SUMMARY REPORTS OF EXCHANGE SCIENTISTS

(1) Takashi Tsuruo
Japanese Foundation for Cancer Research

Sponsor and Host Institution:
Drs. R. Ozols and B.A. Chabner
Clinical Oncology Program
National Cancer Institute
Dates of Visit: April 1984 - April 1985

Summary of Activities
1. Establishment of vincristine- and adriamycin-resistant sublines from human lung, breast, and ovarian cancers, and the elucidation of resistance mechanisms. Cultured cell lines from refractory patient established. Mechanism of resistance studied by measuring drug efflux function, changes in targets and changes in affinity to the drugs.
2. Overcoming of drug resistance by calcium channel blockers. The effects of calcium channel blockers, such as verapamil, nicardipine, diltiazem, studied in these resistant tumor lines and the mechanisms of action of calcium channel blockers elucidated.
3. Elucidation of the variety of mechanisms of inherent drug resistance.
4. Clinical application of the approach against drug refractory patients.



(2) Toshitada Takahashi
Aichi Cancer Center Research Institute

Sponsor and Host Institution:
Dr. H. F. Oettgen
Memorial Sloan-Kettering Institute
Dates of Visit: October 8-23, 1984

Summary of Activities
The purpose of this visit to the USA was to exchange information regarding the present status of the application of mouse monoclonal antibodies produced against human tumors for the diagnosis and treatment of cancer patients. The following investigators provided me with their recent progress in this field: Drs. Oettgen, Old, and Dupont of Memorial Sloan-Kettering Institute, New York; Dr. Hansen of Fred Hutchinson Cancer Research Center, Seattle; and Dr. Bean of Virginia Mason Research Center, Seattle.
1. Discussion with Dr. Dupont about basic studies on the use of toxin-conjugated anti-T cell antibody for allogeneic bone marrow transplantation.
For T lymphocyte depletion of donor bone marrow for graft-versus-host disease (GvHD) prophylaxis, the use of a monoclonal instead of an antibody and complement might be more efficient as suggested by a rodent model. Thus, the toxic A chain of ricin was conjugated by a disulfide bond to a monoclonal anti-body against p67kD pan-T antigen, and the ability to inhibit in vitro T lymphocyte transformation was studied. In the presence of 20 mM NHH₄CL, the immunotoxin decreased lymphocyte proliferation in response in mixed lymphocytic culture, and the development of allocytotoxic T cell was also inhibited. Further treatment of bone marrow cells with the immunotoxin preserved hematopoietic progenitor cells. These in vitro studies suggested the possibility that the immunotoxin as such might be an effective agent for use in clinical bone marrow transplantation. One weak point with the immunotoxin prepared is the instability after lyophilization, indicating that the conjugation method should be improved to be used by various clinics.
2. Discussion with Dr. Hansen and Dr. Bean about the use of anti-T cell monoclonal antibodies for allogeneic bone marrow transplantation in hematopoietic malignancies.
In vitro depletion of T lymphocytes from donor marrow in patients receiving HLA-identical marrow grafts for treatment of hematologic malignancies (20 patients) has been attempted to reduce the incidence of GvHD, which occurs in 35-50% of the patients treated with a mixture of eight monoclonal antibodies against T cells [p50, p19-25, p55, p67, p32-33, and p44 (9.3) antigens] and rabbit complement in order to achieve a 2-3 log depletion of T cells. The incidence and severity of acute GvHD was significantly decreased (3 of 19, grade II) in the group of patients compared to histological controls (32 of 63, grade II, III, IV). However, depletion of T cells was associated with an increased incidence of graft failure which was transient in one patient, but apparently irreversible in four others, in contrast to historical controls (0 of 21).
Their results suggested that a certain fraction of T cells plays an important role in maintaining sustained hematopoietic function in recipients of HLA-identical marrow grafts, i.e., the absence of this T cell fraction in donor marrow allows residual host T cells to survive so that these host immune cells reject donor hematopoietic cells. Thus, it is important to study whether the T cell fraction in donor marrow causing GvHD and that fraction suppressing residual host T cells after the conditioning regimen can be delineated by a certain monoclonal antibody. Clinically, attempts should be made to reduce the residual T cells in the recipient by modification of the present conditioning regimen.
3. Discussion with Dr. Old and Dr. Oettgen about the use of antibodies against colon cancer for serum diagnosis.
Mouse monoclonal antibodies against melanoma, kidney cancer, bladder cancer, astrocytoma, gastrointestinal tract cancer, choriocarcinoma, and other types of cancer have been produced and serological analysis has been extensively carried out in their laboratories. One of the antibodies against melanoma cells (R24 antibody) has already been used for a Phase I trial in patients with malignant melanoma as described below. Preliminary experiments for the possible use of some of these antibodies for serum diagnosis are also in progress.
During the course of analysis of antibodies produced against colon cancer and other adenocarcinomas, complexity of CEA and ABO-Lewis blood group antigen has been elucidated. (a) By radioimmunoprecipitation, 180 kDa of CEA molecule and 50 kDa NCA (normal crossreacting antigen) molecule was defined. Most of the antibodies immunoprecipitate 180/160 kDa or 180/160/50 kDa, and one anti-body detects only 180 kDa of CEA. The latter antibody will probably give lower background if it were used for screening or noncancer population. (b) CA19-9, which is a good tumor marker for colon and pancreas cancer, is now defined as sialylated le^a by Wistar Institute Group. Various determinants of ABO-Lewis blood group antigen have been defined by monoclonal antibodies, and precise analysis of these carbohydrates’ antigen structures will provide us information whether some of the determinants can be used for serodiagnosis of adenocarcinoma.
4. Discussion with Dr. Oettgen and Dr. Old about the use of anti-melanoma antibody for treatment of the patients.
R24 antibody, which identifies GD3 ganglioside on the surface of melanoma cells and other cells of neuroectodermal origin, has been applied for 12 patients with metastatic melanoma in order to investigate the responses of the patients with regard to different dose levels, toxicity, serological parameters, and tumor response. Patients were treated with intravenous infusion of R24 antibody at three dose levels, 8 mg/M², 80 mg/M², and 240 mg/M², over a period of 2 weeks. Inflammatory reactions (urticaria, pruritis, erythema, subcutaneous ecchymoses) were observed around tumor sites in patients treated at doses of 80 mg/M². However, no major side effects were observed thus far. Tumor biopsies during and after treatment showed lymphocyte and mast cell infiltration, mast cell degranulation, and complement deposition.
Major tumor regression has been observed in three patients [one of three (8 mg/M²), one of six (180 mg/M²), one of three (240 mg/M²)]. It is noted that the responses to the antibody treatment were not observed right after the treatment and were observed 5 to 10 weeks later than the treatment. Immunoperoxidase stain showed that tumor cells biopsied or aspirated during therapy in two patients still expressed GD3 antigen in the face of substantial levels of circulating and tumor-bound R24 antibody, indicating no evidence of antigenic modulation during therapy. Another interesting finding is that the progression of tumor after the treatment was not related to outgrowth of GD3 antigen-negative melanoma cells, because biopsies done after tumor persistence or progression showed strongly GD3-positive cells in the lung or skin lesions of four patients. Further studies in melanoma patients and in patients with other types of GD3-positive tumors (astrocytomas, sarcomas) will be necessary to evaluate the potential of R24 antibody in the treatment of human cancer.

Summary
a. The program assisted my research very much. The discussion with Dr. Hansen gave us valuable information on allogeneic bone marrow transplantation, which another group in Nagoya is about to start. In vitro experiments with immunotoxins with anti-T cell activity by Dr. Dupont's group suggested a good possibility that antibodies against null cell acute lymphocytic leukemia produced in our laboratories can be used as immunotoxins for autologous bone marrow transplantation. Discussion with Dr. Oettgen and Dr. Old about a Phase I study of R24 anti-melanoma antibody gave us a hope that some of the mouse monoclonal antibodies might be useful for the treatment of cancer patients.
b. Through the Program, I was able to have a chance for collaborations and discussions with leading scientists in the United States. Up-to-date information and new approaches encouraged and improved our research. The collaborations will be continued by exchange materials and methodologies.
c. It is very important for this Program to support promising young investigators who wish to learn new techniques and researchers who want to obtain precious experimental materials which are difficult to mail.



(3) Tohru Masaoka
Center for Adult Diseases, Osaka

Sponsor and Host Institution: Dr. E. D. Thomas
Fred Hutchinson Cancer Center
Dates of Visit: February 3-17, 1985

Summary of Activities
1. To review and inspect the following:
The usage of laminar air flow room.
Procedures of bone marrow transplantation.
Total body irradiation.
Treatment of bone marrow with monoclonal antibody.
Measures against cytomegalovirus infection.
The organization of BMT team.
Costs and management.
2. To present data for critical review concerning:
BMT results in Japan.
Prevention and treatment of GvHD and interstitial pneumonitis.
Stepwise standards of bioclean nursing.
3. To discuss the following general issues:
Prevention and treatment of GvHD and interstitial pneumonitis.
Application of monoclonal antibody on BMT.
Further study plan on BMT.



(4) Taketo Mukaiyama
Japanese Foundation for Cancer Research

Sponsor and Host Institution:
D. Houseman
Massachusetts Institute of Technology
D. Von Hoff
University of Texas Health Science Center
San Antonio
Dates of Visit: January 24 -February 5, 1985

Summary of Activities
A. M.I.T.
Using "in gel DNA renaturation method," Dr. Igor B. Roninson showed that multidrug-resistant cells (which are resistant to adriamycin, daunomycin, vincristine, actinomycin D, etc.) contain amplified DNA fragments. Furthermore, loss of the multidrug-resistant phenotype on growth in the absence of drugs correlates with the loss of amplified DNA. These results suggest that the DNA amplified in multidrug-resistant cell lines include the gene responsible for common mechanism of multidrug resistance in these cells. Subsequently, Dr. Phillips Gros made cosmid library and plasmids containing small fragments from this cosmid library. Now we are analyzing these plasmids for the presence of transcribed DNA sequences by Northern blotting and determining encoding DNA sequence. Recently, Dr. Roninson showed that one of these plasmids is amplified in two clinical samples taken from patients resistant to adriamycin and vincristine.
As a medical oncologist, I am very interested in this result. Therefore, I would like to return to M.I.T. bringing many clinical samples which are resistant to various antitumor agents and mouse cell lines which are also resistant to these drugs. Then I will examine whether these samples contain amplified DNA fragments or not. Thus, I would like to develop a new drug sensitivity test to use this drug resistance-related gene. Furthermore, I want to make the protein encoded by this multidrug-resistant gene in E. coli and make monoclonal antibody, with plans to target therapy against drug-resistant cells.
B. University of Texas Health Science Center at San Antonio
Recently, Dr. Daniel D. Von Hoff developed a new drug sensitivity test, named "capillary cloning assay". This method has several advantages compared to conventional human tumor clonogenic assay. These are improved growth and plating efficiencies, decreased contamination, and economy, etc. Dr. Von Hoff planned a prospective randomized clinical trial of the capillary cloning system versus clinician's choice. This trial showed that the response rate and survival time of patients with metastatic cancer are similar regardless of whether their single agent chemotherapy treatment is selected by the clinician or by the in vitro capillary cloning assay. This method does not appear to be perfect, but I think it is worth trying.
Dr. Von Hoff also developed a rapid, semiautomated radiometric system, named “BACTEC system.” The radiometric system utilizes inhibition of convention of ¹⁴C-glucose to ¹⁴CO₂ as an index of cytotoxicity. Human tumor cell lines were used to compare the antitumor activity of drugs measured by the BACTEC system versus the antitumor activity of the same drugs measured by a conventional cloning system. For all cell lines tested there was a good agreement in comparison of percent, survival measured by the BACTEC system versus the standard cloning system. The radiometric- BACTEC system is ideal for use as a prescreening for testing a large number of new chemical compounds against a large number of human tumor cell lines.