Sacituzumab govitecan, a Trop-2-directed antibody-drug conjugate, for patients with epithelial cancer: final safety and efficacy results from the phase I/II IMMU-132-01 basket trial.

BACKGROUND: Sacituzumab govitecan (SG), a trophoblast cell surface antigen-2 (Trop-2)-directed antibody-drug conjugate, has demonstrated antitumor efficacy and acceptable tolerability in a phase I/II multicenter trial (NCT01631552) in patients with advanced epithelial cancers. This report summarizes the safety data from the overall safety population (OSP) and efficacy data, including additional disease cohorts not published previously. PATIENTS AND METHODS: Patients with refractory metastatic epithelial cancers received intravenous SG (8, 10, 12, or 18 mg/kg) on days 1 and 8 of 21-day cycles until disease progression or unacceptable toxicity. Endpoints for the OSP included safety and pharmacokinetic parameters with investigator-evaluated objective response rate (ORR per RECIST 1.1), duration of response, clinical benefit rate, progression-free survival, and overall survival evaluated for cohorts (n > 10 patients) of small-cell lung, colorectal, esophageal, endometrial, pancreatic ductal adenocarcinoma, and castrate-resistant prostate cancer. RESULTS: In the OSP (n = 495, median age 61 years, 68% female; UGT1A1∗28 homozygous, n = 46; 9.3%), 41 (8.3%) permanently discontinued treatment due to adverse events (AEs). Most common treatment-related AEs were nausea (62.6%), diarrhea (56.2%), fatigue (48.3%), alopecia (40.4%), and neutropenia (57.8%). Most common treatment-related serious AEs (n = 75; 15.2%) were febrile neutropenia (4.0%) and diarrhea (2.8%). Grade ≥3 neutropenia and febrile neutropenia occurred in 42.4% and 5.3% of patients, respectively. Neutropenia (all grades) was numerically more frequent in UGT1A1∗28 homozygotes (28/46; 60.9%) than heterozygotes (69/180; 38.3%) or UGT1A1∗1 wild type (59/177; 33.3%). There was one treatment-related death due to an AE of aspiration pneumonia. Partial responses were seen in endometrial cancer (4/18, 22.2% ORR) and small-cell lung cancer (11/62, 17.7% ORR), and one castrate-resistant prostate cancer patient had a complete response (n = 1/11; 9.1% ORR). CONCLUSIONS: SG demonstrated a toxicity profile consistent with previous published reports. Efficacy was seen in several cancer cohorts, which validates Trop-2 as a broad target in solid tumors.

Promising clinical performance of pretargeted immuno-PET with anti-CEA bispecific antibody and gallium-68-labelled IMP-288 peptide for imaging colorectal cancer metastases: a pilot study.

INTRODUCTION: This pilot study evaluated the imaging performance of pretargeted immunological positron emission tomography (immuno-PET) using an anti-carcinoembryonic antigen (CEA) recombinant bispecific monoclonal antibody (BsMAb), TF2 and the [68Ga]Ga-labelled HSG peptide, IMP288, in patients with metastatic colorectal carcinoma (CRC). PATIENTS AND METHODS: Patients requiring diagnostic workup of CRC metastases or in case of elevated CEA for surveillance were prospectively studied. They had to present with elevated CEA serum titre or positive CEA tumour staining by immunohistochemistry of a previous biopsy or surgical specimen. All patients underwent endoscopic ultrasound (EUS), chest-abdominal-pelvic computed tomography (CT), abdominal magnetic resonance imaging (MRI) and positron emission tomography using [18F]fluorodeoxyglucose (FDG-PET). For immuno-PET, patients received intravenously 120 nmol of TF2 followed 30 h later by 150 MBq of [68Ga]Ga-labelled IMP288, both I.V. The gold standard was histology and imaging after 6-month follow-up. RESULTS: Eleven patients were included. No adverse effects were reported after BsMAb and peptide injections. In a per-patient analysis, immuno-PET was positive in 9/11 patients. On a per-lesion analysis, 12 of 14 lesions were positive with immuno-PET. Median SUVmax, MTV and TLG were 7.65 [3.98-13.94, SD 3.37], 8.63 cm3 [1.98-46.64; SD 14.83] and 37.90 cm3 [8.07-127.5; SD 43.47] respectively for immuno-PET lesions. Based on a per-lesion analysis, the sensitivity, specificity, positive-predictive value and negative-predictive value were, respectively, 82%, 25%, 82% and 25% for the combination of EUS/CT/MRI; 76%, 67%, 87% and 33% for FDG-PET; and 88%, 100%, 100% and 67% for immuno-PET. Immuno-PET had an impact on management in 2 patients. CONCLUSION: This pilot study showed that pretargeted immuno-PET using anti-CEA/anti-IMP288 BsMAb and a [68Ga]Ga-labelled hapten was safe and feasible, with promising diagnostic performance. TRIAL REGISTRATION: ClinicalTrials.gov NCT02587247 Registered 27 October 2015.

Sacituzumab govitecan in previously treated hormone receptor-positive/HER2-negative metastatic breast cancer: final results from a phase I/II, single-arm, basket trial.

BACKGROUND: Trophoblast cell-surface antigen-2 (Trop-2) is expressed in epithelial cancers, including hormone receptor-positive (HR+) metastatic breast cancer (mBC). Sacituzumab govitecan (SG; Trodelvy®) is an antibody-drug conjugate composed of a humanized anti-Trop-2 monoclonal antibody coupled to SN-38 at a high drug-to-antibody ratio via a unique hydrolyzable linker that delivers SN-38 intracellularly and in the tumor microenvironment. SG was granted accelerated FDA approval for metastatic triple-negative BC treatment in April 2020. PATIENTS AND METHODS: We analyzed a prespecified subpopulation of patients with HR+/human epidermal growth factor receptor 2-negative (HER2-) HR+/HER2- mBC from the phase I/II, single-arm trial (NCT01631552), who received intravenous SG (10 mg/kg) and whose disease progressed on endocrine-based therapy and at least one prior chemotherapy for mBC. End points included objective response rate (ORR; RECIST version 1.1) assessed locally, duration of response (DOR), clinical benefit rate, progression-free survival (PFS), overall survival (OS), and safety. RESULTS: Fifty-four women were enrolled between 13 February 2015 and 1 June 2017. Median (range) age was 54 (33-79) years and all received at least two prior lines of therapy for mBC. At data cut-off (1 March 2019), 12 patients were still alive. Key grade ≥3 treatment-related toxicities included neutropenia (50.0%), anemia (11.1%), and diarrhea (7.4%). Two patients discontinued treatment due to treatment-related adverse events. No treatment-related deaths occurred. At a median follow-up of 11.5 months, the ORR was 31.5% [95% confidence interval (CI), 19.5%-45.6%; 17 partial responses]; median DOR was 8.7 months (95% CI 3.7-12.7), median PFS was 5.5 months (95% CI 3.6-7.6), and median OS was 12 months (95% CI 9.0-18.2). CONCLUSIONS: SG shows encouraging activity in patients with pretreated HR+/HER2- mBC and a predictable, manageable safety profile. Further evaluation in a randomized phase III trial (TROPiCS-02) is ongoing (NCT03901339). TRIAL REGISTRATION: ClinicalTrials.gov NCT01631552; https://clinicaltrials.gov/ct2/show/NCT01631552.

Anti-CEA antibody fragments labeled with [(18)F]AlF for PET imaging of CEA-expressing tumors.

A facile and rapid method to label peptides with (18)F based on chelation of [(18)F]AlF has been developed recently. Since this method requires heating to 100 °C, it cannot be used to label heat-sensitive proteins. Here, we used a two-step procedure to prepare (18)F-labeled heat-labile proteins using the [(18)F]AlF method based on hot maleimide conjugation. 1,4,7-Triazacyclononae-1,4-diacetate (NODA) containing a methyl phenylacetic acid group (MPA) functionalized with N-(2-aminoethyl)maleimide (EM) was used as a ligand which was labeled with [(18)F]AlF and then conjugated to the humanized anti-CEA antibody derivatives hMN-14-Fab', hMN-14-(scFv)2 (diabody), and a Dock-and-Lock engineered dimeric fragment hMN-14 Fab-AD2 at room temperature. The in vivo tumor targeting characteristics of the (18)F-labeled antibody derivatives were determined by PET imaging of mice with s.c. xenografts. NODA-MPAEM was radiolabeled with [(18)F]AlF at a specific activity of 29-39 MBq/nmol and a labeling efficiency of 94 ± 2%. The labeling efficiencies of the maleimide conjugation ranged from 70% to 77%, resulting in [(18)F]AlF-labeled hMN14-Fab', hMN14-Fab-AD2, or hMN14-diabody with a specific activity of 15-17 MBq/nmol. The radiolabeled conjugates were purified by gel filtration. For biodistribution and microPET imaging, antibody fragments were injected intravenously into BALB/c nude mice with s.c. CEA-expressing LS174T xenografts (right flank) and CEA-negative SK-RC-52 xenografts (left flank). All [(18)F]AlF-labeled conjugates showed specific uptake in the LS174T xenografts with a maximal tumor uptake of 4.73% ID/g at 4 h after injection. Uptake in CEA-negative SK-RC-52 xenografts was significantly lower. Tumors were clearly visualized on microPET images. Using a [(18)F]AlF-labeled maleimide functionalized chelator, antibody fragments could be radiofluorinated within 4 h at high specific activity. Here, we translated this method to preclinical PET imaging studies and showed feasibility of [(18)F]AlF-fluorinated hMN-14-Fab', [(18)F]AlF-hMN-14-Fab-AD2, and [(18)F]AlF-hMN-14-diabody for microPET imaging of CEA-expressing colonic cancer.

Development of an imaging-guided CEA-pretargeted radionuclide treatment of advanced colorectal cancer: first clinical results.

BACKGROUND: Radiolabelled antibody targeting of cancer is limited by slow blood clearance. Pretargeting with a non-radiolabelled bispecific monoclonal antibody (bsMAb) followed by a rapidly clearing radiolabelled hapten peptide improves tumour localisation. The primary goals of this first pretargeting study in patients with the anti-CEACAM5 × anti-hapten (HSG) bsMAb, TF2, and the radiolabelled hapten-peptide, IMP288, were to assess optimal pretargeting conditions and safety in patients with metastatic colorectal cancer (CRC). METHODS: Different dose schedules were studied in four cohorts of five patients: (1) shortening the interval between the bsMAb and peptide administration (5 days vs 1 day), (2) escalating the TF2 dose (from 75 to 150 mg), and (3) reducing the peptide dose (from 100 to 25 µg). After confirmation of tumour targeting by (111)In-IMP288, patients were treated with a bsMAb/(177)Lu-IMP288 cycle. RESULTS: Rapid and selective tumour targeting of the radiolabelled peptide was visualised within 1 h, with high tumour-to-tissue ratios (>20 at 24 h). Improved tumour targeting was achieved with a 1-day interval between the administration of the bsMAb and the peptide and with the 25-µg peptide dose. High (177)Lu-IMP288 doses (2.5-7.4 GBq) were well tolerated, with some manageable TF2 infusion reactions, and transient grades 3-4 thrombocytopaenia in 10% of the patients who received (177)Lu-IMP288. CONCLUSION: This phase I study demonstrates for the first time that pretargeting with bsMAb TF2 and radiolabelled IMP288 in patients with CEA-expressing CRC is feasible and safe. With this pretargeting method, tumours are specifically and rapidly targeted.

Novel radiolabeled antibody conjugates.

This article reviews the development of radioimmunoconjugates as a new class of cancer therapeutics. Numerous conjugates involving different antigen targets, antibody forms, radionuclides and methods of radiochemistry have been studied in the half-century since radioactive antibodies were first used in model systems to selectively target radiation to tumors. Whereas directly conjugated antibodies, fragments and subfragments have shown promise preclinically, the same approaches have not gained success in patients except in radiosensitive hematological neoplasms, or in settings involving minimal or locoregional disease. The separation of tumor targeting from the delivery of the therapeutic radionuclide in a multistep process called pretargeting has the potential to overcome many of the limitations of conventional, or one-step, radioimmunotherapy, with initial preclinical and clinical data showing increased sensitivity, specificity and higher radiation doses delivered. Our particular focus in pretargeting is the use of bispecific, trimeric (three Fab's) constructs made by a new antibody engineering method termed 'dock-and-lock.

Improved therapy of non-Hodgkin's lymphoma xenografts using radionuclides pretargeted with a new anti-CD20 bispecific antibody.

A comparison of the therapeutic efficacy of a new bispecific monoclonal antibody (bsMAb)-pretargeting system vs the conventional direct targeting modality was undertaken. A bsMAb was made by coupling the Fab' of a humanized anti-CD20 antibody to the Fab' of a murine antibody directed against the peptide histamine-succinyl-glycine (HSG). The tumor targeting of the bsMAb was separated from the subsequent delivery of the radionuclide-bearing HSG peptide conjugated with (111)In or (90)Y. Nude mice bearing s.c. Ramos human B-cell lymphomas were injected with the bsMAb and then, 48 h later, (111)In/(90)Y-HSG peptide was given. At 3 h postinjection, tumor/blood ratios for pretargeted (111)In-HSG-peptide were similar to that observed with the directly conjugated (111)In-anti-CD20 IgG at its highest level on day 7, but by day 1, tumor/blood ratios were about 10-fold higher than the IgG. Tumors progressed rapidly in animals given 800 microCi of (90)Y-HSG peptide alone, whereas 5/10 animals in the group pretargeted by the anti-CD20 bsMAb were tumor-free 18 weeks later. The antitumor response in animals administered the pretargeted (90)Y-HSG peptide was also significantly superior to treatment with the directly radiolabeled (90)Y-anti-CD20 IgG, whether given as a single injection (P<0.007) or as a divided dose (P=0.016). This bsMAb-pretargeting procedure significantly improves the therapeutic response of targeted radionuclides in non-Hodgkin's lymphoma, warranting further development of this method of radioimmunotherapy.

Dose escalation of radioantibody in a mouse model with the use of recombinant human interleukin-1 and granulocyte-macrophage colony-stimulating factor intervention to reduce myelosuppression.

BACKGROUND: In previous studies in a tumor-bearing hamster model, we demonstrated protection and rescue from radioantibody-induced hematopoietic toxicity by treatment with interleukin-1 (IL-1) before or after radioantibody treatment, as well as attenuation of duration of myelosuppression by administration of granulocyte-macrophage colony-stimulating factor (GM-CSF). PURPOSE: The purpose of this study was to evaluate the ability of recombinant human IL-1 and recombinant murine GM-CSF to reduce myelosuppression and increase survival of non-tumor-bearing, female BALB/c mice while escalating the maximal tolerated dose (MTD) of radioantibody--the highest dose that results in no deaths. METHODS: We administered IL-1 for 7 days at 1 x 10(3) U twice a day and GM-CSF starting on the same day for 12 days at a dose of 0.5 micrograms twice a day, alone or in combination. The doses of iodine 131 (131I)-NP-4 IgG (anti-carcinoembryonic antigen monoclonal antibody) radioantibody used were 270, 340, and 370 microCi; the MTD in mice is 270 microCi. The 12-day schedule of cytokine administration was initiated at various times with respect to the radioantibody dose: on the same day; 6 or 3 days before radioantibody; or 3, 6, or 9 days after radioantibody. Treatment efficacy was measured by survival and white blood cell and platelet counts. RESULTS: A 25% increase to 340 microCi of radioantibody used alone resulted in 100% lethality within 25 days of treatment. The optimal cytokine schedule was a 12-day treatment with the combination of cytokines initiated 3 days before radioantibody. This treatment resulted in 100% survival and significantly reduced the magnitude and duration of hematopoietic toxicity. The increase in radioantibody dose resulted in an 85%-95% decrease in peripheral white blood cells and a 75%-85% reduction in platelets within 14 days of radioantibody administration. Further dose escalation to 370 microCi of radioantibody used alone (37% increase above the MTD) resulted in lethality to 12% of the mice. IL-1 or GM-CSF alone was minimally effective. CONCLUSIONS: These studies are the first demonstration that cytokines could be used to reduce radioantibody-induced leukopenia and thrombocytopenia and to escalate the tolerated dose of radioantibody by 25%. IMPLICATIONS: We plan to evaluate the potential therapeutic benefit of a 25% increase in radioantibody dose in a tumor-bearing mouse model.

Carcinoembryonic antigen in histopathology: immunoperoxidase staining of conventional tissue sections.

A triple-bridge, indirect peroxidase-antiperoxidase method for demonstrating carcinoembryonic antigen (CEA) in frozen, ethanol-fixed or formalin-fixed, paraffin-embedded specimens was evaluated. Examination of 359 tissue specimens--234 malignant tumors, 37 benign neoplasms, 41 nonneoplastic diseased tissues, and 47 normal specimens--showed that CEA could usually be demonstrated in a group of cancers. We could detect CEA in carcinomas of the stomach, colon, rectum, pancreas, lung, and cervix. However, malignant tumors of the breast, prostate, kidney, larynx, brain, lymphoreticular system, soft tissues, and skin proved negative for CEA by the immunoperoxidase test. CEA could be detected in ethanol- or formalin-fixed sections. The only nonmalignant specimens showing CEA staining were a few benign tumors, the mucosae of some cases of colitis, and the resection margins of 2 cases of colon cancer; however, these were commonly very weak reactions. Measurement of tumor CEA content by radioimmunoassay revealed two causes for this relative specificity of the immunoperoxidase test for CEA:1) a quantitative difference existed in tissue CEA among the various specimens, and 2) the threshold for CEA staining in malignant specimens was usually above that in nonmalignant specimens. An analysis of the formalin-paraffin-treated sections showed that immunoperoxidase-tested CEA positivity reflected CEA levels in tissue of at least 3.0-5.0 mug/g; this permitted retrospective estimates of minimal tissue CEA concentrations in older histopathologic specimens by the immunoperoxidase reaction method. Formalin-paraffin-treated sections as old as 10 years still had demonstrable CEA. Although tumor CEA concentration correlated well with immunoperoxidase staining for CEA, plasma CEA titer did not necessarily reflect tumor CEA content. CEA positivity in primary and secondary tumors was strongly correlated; it was less strongly correlated with level of tumor differentiation.

Reduction by anti-antibody administration of the radiotoxicity associated with 131I-labeled antibody to carcinoembryonic antigen in cancer radioimmunotherapy.

Radiolabeled antibodies have been shown to have a therapeutic potential in tumor-bearing animal models. However, treatment with radiolabeled antibodies results in toxic effects to normal tissues, as monitored by losses in body weight and in peripheral wbcs. We have investigated the use of an anti-antibody, or second antibody (SA), as a means of reducing this toxicity. SA rapidly forms a complex with circulating radiolabeled antibody, causing an increase in the clearance rate of the radiolabeled antibody from the blood. Toxicity was significantly reduced in animals given the SA in comparison to the toxicity seen in animals given only the radiolabeled primary antibody (PA). The earlier the SA was administered, the lower was the toxicity. The therapeutic efficacy of radiolabeled antibody was not influenced by the administration of the SA given 48 hours after the PA. Thus, the controlled removal of circulating radiolabeled antibody by an anti-antibody can reduce the toxicity associated with radiolabeled antibody therapy without influencing the antitumor effect.

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

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

Targeting of xenografted pancreatic cancer with a new monoclonal antibody, PAM4.

We have examined the ability of murine monoclonal antibody PAM4, directed against a pancreatic cancer-derived mucin, to target human pancreatic cancers carried as xenografts in athymic nude mice. Four tumor lines were used representing the range of expected differentiation; CaPan1, AsPc1, Hs766T, and BxPc3. In each case tumor uptake of PAM4 (range, 21-48% injected dose/g on day 3) was significantly higher than concomitantly administered, nonspecific, isotype-matched Ag8 antibody (range, 3.6-9.3% injected dose/g on day 3). Based upon the biodistribution data the estimated potential radiation dose delivered to the tumors when normalized to the blood dose as an estimate of dose-limiting myelotoxicity would be 13.1-, 2.2-, 3.4-, and 3.3-fold higher than to blood, respectively. PAM4 showed no evidence of targeting to normal tissues, except within the CaPan1 tumor model, where a small but consistent splenic uptake was observed. Splenic targeting was abolished by use of an increased PAM4 protein dose. Targeting of PAM4 to other normal tissues was not affected by the increased protein dose; however, tumor uptake of PAM4 (percentage of injected dose/g) was significantly increased by as much as 3-fold. The ability of PAM4 to target the CaPan1 tumor compared favorably to that of MN14, an anti-carcinoembryonic antigen murine monoclonal antibody. Tumor uptake of PAM4 was much greater than that for MN14 at days 1 and 3, whereas at later time points equivalent accumulations of activity were noted. Estimates of potential radiation doses to the tumor when normalized to the blood dose were 3.0 for MN14 and 9.6 for PAM4. These studies have shown that PAM4 is able to target pancreatic cancer with high specificity, achieving high concentrations at the tumor site. A rationale exists, then, for the performance of a clinical trial of radiolabeled PAM4 in the detection and localization of pancreatic cancer.

Factors influencing anti-antibody enhancement of tumor targeting with antibodies in hamsters with human colonic tumor xenografts.

The injection of an antiantibody (second antibody, SA) can enhance the clearance rate of a radiolabeled antitumor antibody (primary antibody, PA) from the blood. We have studied how the dose of the SA and the timing of the SA administration influence the rate of PA clearance and thereby improve tumor/nontumor ratios. Adult hamsters bearing the carcinoembryonic antigen-producing, GW-39 human colonic tumor xenograft were given injections of 131I-labeled, goat anti-carcinoembryonic antigen antibody, and after 6, 24, or 48 h, an injection of donkey anti-goat immunoglobulin was given at SA:PA ratios of 25, 50, 100, or 200:1. In comparison to a control group of animals that were only given 131I-PA, the administration of the SA improved tumor/blood ratios regardless of the SA:PA ratio or time the SA was given. The most important factor in optimizing this procedure was the timing of the SA injection. Significantly improved tumor/nontumor ratios were found when the SA was given between 24 and 48 h after the PA in comparison to 6 h. This was because maximum accretion of radiolabeled PA in the tumor was not achieved until 24 h. At SA:PA ratios of 25:1, only tumor/blood ratios were significantly improved in comparison to the control group. In addition, at SA:PA ratios of 25:1 and 50:1, tumor/spleen and tumor/kidney ratios were lower than the control group, whereas at higher SA:PA ratios, all tumor/nontumor ratios were significantly improved. These studies suggest that for this model, a ratio of SA:PA of 100:1 or higher given at 24 to 48 h after the PA is the best combination for maximizing tumor/nontumor ratios.

Use of whole-body autoradiography in cancer targeting with radiolabeled antibodies.

Methods of single-tracer whole-body autoradiography (WBAR) have been developed in our laboratory which allow imaging and measurement of the zonal distribution of radioiodinated antibodies and their fragments within GW-39 colon carcinoma xenografts varying in size from large, cystic masses with necrotic cores to micrometastases. The whole-animal distribution of 90Y-labeled anti-carcinoembryonic antigen monoclonal antibody NP-2 was evaluated by WBAR in nude mice bearing s.c. implants of GW-39 colon cancer and revealed antitumor uptake specifically as well as significant accumulation of 90Y in the bones. Dual-tracer qualitative WBAR methods have also been applied in order to examine the biodistribution of labeled immunoglobulins in the GW-39 animal tumor model as a function of the underlying rapid cell proliferation index ([3H]-thymidine assay) in the same tumor. In addition, extension of the WBAR method was made to permit imaging of the biodistribution of 10B compounds in mice bearing Harding-Passey melanoma implants by using a track-etch procedure to produce alpha-particle WBAR. Further applications of single and multiple radionuclide WBAR are offered and discussed as an effective means of assessing the degree of penetration of immunoglobulins in tumors in which vascular patterns, local glucose metabolism, protein synthesis, and rapid cell proliferation indices may be characterized.

Monoclonal antibody targeting of human non-small cell carcinoma of the lung.

Murine monoclonal antibody RS5-4H6 is an IgG1, which was raised against a crude membrane preparation of Calu-3, a human adenocarcinoma of the lung cell line. MAb RS5-4H6 reacts with the majority (14 of 15) of surgical specimens of non-small cell carcinomas of the lung by immunoperoxidase staining. Reactivity with this antibody was not limited to tumors of the lung but rather exhibited pancarcinoma reactivity, staining 87% of tumors from all organs tested. In this study we examined the potential of radiolabeled RS5-4H6 to target Calu-3 xenografts in nude mice. The monoclonal antibody was found to localize preferentially to the heterotransplanted tumors, with 6.6% of the injected dose/g accreting in the tumor at 7 days, a 3-fold higher level than an anti-carcinoembryonic antigen monoclonal antibody which was used as a negative control.

Rapid blood clearance of immunoglobulin G2a and immunoglobulin G2b in nude mice.

An extremely rapid blood clearance rate of murine IgG2a antibodies was found in all strains tested of outbred Swiss nu/nu mice, including mice from the major commercial suppliers. The clearance half-life was less than 5 h, in comparison to a 4-5-day half-life in BALB/c mice. Therefore, most of the IgG2a antibody injected i.v. in such mice is cleared before it can reach interstitial fluid, which interferes with immunotherapy and immunodetection experiments. Individual nude mice varied greatly in their IgG2a clearance rates, which hampered investigation of the phenomena. In our experience, approximately three-fourths of nude mice had a rapid or intermediate clearance rate, whereas the remainder had an approximately normal clearance rate. The clearance rate in nude mice was age-dependent, at least in some instances, in that a rapid clearance rate was observed at 2 months of age, whereas the same mice retested at 4 months of age had a normal clearance rate. Rapid clearance could be inhibited by increasing the dose injected: 100 micrograms/mouse resulted in a normal clearance rate, whereas 30 micrograms/mouse was insufficient to inhibit rapid clearance. The clearance rate of IgG2b antibodies was affected similarly to that of IgG2a, whereas the clearance rate of IgG1 and IgG3 was not affected. The Fc region of IgG2a was required in order for rapid clearance to occur. Biodistribution experiments demonstrated that rapid blood clearance was due, at least partially, to binding to the liver and spleen. To determine the genetic basis for rapid IgG2a clearance, approximately 20 inbred and outbred mouse strains were tested. Unexpectedly, nu/+ as well as nu/nu outbred Swiss mice displayed rapid clearance, whereas control +/+ mice did not, so this phenotype appears to be a dominant effect of the nu mutation. BALB/c nu/nu and nu/+ mice did not display rapid clearance, which may be due to expression of the Igh-1a gene, which codes for the IgG2a present in BALB/c mice and in the monoclonal antibodies used in these studies. In conclusion, this clearance effect must be considered in experiments involving murine IgG2a or IgG2b antibodies in outbred Swiss nude mice, except those in which high antibody doses of greater than 0.1 mg/mouse are used. One method of circumventing this problem is to increase the antibody dose injected; a better but more long-range method is to develop strains of outbred nude mice that do have this characteristic.

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.

Biological considerations for radioimmunotherapy.

We have examined three methods that may be useful for improving the therapeutic efficacy of antibody-targeted radionuclides. The principal limitation of radioimmunotherapy is myelotoxicity and thrombocytopenia. These are due mainly to the length of time the radioantibody remains in the blood. The clearance time of a radiolabeled immunoglobulin G (IgG) may be decreased by using fragments prepared from the IgG. Using murine monoclonal antibodies against human colonic cancer in an animal model with a transplantable human colonic tumor, we have shown that fractionated doses of 131I-labeled F(ab')2 fragments can provide similar tumoricidal activity as a single injection of IgG, but toxicity to the normal tissues is reduced significantly at this tumoricidal level. Thus, it is expected that improved tumoricidal activity may be achieved by further escalating the dose of F(ab')2 that is administered at each injection. An anti-antibody (second antibody) may also be used to remove an anti-tumor antibody rapidly from the blood. By allowing intact IgG to be used instead of fragments, a higher percentage of the radiolabeled anti-tumor antibodies may be concentrated in the tumor to provide higher tumor doses, yet toxicity to the normal tissues may be controlled by the removal of the radiolabeled antibody from the blood. We have shown that the injection of a second antibody 48 h after 131I-labeled anti-carcinoembryonic antigen antibody is given can reduce toxicity at least 2-fold without affecting the tumoricidal activity of the radioantibody. A third method for reducing the myelotoxicity of radioantibody treatment involves the use of cytokines to increase the production of white blood cells. For example, interleukin 1 may be given prior to, or sometime after, radioantibody treatment to increase the number of circulating white blood cells and thereby reduce myelotoxicity. Thus, modification of some of the biological factors limiting radioimmunotherapy may provide for improvements in cancer treatment with radiolabeled antibodies.

Use of hematopoietic growth factors to control myelosuppression caused by radioimmunotherapy.

Therapeutically efficacious doses of 131I-antibody result in a loss in circulating white blood cells; the granulocyte population is suppressed by 80-85% and the agranulocytes by 60-65% following 2 mCi of 131I-antibody in hamsters. The administration of 100,000 units of human recombinant interleukin 1 24 h prior to radioantibody can prevent the loss in WBC from 1 mCi of radioantibody and reduce the loss from 2 mCi of antibody. Recombinant murine granulocyte-macrophage colony-stimulating factor is also a potent stimulator of myelopoiesis and may also be useful as a method of reducing radioantibody-induced myelosuppression. The tumor uptake of radioantibody in animals treated with recombinant interleukin 1 is reduced by 30% 1 day after injection of radioantibody but returns to levels seen in animals not treated with the cytokine at 96 and 168 h. Therapeutic efficacy is not compromised by doses of interleukin 1 used to prevent myelosuppression. Therefore, the use of cytokines will permit the use of higher doses of radioantibody for greater tumor therapy with less myelotoxicity than in the absence of cytokine treatments.

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.