Pharmacokinetics of anti-ganglioside GD2 mAb 14G2a in a phase I trial in pediatric cancer patients

A phase I trial of a murine anti-ganglioside (GD2) monoclonal antibody (mAb) 14G2a was conducted in 14 neuroblastoma patients and 1 osteosarcoma patient to assess its safety, toxicity and pharmacokinetics in pediatric patients. The pharmacokinetics of mAb 14G2a were biphasic with at ₁ ^2/ of 2.8±2.8 h and at ₁ ^2/ of 18.3±11.8 h. In general,t ₁ ^2/ was dose-dependent with a level of significance ofP=0.036, and it reached a plateau at doses of 250 mg/m² or more. Overall the peak serum levels were dose-dependent atP<0.001. However, they demonstrated an abrupt increase between doses of 100 mg/m² and 250 mg/m². The latter two suggest a saturable mechanism for mAb elimination. In addition, peak serum concentrations were observed earlier at higher mAb doses, which indicates the achievement of a steady state. Thet ₁ ^2/ of mAb 14G2a in children appears to be shorter than in adults. Furthermore, 2 patients demonstrated a considerable decrease int ₁ ^2/ following retreatment with 14G2a. This was paralleled by high human anti-(mouse Ig) antibody levels. This study represents the first comprehensive analysis of murine mAb pharmacokinetics in children and will be useful in the future design of mAb therapy.This work was supported by grants from FDA, FD-R-000377 and NIH U10 CA 28439 and in part by a grant from the general Clinical Research Center program, MOI RR00827, of the National Center for Research Resources, National Institutes of Health. M. M. U.-F. and C.-S. H. were supported in part by a grant from the Children's Cancer Research Foundation, and R. A. R. was supported in part by NIH grant CA 42508     

Biological characterization of a chimeric mouse-human IgM antibody directed against the 17-1A antigen

Summary The pharmacokinetics of ¹¹¹In-labeled 260F9, a murine monoclonal antibody directed against a breast-cancer-associated antigen, was determined in seven patients with advanced breast cancer. Six patients were administered 1 mg antibody containing 1 mCi ¹¹¹In. The seventh patient was administered 20 mg unlabeled antibody followed by 1 mg ¹¹¹In-labeled antibody all via a peripheral vein. Immunoprecipitation, HPLC and SDS-PAGE gels demonstrated the stability of radiolabel on the antibody. The serum clearance of the radiolabel closely fits (r ²>0.95) a two-compartment model for the first six patients. The apparent volume of distribution of the radiolabel approximated to the plasma volume (3 1) and its mean residence time was 23.7 h. The radiolabel had an average t _1/2 of 22.9±12.21 h at the 1-mg dose. At the 20-mg dose one-compartment elimination kinetics were observed with the radiolabel and antibody showing similar mean residence times (36–41 h) and a t _1/2 of 26–28 h. Whole-body imaging showed that the blood-pool:liver ratio of radioactivity increased fourfold (at 48 h postinfusion) at the higher dose and the percentage of the injected dose of radioactivity in the liver decreased from 25% to 8% (24 h postinfusion).In one patient 7–14 times more radioactivity was localized in a breast tumor than in fat (normal breast). Over the first 25 h an average (cumulative) 7.5% of the total dose was excreted in urine. A study of 260F9 in CDF-1 mice demonstrated that the radiolabel remained associated with the antibody in serum. The antibody, however, cleared 60-fold slower in mice than in patients and showed an increased mean residence time of 191 h. The disparity in the pharmacokinetics of the antibody seen in the mouse and in the clinic, points to the different behavior shown by murine monoclonal antibodies in humans. This points to the need for preliminary studies of antibodies in patients for preclinical evaluations of their effectiveness as drug-targeting agents.     

High-dose,unlabeled, nonspecific antibody pretreatment: influence on specific antibody localization to human melanoma xenografts

Summary Nonspecific uptake of radiolabeled monoclonal antibodies in normal tissues is a significant problem for tumor imaging. A potential means of decreasing nonspecific antibody binding is to blockade nonspecific antibody binding sites by predosing with cold, nonspecific isotypematched antibody, before injecting specific antibody. Nontumor-specific murine monoclonal antibody LK2H10 (IgG1) or Ab-1 (IgG2a) was given i.v. at doses of 0 to 3.5 mg to nude mice with xenografts of human melanoma. These mice were then given i.v. 4 g of ¹³¹I anti-high molecular weight antigen of melanoma (HMWMAA) monoclonal antibody 763.24T (IgG1) or 225.28S (IgG2a), respectively. These mice were also given a tracer dose of ¹²⁵I LK2H10 or Ab-1, respectively. Specific tumor uptake of anti-HMWMAA antibodies was see in all cases. No drop in tumor or nontumor uptake was demonstrated for either of the tumor-specific or nonspecific monoclonal antibodies due to nonspecific monoclonal antibody pretreatment. These data suggest that high doses of isotype-matched unlabeled nonspecific monoclonal antibody given before ¹³¹I tumor-specific monoclonal antibody, will not enhance tumor imaging. Present address: Hybritech, San Diego, CA, USA     

A human/mouse chimeric monoclonal antibody against CA125 for radioimmunoimaging of ovarian cancer

Murine monoclonal antibody 196-14 recognizes the ovarian-cancer-associated antigen CA 125, but the epitope it recognizes is different from that of monoclonal antibody OC125. We developed a human/mouse chimeric 196-14 using the variable regions of the murine 196-14 and human heavy-chain (l) and light-chain () constant regions. Cell binding and competitive inhibition assays using chimeric 196-14 labeled with¹²⁵I,¹¹¹In or^99mTc demonstrated that the in vitro immunoreactivity of the chimeric antibody was identical to that of the parental murine monoclonal antibody. However, in mice bearing human ovarian cancer xenografts, the clearance from blood was faster and absolute levels of accumulation in the tumor were lower for the¹²⁵I-labeled or^99mTc-labeled chimeric antibody than for the murine antibody labeled with the corresponding radionuclides. The tumor-to-blood radioactivity ratio was not significantly different between the chimeric antibody and the murine antibody, regardless of the radionuclide used for labeling. Chimeric antibody 196-14 labeled with¹³¹I,¹¹¹In or^99mTc is promising for the radioimmunoimaging of ovarian cancer.     

Differential responses of human tumor cell lines to anti-p185HER2 monoclonal antibodies

The HER2 protooncogene encodes a receptor tyrosine kinase, p185^HER2. The overexpression of p185^HER2 has been associated with a worsened prognosis in certain human cancers. In the present work we have screened a variety of different tumor cell lines for p185^HER2 expression using both enzyme-linked immunosorbent and fluorescence-activated cell sorting assays employing murine monoclonal antibodies directed against the extracellular domain of the receptor. Increased levels of p185^HER2 were found in breast (5/9), ovarian (1/6), stomach (2/3) and colorectal (5/16) carcinomas, whereas all kidney and submaxillary adenocarcinoma cell lines tested were negative. Some monoclonal antibodies directed against the extracellular domain of p185^HER2 inhibited growth in monolayer culture of breast and ovarian tumor cell lines overexpressing p185^HER2, but had no effect on the growth of colon or gastric adenocarcinomas expressing increased levels of this receptor. The most potent growth-inhibitory anti-p185^HER2 monoclonal antibody in monolayer culture, designated mumAb 4D5 (a murine IgG1 antibody), was also tested in soft-agar growth assays for activity against p185^HER2-overexpressing tumor cell lines of each type, with similar results. In order to increase the spectrum of tumor types potentially susceptible to monoclonal antibody-mediated anti-p185^HER2 therapies, to decrease potential immunogenicity issues with the use of murine monoclonal antibodies for human therapy, and to provide the potential for antibody-mediated cytotoxic activity, a mouse/human chimeric 4D5 (chmAb 4D5) and a humanized 4D5 (rhu)mAb 4D5 HER2 antibody were constructed. Both engineered antibodies, in combination with human peripheral blood mononuclear cells, elicited antibody-dependent cytotoxic responses in accordance with the level of p185^HER2 expression. Since this cytotoxic activity is independent of sensitivity to mumAb 4D5, the engineered monoclonal antibodies expand the potential target population for antibody-mediated therapy of human cancers characterized by the overexpression of p185^HER2.     

Curative effects of combination therapy with lentinan and interleukin-2 against established murine tumors,and the role of CD8-positive T cells

The antitumor activity of a combination of an antitumor polysaccharide, lentinan (a 1–3 glucan with 1–6 branches), and interleukin-2 (IL-2) was evaluated against established MBL-2 lymphoma and S908.D2 sarcoma at i.d. sites. Treatment of the MBL-2-tumor-bearing BDF1 mice with lentinan and IL-2 induced complete regression of tumor in 87.5% of mice treated. In contrast, treatments using either lentinan or IL-2 alone failed to induce complete regression of tumor, although temporal growth inhibition of tumor was observed about in half of the mice treated. Improvements of antitumor effects by the combination of lentinan and IL-2 were also observed in the MBL-2/B6 and S908.D2/B10.D2 systems. Expression of the antitumor effects of lentinan/IL-2 treatments required the intact T cell compartment, because the effects were not observed when nude mice were used. In the MBL-2/B6 system, the antitumor action of lentinan/IL-2 treatment was abolished in mice treated with antibody to CD8 antigen, whereas antibodies to CD4 or NK1.1 were ineffective. Furthermore, augmented tumor-specific cytotoxic T lymphocyte (CTL) activity was observed in regional lymph node cells of the mice after lentinan and IL-2 administration. These data indicate that the antitumor effects of lentinan/IL-2 are mediated by CD8⁺ CTL but not by CD4⁺ T cells or NK1.1⁺ NK/LAK cells, and suggest that this combined therapy may be effective against even established tumors that are resistant to IL-2 therapy.Abbreviations B6 C57BL/6 - BDF1 C57BL/6 × DBA/2 F1 - Lyt2 murine CD8, Lyt2.1. allele of murine CD8 - Lyt2.2 allele of murine CD8 - Lyt3 murine CD8 - L3T4 murine CD4     

Minor human antibody response to a mouse and chimeric monoclonal antibody after a single i.v. infusion in ovarian carcinoma patients: a comparison of five assays

The human anti-(mouse Ig) antibody (HAMA) response was measured in serum of 52 patients suspected of having ovarian carcinoma who had received an i.v. injection of either the murine monoclonal antibody (mAb) OV-TL 3 F(ab)₂ (n=28, 1 mg) or the chimeric mouse/human mAb MOv18 (cMOv18;n=24, 3 mg). Serum samples were taken before injection and 2–3 and 6–14 weeks after administration. A double-antigen or bridging assay was developed to detect responses against both murine as well as chimeric antibodies. In addition, an indirect enzyme-linked immunosorbent assay (ELISA) as well as three commercially available assays were used to study antibody response against the murine antibody OV-TL 3. With both the double-antigen (bridging) assay and the indirect ELISA 1 of the 28 patients (4%) injected with murine OV-TL 3 F(ab)₂ showed a HAMA reaction 6 weeks after injection, which was demonstrated to be a mixed anti-isotypic and anti-idiotypic response. None of the 24 patients injected with the chimeric MOv18 showed an anti-chimeric antibody response. The various commercially available assays demonstrated conflicting results. The double-antigen-or bridging assay is a reliable method to detect anti-murine and antichimeric antibodies. The assay can be easily adapted for use with human antibodies. The immunogenicity of OV-TL 3 F(ab)₂ and cMOv18 in patients is low, making both antibodies candidates for immunotherapy.This work was supported by a clinical research grant of the Netherlands Organization for Scientific Research (NWO 900-716-020) and by the Biocare Foundation (grant 92-05).     

A phase II trial of murine monoclonal antibody 17-1A and interferon-γ: Clinical and immunological data

Summary A group of 15 patients with metastatic colorectal adenocarcinoma received a combination of interferon (0.1 mg/m², days 1–15) and the murine monoclonal antibody 17-1A (400 mg, days 5, 7, 9 and 12). The treatment was tolerated with minimal toxicity. Of the 14 evaluable patients, 13 developed human antibody to murine 17-1A, with 11 patients demonstrating antibody to the variable region of 17-1A (anti-idiotype). Antibody to the variable region was inhibited by 17-1A but not by mouse immunoglobulin. Sera from patients with substantial anti-idiotype reactivity were capable of inhibiting the binding of murine 17-1A to antigen expressing LS174-T cells thus indicating the presence of antibody directed against the 17-1A combining site (mirror-image anti-idiotype). The median survival of the whole group was 56 weeks and there was no correlation between clinical response/survival and the development of anti-idiotype antibody.Supported by the Veterans Administration Medical Center and by Public Health Services grant CA 45 232 from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services     

Construction and characterization of the chimeric monoclonal antibody E48 for therapy of head and neck cancer

Data from an ongoing clinical radioimmunoscintigraphy trial indicate that^99mTc-labeled monoclonal antibody (mAb) E48 is highly capable of selectively targeting squamous cell carcinoma of the head and neck (HNSCC). The percentage of the injected dose per gram of tumor tissue was found to be high, rendering mAbE48 a promising candidate mAb for therapeutic purposes. We now describe the construction of a chimeric (moouse/human) mAb E48 by recombinant DNA technology. The genes encoding the variable domains of the heavy and light chain were cloned and ligated into experession vectors containing the human 1 heavy-chain gene and the human k lightchain gene respectively. Biological properties of the resulting chimeric mAb E48 were compared to the murine form in vitro and in vivo. The reactivities of chimeric (c)mAb and murine (m)mAb E48 with HNSCC, as assessed by immunohistochemical staining as well as immuno-blotting were shown to be similar. The affinity constant appeared to be 0.9×10¹⁰ M^–1 and 1.6×10¹⁰ M^–1 for the mmAb and cmAb respectively. The biodistribution of both antibodies was tested by simultaneous injection into nude mice bearing human HNSCC xenografts. cmAb E48 was found to be cleared more rapidly from the blood than mmAb E48, resulting in a 30% lower tumor uptake but similar tumor to non-tumor ratios, 3 days after injection. Moreover, it was shown that cmAb E48 is highly capable of lysing HNSCC targets in ADCC assays in vitro, whereas the mmAb appeared to be almost incative. These data indicate that cmAb E48 has potential as a targeting agent for the eradication of HNSCC in man.     

Phase I trial of combined treatment with ch14.18 and R24 monoclonal antibodies and interleukin-2 for patients with melanoma or sarcoma

Purpose: We conducted a phase I trial of interleukin 2 (IL-2) in combination with chimeric 14.18 (ch14.18) and murine R24 antibodies to determine the maximal tolerated dose (MTD), immunological effects, and toxicity of this treatment combination. Experimental Design: Twenty-seven patients with either melanoma (23 patients) or sarcoma (4 patients) were enrolled to receive a combination therapy with ch14.18 and R24 antibodies together with continuous infusion of Roche IL-2 (1.5×10⁶ U/m²/day, 26 patients) or Chiron IL-2 (4.5×10⁶ U/m²/day, 1 patient) given 4 days/week for 3 weeks. The antibodies ch14.18 (2–7.5 mg/m²/day) and R24 (1–10 mg/m²/day) were scheduled to be administered for 5 days during the second week of IL-2 therapy. Results: When given in combination in this study, the MTD for ch14.18 was 5 mg/m²/day and the MTD for R24 was 5 mg/m²/day. Dose-limiting toxicities were severe allergic reactions to both ch14.18 and R24 as well as pain related to ch14.18. This ch14.18 MTD was lower than the 7.5 mg/m²/day MTD previously determined for ch14.18 given alone with the same dose and schedule of IL-2. Immunological effects included the induction of lymphokine-activated killer (LAK) activity and antibody-dependent cell-mediated cytoxicity (ADCC). Anti-idiotype response to ch14.18 was seen in six patients, including two melanoma patients who had a partial response to treatment. In addition to two partial responses, four patients had a stable disease and one patient remained without any evidence of disease. Conclusions: Immunotherapy with IL-2 in combination with ch14.18 and R24 antibodies augments LAK function and ADCC measured in vitro in all patients. While there exist theoretical advantages of combining these two antibodies, the MTD of ch14.18 and of R24 were lower than the MTD of each antibody in prior studies evaluating single antibody therapy with IL-2. As such, the combination of these two antibodies together with IL-2 therapy appeared to influence the MTD and toxicity of each of the administered antibodies. This work is supported by NIH grants M01-RR03186, R01-CA32685, and P30-CA14520     

Pharmacokinetics of human-mouse chimeric anti-GD2 mAb ch14.18 in a phase I trial in neuroblastoma patients

A comprehensive analysis of the pharmacokinetics of human-mouse chimeric anti-ganglioside GD2 antibody mAb ch 14.18 was performed during a phase I clinical trial of ten children with neuroblastoma and one adult with osteosarcoma. The patients received a total of 20 courses of ch 14.18 at dose levels from 10 mg/m² to 200 mg/m². The plasma clearance of ch 14.18 was biphasic. Following the first course of treatmentt1/2, was 3.4±3.1 h andt1/2, 66.6±27.4 h in 9/10 children. Thet1/2, values were significantly less than those of 181±73 h previously reported in adult melanoma patients (P<-0.001), and 147.5 h in the adult osteosarcoma patient in our trial. The latter suggests different pharmacokinetics of mAb ch 14.18 in children and adults. After a second course of treatment, administered to 5/10 children,t1/2, decreased significantly from 72.9±19.8 h to 31.7±18.4 h (P=0.015). We there-fore conclude that the elimination kinetics of mAbs ch 14.18 in children and adults are different, and furthermore that repeated administration of mAb ch 14.18 to children with neuroblastoma leads to accelerated antibody clearance.This work was supported by grants from FDA, FD-R-000377 and NIH U10 CA 28439, and in part by a grant from the General Clinical Research Center Program, MOI RR00827, of the National Center for Research Resources, National Institutes of Health. M.M.U.-F. and C.S.H. were in part supported by a grant from the Children's Cancer Research Foundation, and M.M.U.-F. was in part supported by a grant of the Kommission für Forschung und wissenschaftlichen Nachwuchs, Charité 95-010/9610766     

Anti-idiotypic monoclonal antibody to a T-cell chronic lymphatic leukemia

Summary A murine anti-idiotypic monoclonal antibody (mAb), F1, (IgG_2a) was produced against the variable part of the T-cell receptor for antigen (T_i, /) on the tumor cells of a patient with T-cell chronic lymphatic leukemia (CD3⁺, 8⁺, 4^–). The molecular weight of the protein reactive with mAb F1, comodulation and coprecipitation with anti-CD3 antibody, and the restricted tumor-cell reactivity strongly support the anti-idiotypic nature of mAb F1. MAb F1 also stained 4% of peripheral blood lymphocytes of healthy donors. MAb F1 did not stimulate the tumor cells to DNA synthesis, but stimulated a fraction of the normal peripheral blood lymphocytes, mAb F1 did not mediate antibody-dependent cellular cytotoxicity or complement lysis to any significant degree in vitro. Three infusions of 1–10 mg anti-idiotypic mAb were given over a period of 4 weeks. The plasma half-life for mAb F1 was 3 h in the first 2 h after infusion and 44 h from 2 h to 120 h after infusion. After each treatment a rapid decrease of circulating tumor cells was seen. During the observation period an 80% reduction of the total circulating tumor cells was noted. After the second infusion, IgM and IgG antimouse antibodies were detected. Side-effects from therapy were fever, chills, nausea, vomiting, diarrhea, tachycardia, increase in systolic blood pressure and shortness of breath. Thus, in T-cell malignancies a major reduction of circulating tumor cells can be accomplished by low doses of anti-idiotypic mAb. Anti-idiotypic mAb might be a therapeutic agent of significant importance.     

A phase I study of neuroblastoma with the anti-ganglioside GD2 antibody 14.G2a

Summary Nine patients with neuroblastoma stage IV were treated with the murine monoclonal antibody 14.G2a, directed against disialoganglioside GD2. The antibody was injected daily for 5–10 days and the total applied dosage ranged between 100 mg/m² and 400 mg/m². The peak serum levels of mAb 14.G2a ranged from 28 µg/ml to 61 µg/ml. Pharmacokinetic data obtained in three patients indicated that the serum elimination of mAb 14.G2a fits a two-compartment model, with an -half-time (t _1/2 ) between 0.66 h and 1.98 h and a -half-time (t _1/2 ) between 30.13 h and 53.33 h. All patients presented with a human anti-(mouse IgG) antibody response either during or shortly after therapy. Eight patients showed a continuous decrease in complement component C4 during therapy, as well as an initial decrease in C3c and an initial increase in C3a, all suggesting an activation of the complement cascade. Side-effects consisted of allergic reactions like pruritus, exanthema, urticaria and of severe pain, predominantly located in the abdomen and lower extremities, which required the use of continuous intravenous morphine. Four patients additionally developed a transient hypertension and one patient experienced a transient nephrotic syndrome. Three patients were treated in an adjuvant setting and are not evaluable for tumor response. Of the remaining six patients, two had a complete remission, two showed a partial remission, and two patients did not respond to treatment.Supported by the Deutsche Krebshilfe     

Treatment of refractory Hodgkin’s disease with an anti-CD16/CD30 bispecific antibody

 A group of 15 patients with refractory Hodgkin′s disease were treated in a phase I/II trial with the natural-killer (NK)-cell-activating bispecific monoclonal antibody HRS-3/A9, which is directed against the Fcγ receptor III (CD16 antigen) and the Hodgkin’s associated CD30 antigen. The antibody was given four times every 3 – 4 days, starting with 1 mg/m². The treatment was well tolerated and the maximum tolerated dose was not reached at 64 mg/m². Side-effects were rare and consisted of fever, pain in involved lymph nodes and a maculopapulous rash. Nine patients developed human anti-(mouse immunoglobulin) antibodies. One complete and one partial remission (lasting 5 and 3 months, respectively), three minor responses (1 to 11+ months), and one mixed response were achieved. There was no clear-cut dose side-effect or dose/response correlation. NK cell activity increased in most of the patients treated with 4 mg/m² or higher doses but lasted no longer than 6 weeks after therapy. Our results encourage further clinical trials with this novel immunotherapeutic approach and emphasize the necessity to reduce the immunogenicity of the antibody to allow retreatment of responding patients. Accepted: 14 October 1997     

Defining the Pharmacodynamic Profile and Therapeutic Index of NHS-IL12 Immunocytokine in Dogs with Malignant Melanoma

Background

Interleukin (IL)-12 is a pro-inflammatory cytokine that mediates T-helper type 1 responses and cytotoxic T-cell activation, contributing to its utility as anti-cancer agent. Systemic administration of IL-12 often results in unacceptable toxicity; therefore, strategies to direct delivery of IL-12 to tumors are under investigation. The objective of this study was to assist the preclinical development of NHS-IL12, an immunocytokine consisting of an antibody, which targets necrotic tumor regions, linked to IL-12. Specifically this study sought to evaluate the safety, serum pharmacokinetics, anti-tumor activity, and immune modulation of NHS-IL12 in dogs with naturally occurring cancers.

Methodology/Principal Findings

A rapid dose-escalation study of NHS-IL12 administered subcutaneously to dogs with melanoma was conducted through the Comparative Oncology Trials Consortium (COTC). Eleven dogs were enrolled in four dose-escalation cohorts; thereafter, an additional seven dogs were treated at the defined tolerable dose of 0.8 mg/m². The expanded cohort at this fixed dose (ten dogs in total) was accrued for further pharmacokinetics and pharmacodynamics assessment. NHS-IL12 levels, serum cytokine concentrations, and peripheral blood mononuclear cell characterization (post-treatment) and draining lymph node immune profiling, and tumor biopsies (pre- and post-treatment) were collected. Adverse events included thrombocytopenia, liver enzymopathies, fever, and vasculitis. Correlation between interferon (IFN)-γ induction, adverse events, and NHS-IL12 exposure (maximum concentration and area under the concentration-time curve) were dose-dependent. Serum IL-10 levels and intratumoral CD8⁺ populations increased after treatment. Partial responses, according to Response Evaluation Criteria in Solid Tumors (RECIST) criteria, were observed in two dogs treated with NHS-IL12 0.8 mg/m² and 1.6 mg/m².

Conclusions/Significance

NHS-IL12 was administered safely to dogs with melanoma and both immunologic and clinical activity was observed. This study successfully defined a narrow therapeutic window for systemic delivery of NHS-IL12 via the subcutaneous route. Results will inform the design and implementation of first-in-human clinical trials of NHS-IL12 in cancer patients.     

Initiation of humoral and cellular immune responses in patients with refractory Hodgkin's disease by treatment with an anti-CD16/CD30 bispecific antibody

Fifteen patients with refractory Hodgkin's disease were treated in a dose-escalation trial with the bispecific monoclonal antibody (bi-mAb) HRS-3/A9, which is directed against the Fcγ receptor III (CD16 antigen) and the Hodgkin's-associated CD30 antigen. Treatment consisted of four cycles of four bi-mAb infusions given over 1 h every 3–4 days at different dose levels ranging from 1 mg/m² to 64 mg/m². Measurable serum levels (above 0.1 μg/ml) of circulating bi-mAb could be detected in patients treated with doses above 4 mg/m², reaching peak levels of 9.5 μg/ml immediately after the end of antibody infusion on the highest dose level. Bi-mAb elimination corresponded to second-order kinetics with a terminal half-life time (t _1/2,β) of 28–32 h. Bi-mAb treatment induced the occurrence of human anti-(mouse Ig) antibodies (HAMA) in 6 out of 13 patients initially testing negative. All 6 patients not only developed anti-isotypic anti-(mouse Ig) but also anti-idiotypic and anti-anti-idiotypic antibodies. While no consistent changes of peripheral blood cell counts, or of any lymphocyte subpopulation including natural killer (NK) cells, has been observed, 4 out of 6 evaluable patients treated with doses of at least 4 mg/m² showed an increase of NK cell activity within 2 weeks after treatment, which lasted for a maximum of 12 weeks. Circulating amounts of soluble CD30 antigen could be detected in the serum of 6 patients. However, like the results and time courses of all the other immunological parameters evaluated, this was not predictive for treatment outcome. Received: 16 September 1999 / Accepted: 6 January 2000     

Construction and biological activity of a recombinant bispecific single-chain antibody designed for therapy of minimal residual colorectal cancer

 Unlike monoclonal antibodies, clinical application of bispecific antibodies has so far lagged behind because of difficult, low-yield production techniques as well as considerable toxicity attributed to bispecific antibody preparations containing immunoglobulin-Fc parts and anti-CD3 homodimers [10, 2]. These difficulties were overcome by recombinant generation of a bispecific single-chain antibody (bscAb) joining two single-chain Fv fragments via a five-amino-acid glycine-serine linker. The anti-CD3 specificity directed against human T cells was combined with another specificity against the epithelial 17-1A antigen. The following domain arrangement was critical in this individual case to obtain a fully functional bscAb: VL_17-1A-VH_17-1A-VH_CD3-VL_CD3. The bscAb was expressed in chinese hamster ovary cells with a yield of 15 mg/l culture supernatant whereas numerous attempts to obtain a functional protein expression in Escherichia coli failed. The low-molecular-mass bispecific construct (60 kDa) could easily be purified by its C-terminal histidine tail. The antigen-binding properties are indistinguishable from those of the corresponding univalent single-chain Fv fragments as shown by enzyme immunoassay and flow cytometry. We could show that the bscAb, which lacks Fc parts and anti-CD3 homodimers is highly cytotoxic for 17-1A positive tumor cells in nanomolar concentrations and in the presence of human T cells. Most remarkably, it does not stimulate T lymphocyte proliferation in the absence of tumor cells and, moreover, does not induce CD25 up-regulation and the secretion of potentially toxic lymphokines such as tumor necrosis factor α, interleukin-6 and interferon γ. Maximal cytotoxicity (⁵¹Cr release) was achieved without notable costimulation and was not further enhanced by adding costimulatory signals such as those delivered by anti-CD28 antibodies. CD8⁺ as well as CD4⁺ T cell subpopulations were recruited to exert cytotoxicity against tumor cells with different kinetics. CD8⁺ cells induced high ⁵¹Cr release within 4 h while CD4⁺ cells required a 20-h incubation. The systemic application of the 17-1A/CD3-bscAb could be a major improvement in therapy against disseminated micrometastatic tumor cells. A prospective, randomized clinical trial showing that an anti-17-1A monoclonal antibody could prolong survival of colorectal cancer patients after 5 and 7 years, warrants an assessment of the clinical efficacy of this bscAb exhibiting a 1000-fold higher specific cytotoxicity against tumor cells in virto. Accepted: 14 October 1997     

A phase I trial with recombinant interferon γ (Roussel UCLAF) in advanced cancer patients

Summary A total of 29 patients with advanced malignancy were treated with recombinant interferon (rIFN, specific activity = 2.10⁷ units/mg, purity >95%) given by intravenous bolus at doses escalating from 0.01 mg/m² to 5 mg/m² (2 × 10⁵–10⁸ IU/m²) in nine successive steps (at least 3 patients/step). Injections of rIFN were repeated every 72 h for 15 days. Toxicity was evaluated according to the WHO scale. Fever and chills occurred in all patients treated without clear dose effect. Nausea and vomiting appeared at the fifth dose level and their frequency seemed to be dose-related. Cardiovascular side-effects (first-degree atrioventricular reversible block) were observed at the 2 mg/m² and 5 mg/m² levels (3 patients). Hematological toxicities were mild (2 grade 1 and 1 grade II cases of granulocytopenia). Minor biological modifications included a transitory rise in hepatic enzymes (12 patients), which correlated with the presence of liver metastasis. Hypocholesterolemia was observed in 18 patients. The appearance of antibodies against rIFN was not detected. One partial clinical response was observed in a patient receiving 2 mg/m². During rIFN therapy this patient had the highest scores in this series for peripheral T lymphocytes with an activated phenotype (HLA DR⁺, TAC⁺) = 15% and for natural killer (NK) cells (NKH1, Leu19⁺) = 17%. rIFN appears as a well-tolerated and promising therapeutic agent with toxicities and mode of action probably distinct from IFN and .     

Photosynthetic activity of phytoplankton in a high altitude lake (Emerald Lake,Sierra Nevada,California)

Photosynthetic activity by phytoplankton was measured during the ice-free seasons of 1984, 1985 and 1987 using the ¹⁴C radioassay in high altitude Emerald Lake (California). Relative quantum yield (^B) and light-saturated chlorophyll-specific carbon uptake (P_m ^B) were calculated from the relationship of light and photosynthesis fitted to a hyperbolic tangent function. Temporal changes in P_m ^B showed no regular pattern. Seasonal patterns of ^B generally had peaks in the summer and autumn. Phytoplankton biomass (as measured by chlorophyll a) and light-saturated carbon uptake (P_m) had peaks in the summer and autumn which were associated with vertical mixing. Estimates of mean daily carbon production were similar among the three years: 57 mg C m^–2 2 d^–1 in 1984, 70 mg C m^–2 2 d^–1 in 1985 and 60 mg C m^–2 d^–1 in 1987. Primary productivity in Emerald Lake is low compared to other montane lakes of California and similar to high-altitude or high-latitude lakes in other regions.     

Biodistributions of intact monoclonal antibodies and fragments of BLCA-38, a new prostate cancer directed antibody

Background: Monoclonal antibodies (MAbs) are used for targeting agents to tumours while minimizing normal tissue exposure. Methods: A new anti–prostate cancer MAb, BLCA-38, was radioiodinated (I¹²⁵) and assessed for its ability to target subcutaneous human prostate cancer (DU-145) xenografts after systemic intraperitoneal administration. For comparison, the profile of J591 MAb (now in clinical trial) against LNCaP-LN3 tumours was examined. Biodistribution profiles were obtained at various times, by assessing injected dose/gram (%ID/g) and xenograft to blood (X/B) ratios. Microautoradiography of xenografts was performed. After conjugation with a melittin peptide toxin, the profiles of BLCA-38 and J591 were compared with that of an irrelevant antibody, DS-1. Results: Xenograft localization by ¹²⁵I-labeled BLCA-38 and J591 MAbs to their relevant antigen-positive tumors was comparable, and there was no unusual localization in nontumour tissues. F(ab)₂ and Fab fragments gave improved X/B ratios, but the %ID/g xenograft was decreased and they accumulated in kidneys, bladder and stomach. In contrast, the conjugates of irrelevant antibody showed no tumour targeting. Microautoradiography showed more tumour accumulation of MAbs than F(ab)₂s or Fabs. Conclusions: BLCA-38 can target prostate cancer in vivo almost as effectively as J591. Given that J591 is used clinically, BLCA-38, which targets a different antigen, has potential for radioimmunoscintigraphy and for therapeutic targeting of prostate cancer.