Amitabha Mazumder

Phase I study of the adoptive immunotherapy of human cancer with lectin activated autologous mononuclear cells

In previous in vitro studies, the authors showed that phytohemagglutinin (PHA) stimulated peripheral blood lymphocytes (PBL) from cancer patients to generate cells that were lytic for fresh autologous tumor but not for lymphocytes or lymphoblasts. Thus, after IRB approval, a phase I clinical protocol was instituted in cancer patients who had failed all other therapy to determine the toxicity and effects, in vivo, of the infusion of large numbers of such PHA activated autologous PBL. Ten patients were treated on the protocol, six with sarcoma, one with melanoma, and three with colorectal cancer. Up to a total of 1.7 × 1011 PBL were obtained from 7 to 15 successive leukaphereses, the cells from each leukapheresis being incubated in vitro in medium containing PHA and human AB serum for 2 days and then reinfused following the next leukapheresis 2 days later. Toxicity encountered included fever and chills in 10/10 patients, headaches in 5/10, nausea and vomiting in 3/10, and requirement for erythrocyte transfusion in 8/10. No evidence for autoimmune disease, abnormal serum chemical or coagulation studies, or pulmonary emboli was found. 111Indium trafficking studies showed distribution of infused cells mainly to the spleen and liver, with some accumulation in the lungs and tumor especially after repeated infusions. In 9/10 patients, activated PBL were detected in the peripheral circulation by the sixth leukapheresis. Evidence for this was found by assaying the incorporation of tritiated thymidine (3H‐Tdr) into, and lysis of fresh tumor cells by, unstimulated PBL from successive leukaphereses. No tumor regression was seen in these patients with bulk disease. These studies demonstrated that large numbers of PHA‐activated PBL can be safely obtained and infused into humans, achieving an increase in the number of circulating activated cells with evidence of migration of cells to tumor, lungs, liver and spleen. Further studies of the use of activated lymphocyte infusion in conjunction with chemotherapy in humans are in progress.

Lysis of fresh human solid tumor cells by autologous lymphocytes activated in vitro by allosensitization

SummaryWe have previously demonstrated that cancer patients peripheral blood lymphocytes (PBL) allosensitized against single or pool normal donor PBL are capable of lysing fresh autologous tumor cells in a 4-h 51Cr-release assay. In this report, we present further investigations into this phenomenon. These alloactivated killer cells (A-AK cells) lysed autologous and allogeneic tumors and allogeneic but not autologous PBL. Furthermore, cold target inhibition studies demonstrated that autologous and allogeneic tumors were lysed by the same effector cells. Multiple metastases from the same patient were equivalently lysed by these A-AK cells. The presence of adherent cells and proliferation of the precursors were necessary to generate A-AK cells, although the effector cell itself was radioresistant and nonadherent. The effects of allosensitization were enhanced by the addition of lectin-free interleukin-2 preparations to the in vitro culture by partial depletion of adherent cells prior to sensitization. The A-AK effector cell was OKT3+, OKT8+, OKT4−, OKM1− and could be generated by just 3 days of allosensitization. The precursors for A-AK cells could be separated from NK cells on percoll gradients and lysis could be generated from thoracic duct lymphocytes, a population devoid of NK cells. The phenotype of the majority of the precursor cells was OKT3+, OKT4−. These allocatived PBL could be expanded in crude or lectin-free interleukin-2 without loss of cytotoxicity for fresh autologous tumor cells. Activated T cells represent a population of non-NK cells with broad lytic specificity for fresh tumor cells. Such cells may be of value in the adoptive immunotherapy of human solid tumors and may play a role in immune surveillance.

In vitro growth of cytotoxic human lymphocytes. V. Generation of allospecific cytotoxic lymphocytes to nonimmunogenic antigen by supplementation of in vitro sensitization with partially purified T-cell growth factor

Abstract Substantial generation of human target-specific cytotoxic lymphocytes (CTL) was exhibited in allogeneic in vitro sensitization (IVS) cultures employing subimmunogenic numbers of allogeneic stimulator lymphocytes when supplemented with partially purified and lectin-freed T-cell growth factor (PP-TCGF). These results indicate that very few allogeneic stimulator cells are able to provide the optimal specificity signal required for allogeneic IVS. Augmentation was unique to PP-TCGF since neither PHA nor the unpurified TCGF exhibited this property. PP-TCGF augmented the generation of cytotoxic lymphocytes when added at the initiation of the 7-day culture (Day 0) or on Day 1, 2, 3, or 4. The level of cytotoxic activity generated was found to be proportional to the quantity of PP-TCGF used and was not due to immune interferon contained in the PP-TCGF preparations. PP-TCGF was also found to augment alloantigen stimulated proliferation as measured by [ 3 H]tdr uptake. Finally, in two model IVS systems in which allogeneic stimulator cells had been rendered nonimmunogenic by uv irradiation or by heat killing, PP-TCGF supplementation was found to totally restore the generation of target specific CTL by providing a proliferative stimulus. These results show that human PP-TCGF contains a soluble factor, functional in controlling the proliferation and development of human CTL in vitro . The use of PP-TCGF in IVS cultures may represent an useful technique for augmenting the in vitro immune response to weak immunogens.

The Use of Lymphoid Cells Expanded in IL-2 for the Adoptive Immunotherapy of Murine and Human Tumors

The adoptive immunotherapy of cancer refers to the transfer, to the tumor-bearing host, of immunologically competent cells capable of mediating responses against tumor. Specific adoptive immunotherapy is a theoretically attractive approach to the treatment of tumors although few examples exist of the effective treatment of established syngeneic solid tumors using this modality. Early reports from Delorme and Alexander (1964) claimed that thoracic duct lymphocytes from immunized rats as well as lymphocytes from immunized xenogeneic animals (Alexander et al., 1966) could mediate the regression of solid methylcholanthrene-induced sarcomas. Borberg et al. (1972) treated mice bearing the Meth-A sarcoma with up to 4 × 109 immunized syngeneic lymphocytes and succeeded in causing the regression of established tumors. Using the Meth-A tumor, but an alternative method of immunization, Berendt and North (1980) have demonstrated that the intravenous infusion of sensitized T cells from immune donors could cause complete regression of established tumors growing in a T-deficient host. They also showed that infusion of splenocytes from tumor-bearing donors could inhibit this regression, suggesting that suppressor T cells existed in the tumor-bearing host. Fernandez-Cruz et al. (1980) showed that intravenous infusion of immune lymphocytes was capable of curing rats bearing a subcutaneous tumor, and Smith et al. (1977) showed that immunized peritoneal exudate cells from syngeneic guinea pigs were capable of curing guinea pigs with established line 10 hepatoma.