John H. Frenster and William M. Rogoway
Division of Oncology, Department of Medicine
Stanford University School of Medicine
Palo Alto, California 94304

Introduction:
Procedure:
Early Effects:
Support:
References:
Additional References:
Links:

Patients with a wide variety of malignant neoplasms may have circulating antibodies (1) or circulating sensitized lymphocytes (2) which are specific for antigens within the neoplastic cells of the patient or of other patients with the same type of neoplasm. In-vitro assays of the cytotoxicity of such lymphocytes against autochthonous neoplastic cells show that prior in-vitro activation of the lymphocytes by phytohemagglutinin (P.H.A.) significantly increases their cytotoxicity (3), which is partially mediated by the release of soluble cytotoxic substances during activation by P.H.A. (4, 5). Preliminary reports suggest that parenteral administration of P.H.A. to cancer patients may result in favorable tumor responses (6), but the repeated systemic use of P.H.A. is limited by the development of antibodies directed against it (7). A procedure has now been developed by which large numbers of autologous lymphocytes from a patient can be activated in-vitro and reinfused into the patient after the P.H.A. used for the activation is removed from the lymphocytes by washing.

The entire procedure can be completed under sterile conditions within 30 hours, utilising a closed system of Fenwal haemorepellent plastic blood-donor and blood-transfer bags and tubes. The patient donates 10 ml. per kg. of whole blood into a heparinised blood-donor bag, in which it is thoroughly mixed by inversion during collection. The bag is then centrifuged at 10 g for 5 minutes at 2^o C, and the cloudy supernate, containing lymphocytes, granulocytes, and platelets, is carefully transferred to a separate plastic bag by pressure from a plasma-extractor. The remaining erythrocytes are then reinfused from the original donor bag into the patient. The transferred cells and plasma are now diluted 1/5 by the addition of sterile mixture 199. Penicillin and streptomycin are each added to a concentration of 300 ug. per ml. of total volume, and the highly purified glycoprotein octamer of P.H.A. (8, 9) is the added to a concentration of 1- ug. per ml. of total volume. The transfer bag containing the diluted leukocyte suspension is then tightly sealed, incubated without agitation at 37^o C for 24 hours, and then centrifuged at 800 g for 5 minutes at 2^o C. The supernate bearing the unabsorbed P.H.A. is carefully transferred to a separate plastic bag by pressure from a plasma extractor, and the remaining lymphocytes and eryhrocytes are resuspended in 300 ml. of a wash solution consisting of heparinised 3/1 mixture of Earle's basic salt solution and phosphate-buffered isotonic saline solution which is added to the bag. The suspension is then centrifuged in the bag at 800 g for 5 minutes at 2^o C, the supernate again carefully transferred to a separate plastic bag, and the washing procedure of the cells repeated twice more to further remove residual P.H.A. and disintegrated granulocytes and platelets, and to further reduce lymphocyte and erythrocyte aggregation. The resultant washed lymphocytes are then resuspended in the bag with a 300 ml. volume of of the wash solution, and the suspension of activated autologous lymphocytes is slowly infused at a rate of 2 ml. per minute through a filtered recipient line into a peripheral vein of the patient. The yield of autologous peripheral-blood lymphocytes during in-vitro activation and washing before reinfusion into a 42 kg. man with Ewing's Sarcoma metastatic to lungs was as follows:
 

Stage in procedure	No. of lymphocytes (x 108)
Whole-blood donation	9.92
While-blood supernate	3.30
Suspension after incubation	2.31
Reinfused washed suspension	1.56

The peripheral-blood lymphocyte-count was 2300 per c.mm. at the time of the 420 ml. donation of whole blood.

The reinfusion of the activated lymphocytes is well tolerated, with no suggestion to date of pyrogenic reactions, febrile responses, transfusion reactions, hemolysis, leucopenia, thrombocytopenia, thromboembolism, or anaphylactic reactions. Tumour responses are now being evaluated.

Supported in part by grants CA-10174 and AM-01006 from the U.S. Public Health Service, and by a research-scholar award from the Leukemia Society.

References:

1. Gold P, Cancer 20: 1663 (1967).

2. Hellstrom IE, Hellstrom KE, Pierce GE, Bill AH, Proc. Natl. Acad. Sci. U.S.A. 60: 1231 (1968).

3. Chu EHY, Stjernsward J, Clifford P, Klein G, J. Natl. Cancer Inst. 39: 595 (1967).

4. Granger GA, Williams TW, Nature 218: 1253 (1968).

5. Williams TW, Granger GA, Nature 219: 1076 (1968).

6. Robinson E, Lancet 2: 753 (1966).

7. Simons MJ, Fowler R, Fitzgerald MG, Nature 219: 1021 (1968).

8. Rigas DA, Johnson EA, Ann. N. Y. Acad. Sci. 113: 800 (1964).

9. Stanley DA, Frenster JH, Rigas DA, J. Cell Biol. (in the press).

Additional References:

0. Electron Microscopy of Human Lymphocytes before and after Activation by PHA (Busch H, 1974).

1. Stanley DA, Frenster JH, Rigas DA, "Localization of H³-Phytohemagglutinin within Human Lymphocytes and Monocytes", in: Proc. Fourth Annual Leukocyte Culture Conf. (edit. by McIntyre OR), pp. 1-11, New York: Appleton-Century-Crofts, 1971.

2. Frenster JH, Rogoway WM, "Immunotherapy of Human Neoplasms with Autologous Lymphocytes Activated in-Vitro", in: Proc. Fifth Annual Leukocyte Culture Conf. (edit. by Harris, J), pp. 359-373, New York: Academic Press, Inc. 1970.

3. Allen BL, Frenster JH, "Low-Dose Combination Chemotherapy of Disseminated Human Neoplasms", Lancet 2: 1324 (December 11, 1971).

4. Frenster JH, "Phytohemagglutinin-Activated Autochthonous Lymphocytes for Systemic Immunotherapy of Human Neoplasms", Ann. N.Y, Acad. Sci. 277: 45-51 (1976).