Characterization of a high-affinity monoclonal antibody specific for CD44v6 as candidate for immunotherapy of squamous cell carcinomas

 Variant isoforms of CD44, a family of cell-surface glycoproteins generated by alternative splicing and post-translational modifications, are expressed in a variety of human tumors and play important roles in tumor progression and metastasis formation. The murine monoclonal IgG1 antibody VFF18, specific for an epitope encoded by human CD44 variant exon 6, binds with high affinity to the recombinant protein (K _d = 1.7×10^–10 M) as well as to tumor cell lines in vitro, and is suitable for immunohistochemical analysis of human tumors. Screening of more than 500 tumor samples of different histogenesis showed that VFF18 most strongly and uniformly reacts with squamous cell carcinomas (SCC). Detailed analysis of 185 SCC (head and neck, lung, skin) confirmed reactivity of the antibody with 99% of the samples, with intense and homogeneous staining of the tumor cells in the majority of cases, whereas reactivity of VFF18 with normal tissues is limited to certain epithelia and activated lymphocytes. When radiolabelled VFF18 was administered to nude mice bearing human epidermoid carcinoma (A-431) xenograft, fast and selective tumor uptake of the radioimmunoconjugate with a maximum of 18% of the injected dose per gram of tissue was observed. Taken together, these data suggest that mAb VFF18 is a promising targeting vehicle for radioimmunotherapy of squamous cell carcinomas in humans. Received: 9 September 1996 / Accepted: 25 September 1996     

Antibody-dependent difference in biodistribution of monoclonal antibodies in animal models and humans

 The study was designed to clarify the difference in pharmacokinetics of monoclonal antibodies (mAb) in animal models and humans, and to elucidate the applicability of animal models. ^99mTc-labeled murine mAb – against carcinoembryonic antigen (designated BW431/26), and neural cell adhesion molecule (NE150) – and one chimeric mouse/human mAb against nonspecific cross-reacting antigen (chNCA) were administered i.v. to normal mice and athymic mice (370 kBq, 400 ng) xenografted with human cancer cells expressing antigens, and into patients with tumor (925 MBq, 1 mg). The biodistribution of two of the three mAb (not ^99mTc-BW431/26) differed clearly in mice and patients. ^99mTc-NE150 showed specific uptake in xenografted tumor and otherwise a normal biodistribution; however, clinical examination showed increased uptake in the liver with rapid blood clearance (mean α half-life = 31.1 min) compared with ^99mTc-BW431/26 (28.4 h). ^99mTc-chNCA demonstrated increased blood clearance and renal excretion in both normal and athymic mice, with accumulation in tumors. Clinical examination showed rapid blood clearance (mean α half-life = 6.4 min) and increased uptake in the liver. High-performance liquid chromatographic analysis of ^99mTc-chNCA revealed the immune complex in blood, suggesting uptake of the complex by the reticuloendothelial cells. The biodistribution of radiolabeled mAb in animal and human models was variable and specific for each of the three mAb. The results of animal studies with mAb should be evaluated carefully before being extrapolated to humans, on the basis of the nature of the mAb and interacting substances. Received: 9 April 1997 / Accepted 3 March 1998     

Complete ablation of small squamous cell carcinoma xenografts with186Re-labeled monoclonal antibody E48

In previous studies we have shown that in mice bearing head and neck squamous cell carcinoma (HNSCC) xenografts, radioimmunotherapy (RIT) with¹⁸⁶Re-labeled MAb E48 resulted in complete regressions in one-third of the tumors (followup >150 d). MAb E48 was labeled with¹⁸⁶Re following a novel labeling procedure developed at our institute. The injected dose was 600 μCi, which was the maximum tolerated dose (MTD; <15% wt loss) in these studies. The mean size of the tumors was 140±60 mm³. To investigate whether the therapeutic efficacy of RIT in our xenograft model would be improved when treating smaller xenografts, mice bearing 2 HNSCC xenografts with a vol of 75±17 mm³ (number of mice,n=6; number of tumors,t=12) were treated with 600 μCi of¹⁸⁶Re-labeled MAb E48 IgG. All tumors completely regressed and did not regrow during followup (>150 d). In all mice, weight loss did not exceed 10%. to obtain biodistribution data, mice bearing two xenografts with a vol of 58±31 mm³ were injected with 100 μCi of¹⁸⁶Re-labeled MAb E48 IgG. The maximum uptake in blood was 26.4% injected dose/g (%ID·g⁻¹) at 2 h pi and was 53.1%ID·g⁻¹ in the tumor at d 7 pi. In normal tissues, no nonspecific accumulation was observed. Based on these biodistribution data, the absorbed cumulative radiation dose was calculated. The accumulated dose in blood and tumor was 2004 cGy and 8580 cGy, respectively. In other tissues, the dose was less than 8.1% of the dose delivered to tumor. These data implicate that RIT with¹⁸⁶Re-labeled MAb E48 may be especially suited to be used as adjuvant therapy for the treatment of head and neck cancer patients with minimal residual disease.     

Treatment of non-small cell lung cancer patients with the trifunctional monoclonal antibody catumaxomab (anti-EpCAM <Emphasis Type="Bold">×</Emphasis> anti-CD3): a phase I study

Purpose Catumaxomab is a trifunctional monoclonal antibody with binding sites directed to human EpCAM and the human T cell antigen CD3 (anti-EpCAM × anti-CD3). Catumaxomab demonstrated efficacy when administered intraperitoneally in patients with EpCAM positive malignant ascites from ovarian cancer in terms of tumor cell killing and reduction of ascites generation. As EpCAM is also overexpressed in NSCLC, the present study was conducted in order to evaluate safety and tolerability of intravenous treatment with catumaxomab. Patients and methods UICC stage IB–IV NSCLC patients were eligible, if they had at least one prior therapy. Other inclusion criteria were: age 18–75 years, adequate bone marrow function and AST or gamma-GT ≤ 2.5 ULN. Escalating doses of catumaxomab were administered over 8 h as a continuous intravenous infusion. Dose escalation started at 2 μg and was increased to 7.5 μg. In addition various doses of dexamethasone as premedication were investigated. All patients were pretreated with antihistamines. Results Dose limiting toxicity (DLT) was a grade 3 and 4 elevation of ALT, AST and gamma-GT, which was observed in dose level IV (10 mg dexamethasone premedication, 5 μg catumaxomab) and V (40 mg dexamethasone followed by 7.5 μg catumaxomab). Elevated liver enzymes decreased to grade 2 within 3–7 days and were at baseline level within 14 days after infusion. The maximum tolerated dose (MTD) was defined in dose level III (40 mg of dexamethasone followed by 5 μg of catumaxomab). Conclusions Five micrograms of catumaxomab with a pre-medication of 40 mg dexamethasone and antihistamines can be recommended as first dose for i.v. therapy consisting of multiple catumaxomab infusions for patients with NSCLC.     

Mécanisme de fixation du 99mTc-HMDP sur les carcinomes urothéliaux calcifiés de vessie

We report a case of transitional cell carcinoma of the bladder visualized on a ^99mTc-HMDP bone scintigraphy. CT demonstrated irregular tumor of the bladder with curvilinear calcifications on the surface areas and multiple bilateral pulmonary metastases. Bone scintigraphy showed intense uptake corresponding to the bladder tumor and two bone metastases on the left femur. A few days later, the patient underwent retrograde injection of ^99mTc-HMDP into the bladder. Imaging made after voiding showed a tumour uptake of the skeletal labelled agent. Through this case report, we debate ^99mTc-biphosphonate uptake mechanisms in transitional cell carcinoma.     

Safety,biodistribution, pharmacokinetics,and immunogenicity of <Superscript>99m</Superscript>Tc-labeled humanized monoclonal antibody BIWA 4 (bivatuzumab) in patients with squamous cell carcinoma of the head and neck

Previous studies have shown the potential of murine and chimeric anti-CD44v6 monoclonal antibodies (MAbs) for radioimmunotherapy (RIT) of head and neck squamous cell carcinoma (HNSCC). A limitation of these MAbs, however, appeared to be their immunogenicity. Therefore, humanized monoclonal antibody BIWA 4 (bivatuzumab), with an intermediate affinity for CD44v6, was recently selected. As a prelude to RIT, we evaluated the safety, tumor-targeting potential, pharmacokinetics, and immunogenicity of technetium-99m-labeled BIWA 4 in patients undergoing operations for primary HNSCC in this study. Ten patients were treated at BIWA 4 dose levels of 25 mg (n=3), 50 mg (n=4), and 100 mg (n=3). Patients received 2 mg of 750 MBq 99mTc-BIWA 4, together with 23-, 48-, and 98-mg unlabeled BIWA 4, respectively. Radioimmunoscintigraphy (RIS) was performed within 1 h and after 21 h, and patients underwent surgery at 48 h after injection. Biodistribution of 99mTc-BIWA 4 was evaluated by radioactivity measurements in blood, bone marrow, and in biopsies of a surgical specimen obtained 48 h after injection. BIWA 4 concentration in blood was assessed by ELISA and high performance liquid chromatography and related to soluble CD44v6 levels in serum samples. The development of human anti-human antibody (HAHA) responses was determined. Administration of 99mTc-BIWA 4 was well tolerated by all patients and no HAHA responses were observed. A mean t1/2 in plasma of 54.8 +/- 11.5 h, 76.1 +/- 21.8 h, and 68.5 +/- 21.2 h was found for the 25-, 50-, and 100-mg dose group, respectively. No complex formation of BIWA 4 with soluble CD44v6 in blood was observed. RIS showed targeting of primary tumors and lymph node metastases in 8 of 10 and 1 of 5 patients, respectively. The highest tumor uptake and tumor to nontumor ratios were observed for the 50-mg dose group. Tumor uptake was 12.9 +/- 5.9, 26.2 +/- 3.1, and 15.4 +/- 1.9% of the injected dose (ID)/kg for the 25-, 50-, and 100-mg dose group, respectively, while the tumor to bone marrow ratios for these groups were 1.7 +/- 0.5, 3.2 +/- 1.1, and 2.0 +/- 0.6, respectively. CONCLUSION: 99mTc-BIWA 4 can safely be administered to patients with HNSCC, with absence of detectable HAHA responses. The 50-mg dose level showed the highest tumor uptake and tumor to nontumor ratios. These findings support the use of BIWA 4 for RIT studies in patients with HNSCC.     

Production and validation of the pharmacokinetics of a single-chain Fv fragment of the MGR6 antibody for targeting of tumors expressing HER-2

The HER-2 antigen, which is overexpressed in many breast carcinomas, is an ideal target for monoclonal antibodies due to its low expression in normal tissue and its homogeneous distribution in the tumor mass. We have developed and characterized the murine MAb MGR6 against HER-2, which is able to inhibit proliferation of tumor cells overexpressing HER-2. On the basis of these preclinical results, phase I studies in breast carcinoma patients were conducted and radiolocalization data indicated an antibody half life which directly paralleled that of other whole antibodies and thus resulting in a limited in vivo diagnostic capacity. To obtain a smaller reagent with possibly improved in vivo properties, a single chain variable fragment (scFv) of the original MGR6-producing hybridoma was generated by phage display technology. Biologically active MGR6 scFv was purified rapidly and at high yield by metal affinity chromatography. Competition FACS and ELISA analyses identified an epitope on the HER-2 extracellular domain that was shared by the scFv and the parental MAb. BIAcore analysis indicated a K_off of 9.3 × 10⁻⁴ s⁻¹, similar to that of the intact MGR6 MAb. Distribution and elimination half-lives of MGR6 scFv, calculated from in vivo preclinical evaluations, were much faster (13 min and 6.2 h, respectively) than previously published results for the intact MAb (mean t1/2β of 46 h). This represents a theoretical improvement in pharmacokinetics with respect to the parental murine MAb and points to the potential for utilizing this fragment in redirecting therapeutic agents, such as radioisotopes, to different human carcinomas overexpressing HER-2. Received: 10 August 2000 / Accepted: 19 October 2000     

Linear bulk diffusion into heterogeneous tissue

In this paper an expression is derived which describes the transient overall uptake of an inert solute by a section of tissue excised with parallel faces and placed upon an impermeable base. The approach diverges from the conventional analyses for perfused tissue (Morales and Smith,Bull. Math. Biophysics,6, 125–141, 1944;7, 47–99, 1945) because the extravascular zone is regarded as a heterogeneous diffusion medium. Account for this is taken by regarding tissue as effectively composed of two phases—a continuous (extracellular) phase similar to water, and a dispersed phase comprising cells of irregular profile. In both phases the relevant mode of uptake is taken as bulk diffusion rather than surface permeation, thus emphasizing the influence of the internal geometry of the tissue upon its overall exchange response.     

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.     

Syngeneic anti-idiotypic antibodies eliminate excess radiolabeled idiotypes at experimental radioimmunolocalization

Significant improvements in tumor/nontumor ratio can be achieved by injections of nonlabeled anti-idiotypic monoclonal antibodies (MAbs) during radioimmunolocalization and radioimmunotherapy using MAbs to target experimental tumors. The in vivo effects of an anti-idiotypic MAb (αH7) against a radioiodinated, high affinity, low dissociation rate, monoclonal antiplacental alkaline phosphatase antibody (H7) was investigated. Following in vivo injection of the anti-idiotypic MAb, the radioactivity in experimental tumors was found to decrease only 25% while the reduction of corresponding radioactivity in nontumor tissues amounted to 65–85%, compared to the group receiving no anti-idiotypic MAbs. These results indicate that it is possible to partially clear the circulation and nontumor tissues from excess of radiolabeled idiotypic antibody, without significant decrease in specific tumor localization, increasing the tumor/ nontumor ratio three- to fourfold. Circulating nontumor targeting radiolabeled antibodies is one of the major limiting factors in radioimmunotherapy today. Injection of anti-idiotypic MAbs could selectively significantly reduce the radiation dose to radiosensitive tissues, i.e., bone marrow and intestine, thus improving efficiency in radioimmunoscintigraphy and radioimmunotherapy.     

A phase I study of a HER2/neu bispecific antibody with granulocyte-colony-stimulating factor in patients with metastatic breast cancer that overexpresses HER2/neu

A phase I study of escalating doses of humanized bispecific antibody (bsAb) MDX-H210 with granulocyte-colony-stimulating factor (G-CSF) was conducted in patients with metastatic breast cancer that overexpressed HER2/neu. The main objectives of the study were to define the maximal tolerated dose (MTD) of MDX-H210 when combined with G-CSF, to measure the pharmacokinetics of MDX-H210 when administered with G-CSF, and to determine the toxicity, biological effects and possible therapeutic effect of MDX-H210 with G-CSF. MDX-H210 is a F(ab)′ × F(ab)′ humanized bispecific murine antibody that binds to both HER2/neu and the FcγR1 receptor (CD64), and was administered intravenously weekly for three doses followed by a 2-week break and then three more weekly doses. A total of 23 patients were treated, and doses were escalated from 1 mg/m² to 40 mg/m² with no MTD reached. The toxicity of the bsAb + G-CSF combination was modest, with no dose-limiting toxicity noted: 19 patients had fevers, 7 patients had diarrhea, and 3 patients had allergic reactions that did not limit therapy. The β-elimination half-life varied from 4 h to 8 h at doses up to 20 mg/m². Significant release of cytokines interleukin-6, G-CSF, and tumor necrosis factor α was observed after administration of bsAb. Circulating monocytes disappeared within 1 h of bsAb infusion, which correlated with binding of bsAb, noted by flow-cytometric analysis. Significant levels of human anti-(bispecific antibody) were measured in the plasma of most patients by the third infusion. No objective clinical responses were seen in this group of heavily pre-treated patients. Received: 23 July 1998 / Accepted: 6 November 1998     

Detection of invasive Candida albicans infection using a specific 99mTc-labeled monoclonal antibody for the C. albicans germ tube

Accurate diagnosis is critical for effective treatment of the invasive infection by Candida albicans. Here, we investigated whether a ^99m technetium (Tc)-labeled Fab’ fragment of the monoclonal antibody specific for the C. albicans germ tube could specifically identify an invasive C. albicans infection. The germ tube of C. albicans was used as an immunogen to obtain monoclonal antibodies and the Fab’ fragment of MAb03.2 C1–C2 with highest affinity and specificity was labeled with ^99mTc. In vitro binding assays showed that the labeled Fab’ preferentially bound to the germ tubes of C. albicans (4.23 ± 0.17 × 10² Bq per 1 × 10⁷ cells). These values were significantly higher than those for blastospores of C. albicans, blastospores of heat-killed C. albicans, Aspergillus fumigatus, Staphylococcus aureus, and Escherichia coli (P < 0.05). By using in vivo biodistribution and planar imaging with single photon emission computed tomography, we demonstrated a significant specific accumulation of radioactivity in C. albicans-infected tissues. In summary, ^99mTc-MAb03.2 C1–C2 Fab’ is able to specifically accumulate in C. albicans-infected tissues, but not in tissue infected with A. fumigatus or bacteria or in a sterile inflammation. This study provides a new and specific radiopharmaceutical for the diagnosis of invasive C. albicans infections.     

A phase-I trial of the epidermal growth factor receptor directed bispecific antibody MDX-447 without and with recombinant human granulocyte-colony stimulating factor in patients with advanced solid tumors

Introduction MDX-447 is a bispecific antibody directed against the epidermal growth factor receptor (EGFR) and the high affinity Fc receptor (FcγRI). Preclinical data suggest that co-administration of granulocyte-colony stimulating factor (G-CSF) may enhance the tumor cytotoxicity of bispecific antibodies. Methods In group 1, patients received MDX-447 intravenously (IV) weekly. Dose levels of MDX-447 evaluated in group 1 were 1, 3.5, 7, 10, 15, 20, 30, and 40 mg/m². In group 2, patients received MDX-447 IV weekly with G-CSF (3 mcg/kg/day) subcutaneously (days −3 to +2, 5–9, 12–16, etc.). Dose levels of MDX-447 evaluated in group 2 were 1, 3.5, 7, 10, and 15 mg/m². Results Sixty-four patients with advanced solid tumors were treated. Forty-one patients received MDX-447 alone (group 1); 23 patients received MDX-447 + G-CSF (group 2). Hypotension was the predominant dose-limiting toxicity (DLT) in both treatment groups, with seven patients experiencing ≥grade 3 events. MDX-447 half-life (T_1/2) ranged from 1.9 to 8.4 h, with no obvious differences between the two treatment groups. MDX-447 binding to neutrophils and peak levels of circulating tumor necrosis factor α (TNFα) and interleukin-6 (IL-6) were higher in group 2. The MTD for MDX-447 alone was 30 mg/m². When G-CSF was given with MDX-447, treatment was not well tolerated and group 2 was closed early because of safety concerns, with the last patient being treated at the 7 mg/m² dose level. There were no objective complete or partial responses in either group. Conclusion MDX-447 alone was generally well tolerated, but did not achieve objective tumor responses. The MTD for MDX-447 alone was 30 mg/m² weekly. Co-administration of G-CSF with MDX-447 precluded meaningful dose escalation.     

Preclinical studies of monoclonal antibodies for intravesical radioimmunotherapy of human bladder cancer

Eighty percent of bladder cancers present as superficial disease. Many are multifocal, and apparently successful treatment is frequently followed by recurrence. The use of monoclonal antibodies (MAbs) to target radiotherapy to these tumors offers great potential, especially since they can be administered directly into the bladder (intravesically) bypassing many of the side effects encountered to date with systemic MAb-based therapy. Implantation of human bladder cancer cell lines in the bladder wall of nude rats results in tumor formation, providing an excellent model to test this. Tumor size can be monitored by X-ray analysis after administration of urograffin. Comparative studies of two murine MAbs, BLCA-8, IgG3, and C1-137, IgG1, against malignant human bladder cancer cells have been performed. Radio-immunoconjugates produced with¹²⁵Iodine (¹²⁵I) have been used for biodistribution studies following administration directly into rat bladder. Radioiodinated intact MAbs or Fabs administered intravesically into nontumor bearing rats did not leak into the systemic circulation and were stable in urine for up to 100 h. Biodistribution studies carried out following intravesical administration of radio-immunoconjugates to tumor-bearing nude rats indicate better tumor uptake of C1-137 than BLCA-8. Further studies to test two-step intravesical administration of biotinylated MAb followed by radioiodinated streptavidin are in progress. Our studies indicate that the C1-137 MAb may have considerable potential for intravesical radioimmunotherapy of patients with superficial bladder tumors.     

Various routes of administration of (99m)Tc-labeled synthetic lactoferrin antimicrobial peptide hLF 1-11 enables monitoring and effective killing of multidrug-resistant Staphylococcus aureus infections in mice

The synthetic antimicrobial peptide representative of the first 11 N-terminal amino acids of human lactoferrin (hLF 1-11) kills multidrug-resistant Staphylococcus aureus (MRSA). This study displays antimicrobial activity of hLF 1-11, via various routes of administration, against MRSA infections in mice. Radiolabeling hLF 1-11 with technetium-99m ((99m)Tc-hLF 1-11) enables scintigraphic monitoring directly after administration. (99m)Tc-hLF 1-11 was taken up by the gall bladder, intestines, and kidneys. Most of the radioactivity was captured in the urinary bladder and about 1% of the injected dose accumulated into infected thigh muscles. At 2 or 24h after either intravenously, subcutaneously, intraperitoneally, or orally injected a single dose of 0.04 mg/kg hLF 1-11 in mice significantly reduced (20-60 times) the number of viable MRSA. In a dose-response setting in immunocompetent mice maximum bactericidal effects (10,000 times reduction) of intravenously injected (99m)Tc-hLF 1-11 was seen with 40 mg/kg whereas the same dose of orally administered (99m)Tc-hLF 1-11 induced about approximately 100 times reduction. In conclusion, intravenously and orally administrated (99m)Tc-hLF 1-11 accumulates in infected tissues and is highly effective against experimental infections with MRSA. Moreover, scintigraphy is an excellent tool to study the pharmacology of experimental compounds and to determine the uptake in infected tissues.     

Mechanisms in removal of tumor by antibody

We report two preliminary trials of antibody treatment of B-cell lymphoma. Advanced lymphoma was treated with chimeric FabFc₂, in which mouse Fab'γ is linked to two human Fcγ1 fragments so as to recruit natural effectors to tumor targets. Terminal lymphoma was treated with bispecific antibody (BsAb) which recruits the ribosome-inactivating protein saporin. These different mechanisms led to interesting differences in patterns of tumor clearance. Eight patients were treated with chimeric antibody of two specificities, each at 12 mg/kg: anti-CD37, plus either anti-CD38 or anti-CD19 according to tumor phenotype. On completion of the 3-wk treatment, residual plasma antibody had a half-life exceeding 10 d. Tumor cells in blood disappeared rapidly. However significant reductions in solid masses occurred in only three patients, becoming apparent 3–4 wk after beginning treatment and then continuing slowly. Five patients were treated with preformed immune complexes of saporin and F(ab'γ)₂ BsAb. Although doses of saporin reached 10 mg weekly, contact with the tumor can only have been fleeting: plasma antibody was undetectable (<0.5 μg/mL) 48 h after infusion, whereas the saporin disappeared even faster and was undetectable (<4 ng/mL) at 24 h. Tumor cells disappeared from the blood more slowly than occurred with chimeric antibody. In contrast shrinkage of extravascular tumor was more rapid, and occurred in all patients, but proved less durable.     

Internal efficiency, nutrient uptake, and the relation to field water resources in rainfed lowland rice of northeast Thailand

Rice-based (Oryza sativa L.) rainfed lowlands are the major cropping system in northeast Thailand. Average yields are low, which is generally explained by frequent drought events, low soil fertility, and poor fertilizer response. However, neither the relative importance of these factors nor their interaction is well understood. Therefore, we analyzed an existing database on fertilizer trials conducted between 1995 and 1997 at eight different sites in northeast Thailand with the objective to determine indigenous nutrient supplies, internal efficiencies, and recovery efficiencies of applied nutrients in rainfed lowland rice. Of particular interest was the effect of variety type (traditional) and water supply on these components. Comparison of N, P, and K concentrations in grain and straw (average N–P–K grain concentration of 11.0–2.7–3.4 g kg⁻¹; average N–P–K straw concentration of 5.2–0.9–16.4 g kg⁻¹) in the traditional-type varieties used at all trial sites with literature values showed no differences for these parameters between traditional and modern-type varieties or between irrigated and rainfed environments. In contrast, internal efficiencies of N, P, and K (average IEN: 46 kg grain per kg N uptake; IEP: 218 kg grain per kg P uptake; IEK: 25 kg grain per kg K uptake) were much lower than reported for irrigated systems, and the difference was greatest for K, which is mainly accumulated in the straw. Indigenous nutrient supply (average INS: 38 kg ha⁻¹; IPS: 10 kg ha⁻¹; IKS: 89 kg ha⁻¹) and recovery efficiency (average REN: 0.28 kg kg⁻¹; REP: 0.13 kg kg⁻¹; REK: 0.49 kg kg⁻¹) were low but comparable to the lower values reported from irrigated systems. Average seasonal field water resources seemed to reduce the indigenous nutrient supply but had no or little effect on internal efficiency and recovery efficiency. We concluded that the main reason for the low system productivity without and with fertilizer in northeast Thailand is the dominant use of traditional-type varieties with low harvest indices, which was the dominant cause for the observed low internal nutrient efficiency. Therefore, intensification of rainfed systems through substantially increased nutrient inputs can be recommended only where varieties with an average harvest index of close to 0.4 or higher are available.     

Melanoma targeting with α-melanocyte stimulating hormone analogs labeled with fac-[99mTc(CO)3]+: effect of cyclization on tumor-seeking properties

Early detection of primary melanoma tumors is essential because there is no effective treatment for metastatic melanoma. Several linear and cyclic radiolabeled α-melanocyte stimulating hormone (α-MSH) analogs have been proposed to target the melanocortin type 1 receptor (MC1R) overexpressed in melanoma. The compact structure of a rhenium-cyclized α-MSH analog (Re-CCMSH) significantly enhanced its in vivo tumor uptake and retention. Melanotan II (MT-II), a cyclic lactam analog of α-MSH (Ac-Nle-cyclo[Asp-His-dPhe-Arg-Trp-Lys]-NH₂]), is a very potent and stable agonist peptide largely used in the characterization of melanocortin receptors. Taking advantage of the superior biological features associated with the MT-II cyclic peptide, we assessed the effect of lactam-based cyclization on the tumor-seeking properties of α-MSH analogs by comparing the pharmacokinetics profile of the ^99mTc-labeled cyclic peptide βAla-Nle-cyclo[Asp-His-d-Phe-Arg-Trp-Lys]-NH₂ with that of the linear analog βAla-Nle-Asp-His-dPhe-Arg-Trp-Lys-NH₂ in melanoma-bearing mice. We have synthesized and coupled the linear and cyclic peptides to a bifunctional chelator containing a pyrazolyl-diamine backbone (pz) through the amino group of βAla, and the resulting pz–peptide conjugates were reacted with the fac-[^99mTc(CO)₃]⁺ moiety. The ^99mTc(CO)₃-labeled conjugates were obtained in high yield, high specific activity, and high radiochemical purity. The cyclic ^99mTc(CO)₃-labeled conjugate presents a remarkable internalization (87.1% of receptor-bound tracer and 50.5% of total applied activity, after 6 h at 37 °C) and cellular retention (only 24.7% released from the cells after 5 h) in murine melanoma B16F1 cells. A significant tumor uptake and retention was obtained in melanoma-bearing C57BL6 mice for the cyclic radioconjugate [9.26 ± 0.83 and 11.31 ± 1.83% ID/g at 1 and 4 h after injection, respectively]. The linear ^99mTc(CO)₃-pz–peptide presented lower values for both cellular internalization and tumor uptake. Receptor blocking studies with the potent (Nle⁴,dPhe⁷)-αMSH agonist demonstrated the specificity of the radioconjugates to MC1R (74.8 and 44.5% reduction of tumor uptake at 4 h after injection for cyclic and linear radioconjugates, respectively).     

Detection of hepatic metastases from colorectal carcinoma using indium-111 (111In) labeled monoclonal antibody (mAB): MSKCC experience with mAb 111In-C110

Sixteen patients with colorectal carcinoma and a rising serum carcinoembryonic antigen (CEA) level and no evidence of extra-abdominal disease were administered 5 mg of an anti-CEA monoclonal antibody (mAb), C110, labeled with approx. 5 mCi of ¹¹¹In. All patients subsequently underwent exploratory laparotomy, and samples of tumor and normal tissue were obtained. Hepatic lesions (confirmed by histopathology) were visualized as areas of increased radiotracer uptake in 13 of 16 patients. Single photon emission computed tomography (SPECT) considerably aided detection, being positive in two patients with normal planar images. Ten of the 16 patients had positive x-ray computed tomographic (CT) images. The radioimmunodiagnostic study was falsely negative in 3 of 16 patients with subsequently proven hepatic disease, in one of whom CT was also normal. The antibody study was positive in 80% of lesions, thus being, in this small series, significantly more sensitive (P < 0.01) than CT. ¹¹¹In-C110 is a promising monoclonal antibody for the detection of hepatic metastases from colorectal carcinoma; this is the first study to show consistently greater concentration of ¹¹¹In-labeled antibody in hepatic lesions than in surrounding normal hepatic parenchyma.     

Biodistribution of 111In-labelled engineered human antibody CTM01 (hCTM01) in ovarian cancer patients: influence of prior administration of unlabelled hCTM01

mAb hCTM01 binds a carcinoma-associated antigen, the MUC1 gene product. The antigen is also present in the circulation, and administration of ¹¹¹In-labelled hCTM01 results in the formation of immune complexes with enhanced accumulation in the liver. To avoid the unwanted effect of circulating radioactive immune complexes, a strategy to remove the circulating antigen was investigated using a split-dosage schedule. Eleven patients suspected of having ovarian carcinoma were injected with 1 mg/kg unlabelled hCTM01, 1 h before receiving 0.1 mg/kg ¹¹¹In-labelled hCTM01 (100 MBq). The amount of radioactivity was determined in resected tumour tissue, various normal tissues and blood samples obtained at laparotomy 6 days postinjection (p.i.). In all patients, the circulating antigen decreased to its nadir after the unlabelled antibody infusion and immune complex formation was demonstrated. Uptake in tumour deposits 6 days p.i. was 11.1 times higher than in normal tissues (P<0.0001) and 5.9 times higher than in blood (P<0.0001). ¹¹¹In activity in liver tissue was comparable to ¹¹¹In uptake in tumour tissue, and considerably lower than previously reported in patients not pretreated with unlabelled antibody. The split-dosing strategy would appear to be advantageous for use of hCTM01 as a specific carrier for the delivery of cytotoxic agents to patients with ovarian cancer. Received: 12 February 1998 / Accepted: 30 April 1998