Her2 ADC and a cysteine conjugated anti-Her2 ADC. We report that the site specific non-natural amino acid anti-Her2 ADCs have superior in vitro serum stability and preclinical toxicology profile in rats as compared to the cysteine conjugated anti-Her2 ADCs. We also demonstrate that the site specific non-natural amino acid anti-Her2 ADCs maintain their in vitro potency and in vivo efficacy against Her2 expressing human tumor cell lines. Our data suggests that site specific non-natural amino acid ADCs may have a superior therapeutic window than cysteine conjugated ADCs.