melphalan and Thymoma

The heterogeneity of tumour antigen expression, the differential sensitivity of individual cells to drugs and the use drug--antibody immunoconjugates with limited potency can limit the antitumour effects of immunoconjugate therapy. In this study we have used two different antibodies linked to two different drugs Melphalan (Mel) and Idarubicin (Ida), each with a different site action, to evaluate the potential of using cocktails of immunoconjugates. A series of drug combinations were screened for their synergistic activity in vitro using the inhibition of [3H]-thyrmidine uptake by E3 cells, and constructing isobolagrams: Mel plus Ida was the only combination found to be synergistic in vitro and this synergism extended to the drugs after conjugation to antibodies. In addition, in vivo studies in mice bearing E3 tumours showed that synergy between both free drugs and between Ida-anti-Ly-2.1 and N-AcMEL-anti-Ly-3.1 immunoconjugates was time dependent, requiring treatment with Ida or Ida-MoAb conjugates prior to the addition of the second melphalan containing immunoconjugate. The use of two different antibodies, anti-Ly-2.1 and anti-Ly-3.1 against E3 (Ly-2.1+ve, Ly-3.1+ve) gave greater synergy in vitro compared to using only one antibody. Again a cocktail of two antibody immunoconjugates provided significantly greater antitumour efficacy when given to tumour bearing mice, provided that the Ida-anti-Ly-2.1 was given 24 h before injection of N-AcMEL-anti-Ly-3.1. The enhanced antitumour effect was not observed when the immunoconjugates were given simultaneously, or if the same antibody was used in each conjugate. Of importance was the finding that although the anti-tumour effect was synergistic, there was no increase in toxicity noted. The increased therapeutic index observed by using a double cocktail (2 antibodies + 2 drugs) could have major implications for immunoconjugate therapy.

Topics: Animals; Antibodies, Monoclonal; Antineoplastic Agents; Cell Survival; Drug Synergism; Idarubicin; Immunotoxins; Leukocyte Count; Melphalan; Mice; Mice, Inbred Strains; Neoplasm Transplantation; Thymoma; Thymus Neoplasms; Tumor Cells, Cultured

We have previously shown that thymocytes from low-dose melphalan (L-phenylalanine mustard)-treated MOPC-315-tumor-bearing mice (melphalan TuB) are able to generate an enhanced level of anti-MOPC-315 cytotoxicity, as compared to thymocytes from untreated MOPC-315-tumor-bearing mice or thymocytes from untreated or low-dose melphalan-treated normal mice, upon in vitro stimulation with MOPC-315 tumor cells in the presence of a low concentration of recombinant interleukin-2 (rIL-2). Here we show that the generation of enhanced anti-MOPC-315 cytotoxicity by melphalan TuB thymocytes depends on the ability of the thymocytes to proliferate. In addition, the ability of melphalan TuB thymocytes to generate an enhanced level of anti-MOPC-315 cytotoxicity correlated with their ability to proliferate more readily than thymocytes from untreated tumor-bearing mice and thymocytes from untreated or melphalan-treated normal mice in response to stimulation with MOPC-315 tumor cells plus a low concentration of rIL-2. Moreover, although fresh melphalan TuB thymocytes do not contain a higher percentage of phenotypically mature cells (i.e., CD4-/CD8+ or CD4+/CD8-) than do thymocytes from normal mice or untreated tumor-bearing mice, after a 5-day culture with both MOPC-315 tumor cells and a low concentration of rIL-2, cultures of thymocytes from melphalan TuB contained a much higher percentage of CD4-/CD8+ (but not CD4+/CD8-) cells than did cultures of thymocytes from the other two sources. Since CD4-/CD8+ cells were previously shown to be responsible for the exertion of antitumor cytotoxicity by thymocytes stimulated with MOPC-315 in vitro, our results indicate that the enhanced antitumor cytotoxicity exerted by melphalan TuB thymocytes following in vitro stimulation with MOPC-315 tumor cells in the presence of a low concentration of rIL-2 is due, at least in part, to an expansion of the pool of CD4-/CD8+ effector cells.

Topics: Animals; CD4-CD8 Ratio; CD8 Antigens; Cytotoxicity, Immunologic; Female; Interleukin-2; Lymphocyte Activation; Melphalan; Mice; Mice, Inbred BALB C; Plasmacytoma; Recombinant Proteins; T-Lymphocyte Subsets; Thymoma

We have previously shown that while spleen cells from untreated mice bearing a large MOPC-315 tumor are not cytotoxic in vitro for MOPC-315 tumor cells, spleen cells obtained from such mice on day 7 after low-dose melphalan (L-phenylalanine mustard); L-PAM therapy exert a substantial anti-MOPC-315 cytotoxicity [Mokyr et al. (1989) Cancer Res 49: 4597]. Here we show that this anti-MOPC-315 lytic activity is evident by day 5, and peaks on day 7 after the low-dose chemotherapy, at a time when the mice are actively engaged in tumor eradication. Short-term exposure of spleen cells from mice bearing a MOPC-315 tumor and treated with low-dose L-PAM (L-PAM TuB mice) to phorbol 12-myristate 13-acetate (PMA) was found to enhance greatly the ability of these spleen cells to lyse MOPC-315 tumor cells. The highest level of anti-MOPC-315 cytotoxicity was obtained when spleen cells from tumor-bearing mice that had received chemotherapy 7 days earlier were exposed to PMA at a concentration of 1-10 ng/ml. The exertion of the enhanced anti-MOPC-315 lytic activity by L-PAM TuB spleen cells exposed to PMA was found to require CD8+, but not CD4+, T cells. The apparent specificity of the lytic activity exerted by the PMA-stimulated L-PAM TuB spleen cells was illustrated not only by the inability of the spleen cells to lyse an allogeneic, antigenically unrelated thymoma (EL4), but also by their relatively weak lytic activity for two antigenically related syngeneic plasmacytomas. In addition, when EL4 target cells were admixed with MOPC-315 tumor cells, the lytic activity triggered in the L-PAM TuB spleen cells by the MOPC-315 tumor cells plus PMA was not effective in lysing the antigenically unrelated target cells. Moreover, even in the presence of the calcium-specific ionophore, ionomycin, L-PAM TuB spleen cells exposed to PMA were unable to lyse the EL4 target cells. Thus, fresh CD8+ splenic T cells from L-PAM TuB mice that are in the process of eradicating a large MOPC-315 tumor as a consequence of low-dose L-PAM therapy can be triggered with PMA to exert enhanced lytic activity against MOPC-315 tumor cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: Animals; Antigens, Differentiation, T-Lymphocyte; CD8 Antigens; Cell Line; Cytotoxicity, Immunologic; Dose-Response Relationship, Drug; Drug Synergism; Female; In Vitro Techniques; Ionomycin; Lymphocyte Depletion; Melphalan; Mice; Mice, Inbred BALB C; Phagocytes; Plasmacytoma; T-Lymphocytes; Tetradecanoylphorbol Acetate; Thymoma; Time Factors

We have previously demonstrated that depletion of CD8+ T-cells by the use of a monoclonal anti-Lyt-2.2 antibody abolishes the curative effectiveness of low-dose melphalan (L-phenylalanine mustard; L-PAM) therapy for BALB/c mice bearing a large (greater than or equal to 20 mm) s.c. MOPC-315 tumor and extensive metastases (Mokyr et al., Cancer Res., 49: 4597-4606, 1989). Here we show that as a consequence of low-dose L-PAM therapy, CD8+ T-cells accumulate in the s.c. tumor nodules of MOPC-315 tumor bearers. Specifically, an 80-fold increase in the number of CD8+ T-cells was seen within 5 days after the chemotherapy. Treatment of MOPC-315 tumor bearers with low-dose L-PAM in conjunction with monoclonal anti-Thy-1.2 or anti-Lyt-2.2 antibody, in contrast to treatment with monoclonal anti-L3T4 antibody, prevented the appearance of the massive CD8+ T-cell infiltrate in the s.c. tumor nodules. Fresh CD8+ T-cells derived from s.c. MOPC-315 tumor nodules that were regressing as a consequence of low-dose L-PAM therapy exhibited a potent direct lytic activity against the MOPC-315 plasmacytoma in a short-term in vitro assay. The specificity of the lytic activity exhibited by the CD8+ T-cells was illustrated not only by the inability of the CD8+ T-cells to lyse two antigenically unrelated thymomas (the WEHI 22.1 and the EL-4) and a natural killer-sensitive lymphoma (the YAC-1), but also by their relatively weak lytic activity against an antigenically related plasmacytoma (the MOPC-104E). Thus, CD8+ T-cells that infiltrate the s.c. tumor nodules of MOPC-315 tumor bearers following low-dose L-PAM therapy most likely exploit a CTL-type lytic mechanism to eradicate at least part of the large tumor burden not eliminated by the direct antitumor effects of the drug.

Topics: Animals; Antibodies, Monoclonal; Antigens, Differentiation, T-Lymphocyte; CD8 Antigens; Female; Immunohistochemistry; In Vitro Techniques; Lymphoma; Melphalan; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Plasmacytoma; T-Lymphocytes, Cytotoxic; Thymoma

Twenty-six patients (median age 33 years) with poor-risk malignancies were treated with high-dose combination chemotherapy associating BCNU-etoposide-cytosine arabinoside and melphalan (BEAM) followed by autologous bone marrow transplantation (ABMT). Twenty-one patients had malignant lymphomas, three, acute lymphoblastic leukemia (ALL), and two, malignant thymomas. Eleven patients (group 1) were not in complete remission (CR) at the time of BEAM, and fifteen patients (group 2) were in CR. Hematological recovery occurred in all patients. The duration of aplasia and the non-hematological toxicities were similar in both groups. Ten of the eleven patients (group 1) evaluable for response achieved CR and one achieved partial remission (PR). Five patients relapsed, and five are in continuous CR with a short follow-up (median 8 months). Among the fifteen patients in CR at the time of BEAM (group 2), four patients relapsed and ten patients are in unmaintained continuous CR with a median follow-up of 15 months (one patient died in CR). The disease-free survival is 53%, with 29% for patients receiving BEAM while in relapse (group 1) and 65% for patients receiving BEAM while in CR (group 2). These data indicate that BEAM followed by ABMT can produce a high antitumor response with an acceptable toxicity in patients with poor-risk malignancies.

Topics: Adolescent; Adult; Antineoplastic Combined Chemotherapy Protocols; Bone Marrow Transplantation; Carmustine; Child; Child, Preschool; Combined Modality Therapy; Cytarabine; Etoposide; Female; Hematologic Diseases; Humans; Leukemia, Lymphoid; Lymphoma; Male; Melphalan; Middle Aged; Recurrence; Remission Induction; Thymoma; Thymus Neoplasms

BALB/c mice cured of a large MOPC-315 or MOPC-104E plasmacytoma following treatment with a low dose (2.5 mg/kg) of melphalan (L-PAM) were resistant to challenge with the other plasmacytoma but to a much lesser extent than to challenge with the autochthonous plasmacytoma. The resistance of the L-PAM-cured MOPC-315-tumor bearers to challenge with MOPC-104E tumor cells was increased when the MOPC-104E tumor cells were admixed with MOPC-315 tumor cells prior to their inoculation. This enhanced resistance to MOPC-104E cells was due to elimination of the MOPC-104E tumor cells through an innocent bystander killing effect since it did not render the mice more resistant to a subsequent challenge with MOPC-104E tumor cells alone. Administration of carrageenan to L-PAM-cured MOPC-315-tumor bearers 1 day after the challenge with the mixture of MOPC-104E and MOPC-315 tumor cells drastically reduced the ability of the mice to resist the tumor challenge. All of the tumors that developed in such mice were of MOPC-104E origin only (as judged by the binding specificity of the myeloma proteins secreted by the tumor cells as well as that present on their surface) even though (a) the tumor inoculum used consisted of up to 10-fold more MOPC-315 than MOPC-104E tumor cells and (b) the MOPC-315 tumor cells divide more rapidly. The same protocol of carrageenan treatment did not reduce the ability of normal BALB/c mice to develop in vivo a primary cell-mediated cytotoxic response nor a primary antibody response indicating that it has no effect on the initiation of an immune response. Therefore, it is conceivable that carrageenan treatment reduced the ability of L-PAM-cured MOPC-315-tumor bearers to reject a challenge with MOPC-315 and MOPC-104E tumor cells by interfering at the effector stage. The ability of the L-PAM-cured MOPC-315-tumor bearers to reject the MOPC-315 cells present in the challenge mixture was reduced when the mice were treated with anti-Thy 1.2 antibody but not with carrageenan, indicating that T-cells independent from carrageenan-sensitive effector cells are required for the rejection of the MOPC-315 tumor cells. Thus, at least two different effector mechanisms participate in the rejection of a challenge composed of MOPC-315 and MOPC-104E tumor cells by L-PAM-cured MOPC-315-tumor bearers.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: Animals; Carrageenan; Depression, Chemical; Female; Graft Rejection; Immunity, Cellular; Immunity, Innate; Melphalan; Mice; Mice, Inbred BALB C; Neoplasm Transplantation; Plasmacytoma; Thymoma; Thymus Neoplasms

To increase the accessibility of drug-antibody complexes to tumours and to decrease non-specific binding via Fc receptors N-acetyl-melphalan (N-AcMEL) was conjugated to F(ab')2 fragments. These fragments were synthesised by pepsin degradation of IgG MoAb. Up to 20 molecules of N-AcMEL could be successfully coupled to each F(ab')2 fragment (compared with 25 molecules/intact IgG) with retention of both drug and antibody activity. The N-AcMEL-F(ab')2 conjugates demonstrated specific cytotoxicity in vitro however despite the absence of non specific Fc receptor binding and greater permeability when using F(ab')2 fragments, the N-AcMEL-F(ab')2 and N-AcMEL-IgG conjugates had similar anti-tumour activity in vivo. Conjugates made with whole IgG and F(ab')2 were equally effective in eradicating subcutaneous solid tumours in mice when injected intravenously. The lower immunogenicity of F(ab')2 fragments compared with whole IgG and the similar cytotoxicity of their conjugates, suggests that the F(ab')2 conjugate has greater clinical utility.

Topics: Animals; Antibodies, Monoclonal; Antigens, Ly; Antineoplastic Agents; Immunoglobulin Fab Fragments; Lymphoma; Melphalan; Mice; Mice, Inbred Strains; Neoplasms, Experimental; Thymoma; Thymus Neoplasms

We have shown previously that low-dose melphalan (L-PAM) therapy of mice bearing a large MOPC-315 plasmacytoma enables their hitherto immunosuppressed spleen cells to exert potent anti-MOPC-315 cytotoxicity following in vitro immunization with MOPC-315 tumor cells. Here we show that, following in vitro immunization with MOPC-315 tumor cells, spleen cells from such L-PAM-treated MOPC-315 tumor bearers exhibited enhanced T-cell-mediated cytotoxicity not only against the MOPC-315 tumor, but also against another plasmacytoma (MOPC-104E) possessing surface immunoglobulin (SIg) of a different idiotype than the MOPC-315 cells, as well as against a variant of the MOPC-315 tumor which does not produce nor possess SIg (SIg- MOPC-315). The enhanced cytotoxicity was directed against target antigens which are not expressed on the surface of the syngeneic WEHI 22.1 thymoma or the natural killer-sensitive YAC-1 cells. Plasmacytoma shared antigens, other than immunoglobulins, were able to stimulate spleen cells from L-PAM-cured MOPC-315 tumor bearers to generate in vitro a secondary type anti-plasmacytoma cytotoxic response. L-PAM-cured MOPC-315 tumor bearers exhibited in vivo immunity against SIg- MOPC-315 tumor cells, which was sufficiently triggered by the SIg- cells to bring about the rejection of a challenge of at least 100-fold the minimal lethal tumor dose of the SIg- MOPC-315 cells. Thus, SIg- MOPC-315 tumor cells present among SIg+ tumor cells in the parental MOPC-315 tumor inoculum can be eradicated in the L-PAM-treated MOPC-315 tumor bearers by the immune response to SIg+ tumor cells as well as by the immune response to SIg- tumor cells themselves.

Topics: Animals; Antibody Specificity; Antigens, Neoplasm; Cell Line; Cytotoxicity, Immunologic; Female; Immunocompetence; Lymphocyte Depletion; Macrophages; Melphalan; Mice; Mice, Inbred BALB C; Plasmacytoma; Spleen; T-Lymphocytes; Thymoma

Topics: Female; Lymphoma, Large B-Cell, Diffuse; Lymphoma, Non-Hodgkin; Male; Melphalan; Multiple Myeloma; Neoplasms; Osteosarcoma; Ovarian Neoplasms; Research; Sarcoma; Sarcoma, Ewing; Testicular Neoplasms; Thymoma