Inhibition of murine Lewis lung carcinoma metastases by combined chemotherapy and intranasal THF-gamma 2 immunotherapy.

Previous research in our laboratories has shown that the immunoregulatory octapeptide, THF-gamma 2, potentiates the efficacy of anticancer chemotherapy in experimental animal models of local plasmacytoma and repairs drug-induced defects in immunocompetence. The highly metastatic, murine D122 lung carcinoma model has been shown to be useful for evaluating the efficacy of experimental antimetastatic therapeutic modalities. The goal of the present study was to determine whether intranasal thymic humoral factor-gamma 2 (THF-gamma 2) immunotherapy, after a single dose of chemotherapy, could inhibit the development of lung metastases, restore immunocompetence, and increase survival in syngeneic C57BL/6 mice bearing highly metastatic Lewis lung carcinoma (D122) solid footpad tumors. Relative to untreated mice and those receiving chemotherapy alone, mice receiving combined chemoimmunotherapy showed the following significant differences: (a) decreased lung metastatic load as assessed by lung weight, (b) prolonged survival time, (c) massive infiltration of lymphoid cells in the lungs, and (d) restoration of impaired immune parameters to normal values in melphalan-treated mice. THF-gamma 2 prevented tumor emboli from colonizing the target tissue, probably by inducing expansion of the lymphoid cell compartment. When used as an adjunct to anticancer chemotherapy, intranasal THF-gamma 2 immunotherapy is a simple and safe treatment modality that seems to be promising for inhibiting lung metastases.

Adjuvants--a balance between toxicity and adjuvanticity.

Adjuvants have been used to augment the immune response in experimental immunology as well as in practical vaccination for more than 60 years. The chemical nature of adjuvants, their mode of action and the profile of their side effects are highly variable. Some of the side effects can be ascribed to an unintentional stimulation of different mechanisms of the immune system whereas others may reflect general adverse pharmacological reactions. The most common adjuvants for human use today are still aluminium hydroxide, aluminium phosphate and calcium phosphate although oil emulsions, products from bacteria and their synthetic derivatives as well as liposomes have also been tested or used in humans. In recent years monophosphoryl lipid A, ISCOMs with Quil-A and Syntex adjuvant formulation (SAF) containing the threonyl derivative of muramyl dipeptide have been under consideration for use as adjuvants in humans. At present the choice of adjuvants for human vaccination reflects a compromise between a requirement for adjuvanticity and an acceptable low level of side effects.

Therapeutic effectiveness against MOPC-315 plasmacytoma of low or high doses of the synthetic thymic hormone THF-gamma 2 in combination with an "immunomodulating" or a "non-immunomodulating" drug.

We reported previously that treatment of mice bearing MOPC-315 plasmacytoma with the drugs L-PAM (phenylalanine mustard) or 5-FU (5-fluorouracil), in combination with low doses of THF-gamma 2, was more effective in increasing their survival time than treatment with the drug alone. We show here that in the combined treatment using a single injection of 5-FU followed by multiple (8-15) injections of THF-gamma 2, the megadoses were more effective than the low doses in increasing the survival time of MOPC-315 tumor-bearing mice. On the other hand, in combination with L-PAM, both low and high doses of THF-gamma 2 were equally effective. The need for high doses of THF-gamma 2, when used in combination with 5-FU, could be due to the fact that 5-FU acts as a "non-immunomodulating" drug and has to be used at a high, immunosuppressive dose.

A synthetic thymic hormone, THF-gamma 2, repairs immunodeficiency of mice cured of plasmacytoma by melphalan.

BALB/c mice cured of large MOPC-315 plasmacytomas by melphalan remain deficient in their spleen T-cell functions. This was manifested by impairment of the allogeneic and the antibody responses in vitro to SRBC and in decreased numbers of T-cells including their subsets CD4 and CD8. IL-2 production and specific cytotoxicity against MOPC-315 tumor cells were, on the other hand, maintained. Treatment of these cured mice by in-vivo administration of THF-gamma 2, an octapeptide from calf thymus, repaired these deficits. This was evidenced by in vitro tests with spleen cells which manifested an increased allogeneic response and elevated generation of primary antibody response, restoration of T-cell subpopulations to normal and an enhanced IL-2 production above normal levels. The potential use of THF-gamma 2 as supportive therapy in cancer treatment is suggested.

Effectiveness of low-dose melphalan treatment against a nonimmunogenic primary induced mouse plasmacytoma.

The fourth i.p. passage of the plasmacytoma "PR" induced by repeated i.p. injections was used for testing chemotherapy with melphalan. The development of "PR" ascitic tumours was slower and the survival time of inoculated mice was longer than that of MOPC-315 inoculated mice. Moreover, the myeloma protein secreted by the "PR" tumour cells, differed from MOPC-315 secreted myeloma protein in its electrophoretic mobility (fast gamma-2) and its characteristics as IgG immunoglobulin. Chemotherapy by a single injection of melphalan 7.5 mg/kg was effective in preventing the development of both MOPC-315 ascitic tumours and "PR" ascitic tumours. Mice cured from MOPC-315 tumours, however, developed antitumour response as shown by resistance to challenge with a high tumourigenic dose and by development of cytotoxic response in vitro against MOPC-315 tumour cells by spleen cells taken from the cured mice. On the other hand, mice cured from "PR" tumour by melphalan were highly susceptible to challenge and their spleen cells were not able to develop a cytotoxic response in vitro against target "PR" tumour cells.

Classification of anticancer chemotherapeutic drugs according to their immunopotentiating activity.

Both melphalan (L-PAM: L phenylalanine mustard) and 5-fluorouracil (5-FU) expressed a cytotoxic effect in vitro on MOPC-315 plasmacytoma tumour cells. However, they differed in their chemotherapeutic effectiveness in BALB/c mice bearing large MOPC-315 plasmacytoma tumours. Therapy with L-PAM, 7.5 mg/kg induced permanent regression of tumours, whereas regression induced by 5-FU, 200 mg/kg, was only transient and the mice dies finally with tumours. Moreover, spleen cells of tumour bearing mice treated with L-PAM exhibited high specific cytotoxic potential in vitro whereas spleen cells from tumour bearing 5-FU treated mice were devoid of cytotoxic potential. Effectiveness of chemotherapy with L-PAM was antagonized by treatment in combination with 5-FU. L-PAM, but not 5-FU potentiated cell mediated contact sensitivity response in vivo and impaired induction of T-suppressor cells by ConA. The parameters mentioned above indicate that L-PAM behaves as an "immunopromoting" drug and 5-FU as a "nonimmunopromoting" drug.

The difference between 5-fluorouracil and melphalan in their ability to promote antitumor immune response against MOPC-315 plasmacytoma.

The anticancer chemotherapeutic drugs melphalan (L-phenylalanine mustard; L-PAM), 5-fluorouracil (5-FU), methotrexate (MTX), and daunorubicin (DAU) were tested for their toxic activity against MOPC-315 tumor cells in vitro. L-PAM, 5-FU, and DAU had a marked toxic effect whereas MTX did not affect the rate of thymidine incorporation in the tumor cells. L-PAM (7.5 mg/kg) induced permanent regression of large s.c. MOPC-315 plasmacytoma tumors, 5-FU (200-250 mg/kg) induced transient regression of MOPC-315 tumors with reappearance starting on the 6th day after the 5-FU injection and DAU (5 mg/kg) was not effective. L-PAM treatment restored the cytotoxic potential of spleen cells of tumor-bearing mice against target MOPC-315 tumor cells whereas spleen cells from tumor-bearing mice treated with 5-FU were unable to mount a cytotoxic response. L-PAM and 5-FU were also assayed for their effect in vitro on induction of suppressor T cells by ConA. L-PAM treatment in vitro markedly reduced the induction of suppressor T cells by ConA whereas 5-FU had no effect. It is suggested that anticancer chemotherapeutic drugs can be classified in "immunopromoting" (L-PAM as prototype) and "nonimmunopromoting" (5-FU as prototype) on the basis of their effect in vivo on established tumors and their effect on induction of suppressor T cells by ConA.