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A review of the literature on cell-mediated immunity to cancer revealed that many in vitro techniques have been used to investigate this subject. The techniques had been introduced to detect reactions between lymphocytes and tumour antigens, and serum factors which modify these reactions. Most of the methods were applicable to both animals and man and clinical uses in human cancer have been suggested, but the many disadvantages of existing methods seem to make them unsuitable for routine use.The leucocyte adherence inhibition (LAI) test was introduced as an improved technique in tumour immunology, and the original work of this thesis is concerned with the development, investigation and application of this techniqueAnti-tumour immunoreactivity to methylcholanthrene-induced tumours in mice was studied and specific cell-mediated immunity was demonstrated. Murine tumours showed individual tumour specificity. The LAI test was shown to depend on the production of a soluble factor which had characteristics in common with known lymphokines. It was produced by T-lymphocytes and its action was directed against adherent cells in the leucocyte-antigen mixtures. An indirect or 2-stage LAI test which depended on the presence of a soluble mediator was developed and this technique was used to study the kinetics of cell-mediated immunity in mice after tumour transplantation.Specific blocking of lymphocyte-antigen interaction was demonstrated in the serum of tumour-bearers and the kinetics of blocking in mice before and after tumour removal were investigated. Serum from mice after tumour removal was found to contain unblocking factors which interfered with the action of blocking factors in tumour-bearer serum.The LAI test was used to study immunoreactivity in human cancer. Four types of tumours were used: melanoma, colorectal carcinoma, medullary carcinoma of the thyroid and hepatoma. Patients were studied for reactivity to tumour antigens before and after tumour removal.The specificity of the LAI reaction with human tumours was related to the histological type of tumour rather than individual tumours. In hepatoma the situation was more complex and the LAI test was used to analyse the antigens involved. Serum blocking factors were detected consistently in patients with progressing tumours.The LAI test correlated well with the presence of tumour in both animals and man, and was shown to be a useful diagnostic aid in human cancer.

Blood leukocytes from patients with cancer (malignant melanoma; adenocarcinoma of colon, rectum, and breast) reacted with aqueous extracts of tumors of the corresponding type, with the result that adherence of the leu kocytes to glass was diminished. Leukocytes from normal individuals did not react. The leukocyte adherence inhibition (LAI) test could be completed in a few hours. Sera from tumor-bearing patients blocked the LAI reaction of their own leukocytes or leukocytes from other patients with the same type of tumor. Serum blocking activity was lost soon after surgical removal of the tumor; the patient’s serum then became unblocking. The LAI technique gave consistent results in a series of patients, analogous to those reported with the lymphocyte cytotoxicity test, and was easier and quicker.

Three patients with medullary carcinoma of the thyroid (MCT) were tested for antitumor cell-mediated immunity and serum blocking factors by the leukocyte adherence inhibition (LAI) test. Leukocyte reactivity with both autochthonous and allogeneic MCT extracts was detected after surgical removal of tumor. Blocking factors were present in patients with progressing MCT but disappeared after tumor removal; in one patient the serum then became unblocking. The leukocytes of seven control subjects did not react in the LAI test with MCT extracts. The value of the LAI test in MCT is discussed as an adjunct to the serum calcitonin assay.

The leucocyte adherence inhibition test provides a rapid, reliable, and specific technique for the immunodiagnosis of primary hepatocellular carcinoma (malignant hepatoma). The patient's blood leucocytes are tested in vitro for cell-mediated immunity against tumour-associated antigens and the serum is tested for blocking factor which interferes with the immunological reaction. Specific reactivity of both leucocytes and serum was consistently detected in patients with malignant hepatoma, and negative reactions were obtained in other liver diseases including secondary tumours of the liver. The test has provided positive evidence for the presence of hepatoma when more conventional methods gave doubtful or negative results. A positive result preceded the clinical appearance of tumour by up to three years in two patients.

Hairy cell leukemia is a lymphoproliferative disorder characterized clinically by cytopenias. Standard therapy following variable periods of disease stability consists of splenectomy that often restores normal hematologic parameters for periods ranging from weeks to years. Fifteen patients (five without prior splenectomy or chemotherapy) were treated with 3 X 10(6) units per day of recombinant leukocyte A interferon and 14 of 15 patients completed eight weeks of therapy and were evaluated for response. There was one complete and 12 partial responses for an overall response rate of 93 percent. All of these patients' conditions have remained in complete or partial remissions and they continue to receive interferon with a median follow-up of six months. Coincident with the normalization of peripheral blood counts was a return of natural killer activity and normalization of immunologic surface markers as determined by monoclonal antibodies. This study confirms and extends earlier observations with natural alpha-interferon and indicates that recombinant leukocyte A interferon in low daily doses is also very effective treatment for hairy cell leukemia. In fact, it may be the best single modality of therapy for inducing both hematologic and immunologic recovery of these patients and deserves consideration as initial therapy.

The ability of tumour extracts to augment E-rosette formation by cancer patients' lymphocytes was demonstrated for colorectal carcinoma, breast carcinoma and melanoma. Positive augmentation reactions were obtained with 14 to 20 patients tested with extracts related to their own tumour types. Some lack of specificity was suggested by 5 positive reactions in the same patients tested with unrelated extracts. No false positives were found in normal subjects. The technique is simple, rapid and appears to depend on tumour-associated antigens in the extracts. Simultaneous leucocyte adherence inhibition tests on split samples of blood had a high degree of sensitivity and specificity, confirming the potential reactivity of the leucocytes and extracts used.

Patients with hairy cell leukemia (HCL) and chronic lymphocytic leukemia (CLL) were treated with recombinant interferon alpha A (rIFN- alpha A). The binding of iodinated recombinant interferon-alpha to baseline samples of peripheral blood mononuclear cells (PBMCs) from the leukemia patients was compared with clinical responsiveness to rIFN- alpha A. HCL patients (8/10) responded to rIFN-alpha A therapy, whereas none (0/10) of the CLL patients studied responded. The PBMCs from the eight responsive HCL patients bound approximately twice as much iodinated interferon as the PBMCs from nonresponsive CLL patients. This difference was due to more high-affinity receptors per cell with no difference in the affinity of the interferon-receptor interaction. However, because PBMCs from HCL patients were larger than PBMCs from CLL patients, the cell surface receptor density was similar. The leukemic cells from one of the two nonresponsive HCL patients bound iodinated interferon similarly to the cells from the responsive HCL patients, whereas the leukemic cells from the other nonresponsive HCL patient bound considerably less. The rapidity of response of the HCL patients did not correlate with the level of binding of iodinated interferon. Our results suggest that the absolute number of interferon receptors per cell may be only one of several important parameters in the response to rIFN-alpha A therapy, and that the responsiveness of a particular lymphoproliferative disease or a particular patient to rIFN- alpha A therapy cannot be predicted or explained solely by the degree of interaction between IFN and its cell surface receptor.

Lymphokine-activated killer (LAK) cells and interleukin-2 (IL-2) were administered by the ip route to patients with intra-abdominal malignancies. Pharmacokinetic studies of IL-2 revealed 10- to 100-fold higher concentrations of IL-2 in peritoneal fluid versus serum. Ip levels of IL-2 were maintained well above those required to generate and maintain LAK cells in vitro. LAK cell activity was detectable in the peritoneal fluid for the duration of each treatment cycle and did not disappear until IL-2 was discontinued. Detection of interferon-gamma (IFN-gamma) in the peritoneal fluid of all patients was consistent with production in situ by activated lymphocytes. In some patients, low but detectable levels of IFN-gamma were also found in the serum. In vivo activation of monocytes in the peritoneal fluid as measured by in vitro production of hydrogen peroxide was documented in the majority of patients. Neither interleukin-1 nor tumor necrosis factor-alpha was detected in the peritoneal fluid. We found no correlation between the presence or levels of IL-2, IFN-gamma, or LAK cell lytic activity in peritoneal fluid or serum and response or nonresponse to therapy.

Eighty-one patients with a variety of refractory disseminated malignant neoplasms have been treated in the first multiple fixed-dose phase I trial of recombinant leukocyte A interferon (IFL-rA). Each patient received IFL-rA by intramuscular injection, three times weekly for 28 days. Dosages were escalated in different patients from 1 to 136×106units per injection. The toxic reactions seen with IFL-rA resembled those of nonrecombinant leukocyte interferon and included fever, chills, fatigue, anorexia, myalgia, headache, occasional nausea and vomiting, and dose-dependent reversible leukopenia and hepatic transaminase elevations. The pharmacokinetics of IFL-rA were also comparable with nonrecombinant leukocyte interferon. Objective evidence of antitumor activity was seen in non-Hodgkin's lymphoma, chronic lymphocytic leukemia, Hodgkin's disease, breast cancer, and melanoma, indicating that IFL-rA, the first genetically engineered biological response modifier available for testing in cancer patients, is biologically active in vivo. (JAMA1982;248:2461-2466)

The susceptibility of human papillomavirus infection to polyclonal human leukocyte interferon (IFN-alpha) has been evaluated in patients with epidermodysplasia verruciformis (EV), a disease with extensive chronic papillomavirus-induced warts. In a double-blind, placebo-controlled study with intralesional IFN-alpha, four of five IFN-alpha-treated warts regressed; none of the placebo-treated warts responded (p = 0.024). Three patients with EV were treated with systemic IFN-alpha for 4 weeks in an open study, achieving partial regression of warts in all three. In a double-blind, placebo-controlled study, warts in two children with EV regressed with systemic IFN-alpha while two who received placebo showed no improvement. The lesions recurred following cessation of therapy. At the completion of therapy with IFN-alpha, histologic normalization was accompanied by a 95% decrease in the number of viral antigen-containing cells in the warts (p less than 0.001). We conclude that warts in EV respond to systemic and intralesional IFN-alpha.

PATIENTS RECEIVING freeze-dried skin (FDS) allografts were evaluated for cell-mediated response by a lymphocytotoxicity test. Eleven patients received single or multiple FDS allografts from a donor typed for human leukocyte antigen (HLA). Heparinized blood samples were drawn before the procedure and at 2, 4, and 8 weeks after grafting. Mononuclear cells were isolated by Ficoll-Hypaque gradients. Cell-mediated lymphocytotoxicity tests were conducted, with mononuclear cells from the FDS allograft recipients used as effector cells. Phytohemagglutinin-stimulated blast target cells of the same HLA type as that of the skin donor were labeled with chromium 51. Lympholysis was evaluated by measuring the amount of 51Cr release after 18 hours' incubation with target/effector cell ratios of 1:100, 1:50, and 1:25. Negative control wells contained target cells alone. Positive control wells contained target cells and effector cells sensitized in vitro against the target cells. Viability of effector cells was tested by trypan-blud dye exclusion and response to phytohemagglutinin. None of the experimental blood samples showed 51Cr release significantly greater than shown by the negative controls. Our findings of no cell-mediated response and the findings of a previous study showing no production of anti-HLA antibody in response to FDS allografts indicate that allogeneic FDS is an immunologically safe material for use in periodontal surgical procedures.

134 patients with a variety of malignancies were treated in two Phase 1 clinical trials of recombinant leukocyte A interferon (IFN) produced by recombinant DNA methodology by Hoffmann-La Roche Inc. in collaboration with Genentech Inc. IFN was given either twice daily or three times weekly for 28 days. Extensive monitoring of immune function was done on these patients, in an attempt to define the range of doses of interferon and timing of administration that would optimally alter immune functions which might have beneficial anti-tumor effects. These two protocols failed to result in an appreciable increase in NK activity but rather in the majority of patients there was no significant change in NK activity from the pretreatment baseline levels. A particularly unexpected finding was the depresssion in NK activity seen in 30% of the patients. In contrast, the IFN preparation did induce significant augmentation of monocyte function, as measured in a monocyte-mediated cytostatic assay, with increases observed in 80% of the patients. There is some evidence for the presence of a suppressor factor in the sera of these patients which interfered with in vitro boosting of NK activity by IFN and therefore might have been responsible for the paradoxical NK results. These data have also been analyzed in terms of their possible relationship to doses, and protocol of IFN administration, and to tumor response.

A total of 11 patients were treated on an escalating, single dose trial of recombinant gamma interferon (rIFN-gamma), 6 patients by the i.m. and 5 patients by the i.v. route of administration. Dose ranges within each individual were from 0.05 mg/m2 of IFN (1 mg greater than or equal to 10 X 10(6) units of IFN) escalating to 10 mg/m2. All dosages were delivered twice weekly and the i.v. dose was infused over 5 min. The most common toxicities encountered included fever, chills, fatigue, anorexia, and granulocytopenia. The influenza-like symptoms were very similar to those encountered with IFN-alpha but were generally less severe. The granulocytopenia was dose-related and transient with recovery generally seen within 48-72 h following administration of rIFN-gamma. Absolute granulocyte counts only rarely dropped below 1000 mm3. Hepatotoxicity was not observed. IFN levels were determined by both a bioassay and an enzyme-linked immunosorbent assay. By the i.v. route, the peak level of IFN activity could usually be seen at completion of the infusion with a serum half-life of 30 min. By the i.m. route, the peak level of serum activity was generally detected between 4-8 h with a serum half-life of 4.5 h after the initial elimination phase. Peak IFN levels appeared to correlate with maximum toxicity. One patient with melanoma had a 25% reduction in a cutaneous lesion, but there were no other minimal, partial, or complete responses.

This study was undertaken to determine an immunologically active regimen for the administration of recombinant gamma-interferon (rIFN-gamma). The patient population included patients with completely resected melanoma, stage I (Clark's level IV or V) or stage II. All patients exhibited no evidence of disease (NED) at the time of the study. Patients received rIFN-gamma by intramuscular (IM) injection daily for 15 days at 0.0001 mg/m2, 0.001 mg/m2, 0.01 mg/m2, 0.1 mg/m2 (ten patients/group), or 0.25 mg/m2 (five patients). Interferon (IFN) was well tolerated, with non-dose-limiting constitutional symptoms occurring in the majority of patients at 0.1 mg/m2 and 0.25 mg/m2. All five patients receiving 0.25 mg/m2 developed elevated transaminase levels, which led to a discontinuation of therapy in one patient. Immunological activity was assessed by serial measurements of natural killer (NK) cell activity, hydrogen peroxide production by monocytes, and changes in expression of Fc receptors and human leukocyte class II antigen (HLA-DR) on monocytes. These changes were determined at baseline (X2), six to seven time points during rIFN-gamma therapy, and two times after the last dose of rIFN-gamma. No changes were observed at the two lowest doses. At the 0.01 mg/m2 dose, all parameters were elevated but not as consistently nor to the same levels as seen following administration of 0.1 mg/m2. At 0.25 mg/m2, H2O2 production was enhanced, but unlike at 0.1 mg/m2, it declined during the last few days of IFN therapy. Subcutaneous (SC) administration was compared with IM administration using the 0.1 mg/m2 dose. SC administration resulted in enhanced H2O2 production and Fc receptor expression by monocytes. More consistent elevations in peroxide generation and higher levels of Fc receptor expression were seen following SC administration. No significant difference was found between the two routes of administration. A comparison of two schedules, daily and three times weekly, suggested that monocyte activation may return to normal 72 hours after IFN administration. Of the doses tested, 0.1 mg/m2 administered daily appeared to be the most effective biological response modifier (BRM) regimen, and because of ease of administration, we favor the SC route.

A variety of interferons have now been tested in clinical trials. Most of these trials have utilized leukocyte (α) interferon preparations. Early trials were conducted with partially purified material derived from the supernatants of virus-stimulated leukocytes that were of low purity and inconsistent pharmaceutical quality. More recently, trials have been conducted with a lymphoblastoid cell line interferon (α) of high purity and good reproducibility (Knost et al., 1983). Several recent studies have utilized recombinant α-interferon derived by cloning a gene for α-interferon in an E. coli expression system (Sherwin et al., 1982, 1983). Despite the major differences between these α-interferon preparations, many of the toxicities, immunological modulating effects, and therapeutic effects have been similar. Fever, chills, headache, fatigue, and anorexia have been rather constant side effects of these interferon preparations. At higher doses, mild hematological depression and transient hepatic enzyme abnormalities have been seen. Occasional cardiac effects including arrhythmias and ischemic effects have been observed in the context of these trials. Some central nervous toxicity including confusion, decreased ability to concentrate, and rarely seizures at very high doses have been seen in these studies. It is unclear whether all these effects are due to the direct action of the interferon preparation since the induction of fever, tachycardia, and fatigue may have secondary effects (Oldham, 1983a). The phase I trials for the α-interferons are virtually complete.