Role of the H-2 complex in the induction of T cell tolerance to self minor histocompatibility antigens.

The present study has utilized cytotoxic T lymphocyte (CTL) responses specific for minor histocompatibility (minor H) antigens as an experimental approach to determining whether recognition of self MHC determinants is involved in the induction of T cell tolerance to self antigens. It was observed that C3H.SW splenic T cells from C3H.SW leads to B10 X B10.BR radiation bone marrow chimeras contained CTL precursors (pCTL) reactive against self C3H minor H antigens + H-2k but were tolerant to self C3H minor H antigens + H-2b. Precursor CTL with the reciprocal reactivity pattern were observed for C3H leads to B10 X B10.BR chimeras. In addition, it was observed that C3H.SW thymocytes from C3H.SW leads to B10 X B10.BR chimeras could generate minor H-specific CTL responses and were reactive against self C3H minor H antigens + H-2k, but were tolerant to self C3H minor H antigens + H-2b. Thus, the present study demonstrates that for peripheral and intrathymic T cell populations at least a component of T cell tolerance to self antigens is restricted by products of the MHC.

Restoration of interferon alpha potentiation of a recombinant ricin A chain immunotoxin following cytoreduction of xenografts of advanced ovarian tumors.

BACKGROUND: We have demonstrated that, in the human ovarian carcinoma cell line (OVCAR-3), recombinant human interferon alpha (rHuIFN-alpha) potentiated in vitro inhibition of protein synthesis by immunotoxins. The antitumor activity of intracavitary immunotoxin administered to nude mice 5 days after tumor cell injection was enhanced by a nontherapeutic dose of rHuIFN-alpha, as evidenced by increased survival time. PURPOSE: Our purpose was to determine the outcome of treatment with immunotoxin and rHuIFN-alpha in xenografts of more advanced tumors. METHODS: At 10 or 15 days after tumor cell injection, nude mice with peritoneal OVCAR-3 xenografts were treated intraperitoneally with immunotoxin or with 454A12 monoclonal antibody (MAb) recombinant ricin A chain (rRA), alone or combined with a nontherapeutic dose of rHuIFN-alpha. The immunotoxin was composed of rRA covalently bound to an anti-CD71 (transferrin receptor) MAb. In other experiments, mice were treated intraperitoneally with cyclophosphamide and cisplatin to reduce tumor size on days 20 and 27 after tumor cell inoculation and then, beginning on day 40, with immunotoxin alone or combined with rHuIFN-alpha. RESULTS: Initiation of treatment 10 days after OVCAR-3 transplantation significantly increased median survival from 41 to 89 days (10% survivors on day 120) with 454A12 MAb rRA alone and to more than 120 days (70% survivors) with 454A12 MAb rRA combined with rHuIFN-alpha (P < .0001). The increase in survival time between tumor-bearing mice treated with immunotoxin combined with rHuIFN-alpha and those treated with immunotoxin alone was statistically significant (P = .017). In contrast, the 15-day transplant tumors were not curable with immunotoxin therapy (survival, 72 days; 0% survivors) and were refractory to rHuIFN-alpha potentiation (survival, 75 days; 0% survivors). After the second course of chemotherapy to reduce the size of the advanced tumors (day 40), during the ascites cell count nadir, initiation of treatment with 454A12 MAb rRA alone or combined with rHuIFN-alpha resulted in significantly different survival times of 129 and 162 days, respectively (P = .0037). Pathologic examination of surviving mice treated with chemotherapy and 454A12 MAb rRA alone or in combination with rHuIFN-alpha revealed that one (17%) of six mice and 11 (65%) of 17 were tumor free, respectively. CONCLUSIONS: The synergy between immunotoxins and IFN-alpha is dependent on tumor burden. These agents are less effective against large tumor burdens (i.e., advanced stage disease), but their beneficial effects re-emerge after cytoreduction by combination chemotherapy. IMPLICATIONS: The ideal setting for testing the efficacy of intracavitary immunotoxin combined with rHuIFN-alpha after front-line chemotherapy is in patients with residual tumor refractory to additional chemotherapy or in those with toxic effects that prevent delivery of effective doses.

Phase I study of an anti-breast cancer immunotoxin by continuous infusion: report of a targeted toxic effect not predicted by animal studies.

260F9 Monoclonal antibody-recombinant ricin A chain, an immunotoxin reactive with approximately 50% of breast carcinomas, was given by continuous iv infusion at a dose of 50 micrograms/kg per day or 100 micrograms/kg per day. Five patients with refractory breast cancer received treatment for from 6 to 8 days. Severe toxic effects, including marked fluid overload and debilitating sensorimotor neuropathies, occurred in most patients. Immunoperoxidase studies suggested that 260F9 monoclonal antibody targeting of the Schwann cells may have induced demyelination and subsequent neuropathy. This is the first report of a targeted toxic effect due to an immunoconjugate.

Phase I evaluation of an anti-breast carcinoma monoclonal antibody 260F9-recombinant ricin A chain immunoconjugate.

Four women with metastatic breast cancer were treated with monoclonal antibody 260F9-recombinant ricin A chain, a ricin A chain immunoconjugate (IC) which targets a Mr 55,000 antigen expressed by human mammary carcinomas. Patients were treated by daily, 1-h i.v. injections for 6 to 8 consecutive days. Two patients were treated with 10 micrograms/kg daily and the two others were treated with 50 micrograms/kg daily. The trial was suspended after four patients had been treated because patients treated with a continuous infusion schedule with this IC had developed significant neurological toxicity at doses similar to those used in this study. The half-life of the IC showed a t 1/2 alpha of approximately 1.8 h, a t 1/2 beta of approximately 8.3 h, and a peak concentration of about 200 ng/ml, at the lower dose level, and showed a t 1/2 alpha of approximately 2.5 h, t 1/2 beta of about 10.4 h, and a peak concentration of 500 and 850 ng/ml for the two patients at the higher dose level. All four patients developed evidence of a human anticonjugate antibody response within 16 days of the onset of therapy. The treatment was associated with significant toxicity, manifested by a syndrome consisting of weight gain, edema, hypoalbuminemia, and dyspnea. Similar symptoms were observed in patients treated by continuous infusions of the IC. This clinical syndrome, seen at doses of IC which were insufficient to saturate antigen-expressing malignant tumor deposits in this trial, has been seen in other IC therapy trials and in clinical trials using the cytokine interleukin 2. To investigate a possible mechanism responsible for this toxicity, human monocytes were incubated with varying concentrations of IC. There was detectable binding of IC to human monocytes at IC concentrations which were achieved clinically in this trial. Furthermore, the binding appeared to be abrogated by preincubation of the monocytes with pooled human immunoglobulin, thus suggesting that binding occurs via Fc gamma receptor-mediated mechanisms. Binding was not affected if different linkers between recombinant ricin A chain and the antibody were used or if a different antibody moiety was used in the IC preparation. Chemically linked dimers of MOPC-21 bound to human monocytes at least as well as the ICs; this binding was not abrogated by preincubation with pooled human immunoglobulin. Since the IC preparations used in this clinical trial contained small percentages of dimers and/or multimers, the clinical toxicity syndromes which we observed may be related to this series of observations.(ABSTRACT TRUNCATED AT 400 WORDS)

Intravenous administration of recombinant human macrophage colony-stimulating factor to patients with metastatic cancer: a phase I study.

PURPOSE: Recombinant human macrophage colony-stimulating factor (M-CSF) has been shown to stimulate specifically macrophage lineage differentiation in vitro and to induce cells capable of antitumor activity alone or in combination with an antibody. The administration of M-CSF to mice has demonstrated antitumor therapeutic effects in vivo. Therefore, a phase I trial of M-CSF administration to patients with metastatic cancer was undertaken. PATIENTS AND METHODS: M-CSF was given by intermittent intravenous bolus infusion every 8 hours for 7 days; the treatment cycle was repeated once after a week of rest. Cohorts of three patients underwent dose escalation from 10 to 100,000 micrograms/m2/d; 23 patients received 27 courses of M-CSF administration. All patients had metastatic solid tumors refractory to conventional therapy, including renal cell carcinoma (RCC) (nine), melanoma (seven), and colorectal carcinoma (seven). RESULTS: Treatment-related toxicity was minimal; five patients developed transient signs of ocular or periorbital inflammation, with iridocyclitis as the most severe manifestation. At the highest doses, platelet counts decreased with therapy (but remained > 100,000/mm3) and the absolute monocyte count increased during the course of therapy. Only at 30,000 and 100,000 micrograms/m2/d was treatment limited because of toxicity (iritis and malaise). Pharmacokinetic studies demonstrated up to a 1,000-fold increase in circulating serum M-CSF after bolus infusion; half-life varied from 1 to 6 hours. Complete regression of mediastinal adenopathy and multiple pulmonary metastases were observed in one patient with RCC. CONCLUSION: Recombinant M-CSF can be administered safely to patients with metastatic cancer at doses that demonstrate biologic activity.

A phase I trial of recombinant human macrophage colony-stimulating factor by rapid intravenous infusion in patients with refractory malignancy.

Twenty patients with advanced cancer for which there was no effective standard therapy or whose disease was refractory to standard therapy were treated with recombinant macrophage colony-stimulating factor (rM-CSF). The rM-CSF was administered by intravenous bolus infusion for 5 consecutive days every other week for 2 treatment weeks. The doses administered ranged from 30 to 33,000 micrograms/m2/day. There was no intrapatient dose escalation. There were minimal to no systemic side effects seen, except for acute dyspnea noted in three patients. The dyspnea was felt to be related to the rate of infusion and did not recur in one patient given additional rM-CSF at a slower infusion rate. The major hematologic effect seen was a mild decrease in platelet count, which began to recover while the patients continued to receive the rM-CSF. The clearance of rM-CSF was dose dependent. Lower doses resulted in a saturable mechanism felt to represent cellular uptake. Clearance at higher doses demonstrated both a first-order mechanism at high serum rM-CSF concentrations, representing renal clearance, as well as a saturable mechanism at low serum concentrations. The maximum mean serum half-life was reached at dose levels of greater than or equal to 3,690 micrograms/m2 and was in the range of 234-258 min. By this route of administration, rises in absolute monocyte count were slight and seen only at doses of greater than or equal to 450 micrograms/m2 during the second therapy week. The maximum tolerated dose was not reached in this study because of lack of availability of rM-CSF.

Intraventricular immunotoxin therapy for leptomeningeal neoplasia.

OBJECTIVE: The goals of this clinical trial of intraventricular 454A12-rRA therapy were to identify dose-limiting toxicities, to evaluate the pharmacokinetics of single-dose intraventricular 454A12-rRA, and to detect antitumor activity. METHODS: We performed a pilot study of intraventricular therapy with the immunotoxin 454A12-rRA in eight patients with leptomeningeal spread of systemic neoplasia. The immunotoxin 454A12-rRA is a conjugate of a monoclonal antibody against the human transferrin receptor and recombinant ricin A chain, the enzymatically active subunit of the protein toxin ricin. Patients were treated with single doses of 454A12-rRA ranging from 1.2 to 1200 micrograms. RESULTS: The early phase half-life of 454A12-rRA in ventricular cerebrospinal fluid (CSF) averaged 44 +/- 21 minutes, and the late phase half-life averaged 237 +/- 86 minutes. The clearance of the immunotoxin was faster than the clearance of coinjected technetium-99m-diethylenetriamine penta-acetic acid, averaging approximately 2.4-fold greater. No 454A12-rRA degradation was detected by Western blot analysis of ventricular CSF for a period of 24 hours, and bioactivity was retained in CSF paralleling the concentration of immunotoxin. No acute or chronic drug toxicity was identified in patients who received less than or equal to 38 micrograms of 454A12-rRA by intraventricular injection. Doses more than or equal to 120 micrograms caused a CSF inflammatory response that was associated with transient headache, vomiting, and altered mental status. This acute syndrome was responsive to steroids and CSF drainage. No systemic toxicity was detected. In four of the eight patients, a greater than 50% reduction of tumor cell counts in the lumbar CSF occurred within 5 to 7 days after the intraventricular dose of 454A12-rRA; however, no patient had their CSF cleared of tumor, and clinical or magnetic resonance imaging evidence of tumor progression was demonstrated in seven of the eight patients after treatment. CONCLUSION: Tumoricidal concentrations of the immunotoxin 454A12-rRA can be attained safely in the CSF of patients with leptomeningeal tumor spread.

Expression of MEL-14 antigen is not an absolute requirement for dissemination to lymph nodes after adoptive transfer of murine T lymphocyte clones.

The inability of established antigen-specific murine T lymphocyte clones to recirculate well in vivo has been attributed to loss of the surface glycoprotein gp90MEL-14, which is important for specific adherence to post-capillary high endothelial venules in peripheral lymph nodes (LN). Defective recirculation of clones may contribute to inefficient adoptive immunotherapy when compared with fresh immune spleen or LN populations. To optimize models of adoptive immunotherapy, we sought to improve recirculation of Thy-1.2+, L3T4+ clones by inducing reexpression of MEL-14 antigen (gp90MEL-14). Clones were analyzed after treatment with differentiating agents, incubation in the presence or absence of recombinant interleukin 2 (rIL 2), coincubation in vitro with nonirradiated Thy-1.1 LN or thymus cells, or adoptive transfer into Thy-1.1 hosts. We were unable to demonstrate induction of gp90MEL-14 in any case. However, although clones remained MEL-14 negative, they were able to disseminate widely after subcutaneous adoptive transfer in the presence of clone-specific antigen and rIL 2 into Thy-1.1 mice pretreated with cyclophosphamide. Withdrawal of exogenous rIL 2 was associated with rapid disappearance of clones from all sites. We conclude that murine T cell clones undergo a step in terminal differentiation that precludes surface expression of gp90MEL-14 and that these clones would be unlikely to provide a source of long-lived recirculating memory T lymphocytes. However, under appropriate circumstances it is possible for antigen-specific clones to disseminate widely among host LN and mediate short-term immune responses.

Phase I study of antilipopolysaccharide human monoclonal antibody MAB-T88.

Monoclonal antibody MAB-T88 is a human monoclonal immunoglobulin M antibody directed at the lipopolysaccharide of gram-negative bacteria. In this study, nine patients who were expected to become neutropenic from antineoplastic chemotherapy received an infusion of MAB-T88, three patients at each of three doses: 1, 4, and 8 mg/kg of body weight. MAB-T88 was shown to be safe, with an effective half-life in plasma of 25.4 h, and no patient developed immunoglobulin G antibody to MAB-T88.

Suppression and transient induction of lymphokines in cancer patients after administration of polyethylene glycolated interleukin-2.

Polyethylene glycolated (pegylated) interleukin-2 (PEG IL-2) was administered as a weekly i.v. bolus to patients with metastatic cancer in a phase-I trial. Efficacy, toxicity and pharmacokinetics have been described previously. To explore mechanism of IL-2 action and discover predictors of efficacy, the levels of several lymphokines were measured in pharmacokinetic serum samples. IL-1 beta and IL-6 were elevated in many patients before PEG IL-2 administration, forming a continuous, log-normal distribution among patients. The levels of the two lymphokines were strongly correlated. However, no significant correlation could be found between these levels, clinical chemistry, or tumor regression seen after PEG IL-2 administration. Three hours after PEG IL-2 administration, IL-1 beta and IL-6 levels, if elevated, fell to normal. In all patients, independent of initial levels, IL-6 and IFN-gamma, but not IL-1 beta, increased 4 to 6 h after the injection and then fell rapidly, even though PEG IL-2 levels were high and often changed only slightly during this period. This suggests an active shut down of lymphokine synthesis, or an increase in elimination rate. After the fourth administration of PEG IL-2, the peak level of IFN-gamma was 2 to 20 times higher than after the first, while the peak level of IL-6 did not change in a consistent direction. Responding patients had typical peak levels of IL-6 and IFN-gamma. Low levels of TNF and IL-4 were occasionally seen before and after PEG IL-2 administration, but no consistent pattern was evident.

Specific lysis of human tumor cells by T cells coated with anti-T3 cross-linked to anti-tumor antibody.

Heteroaggregates containing anti-T3 cross-linked to anti-target cell antibodies have been shown to cause human T cells to lyse target cells that express antigens recognized by the anti-target cell antibody. In this study, we test targeted human T cells for the ability to lyse human tumor cells as a first step toward the application of this phenomenon to tumor immunotherapy. Several monoclonal anti-human tumor antibodies were assayed for binding to a number of human tumor lines and for the ability to promote specific tumor cell lysis when cross-linked with anti-T3. We found that anti-T3 cross-linked to anti-tumor monoclonal antibodies caused cloned human T cells and fresh peripheral blood T cells to lyse the tumor cells with the same specificity as predicted by the binding studies. Peripheral blood T cells were then tested in the presence of various heteroaggregates for the ability to lyse single cell suspensions prepared from fresh tumor or fresh normal tissue. These studies showed that heteroaggregates containing anti-T3 cross-linked to anti-tumor antibody cause fresh human T cells to specifically lyse fresh tumor cells, but not (with one exception) fresh normal cells.

Adoptive immunotherapy of a syngeneic murine leukemia with a tumor-specific cytotoxic T cell clone and recombinant human interleukin 2: correlation with clonal IL 2 receptor expression.

The successful adoptive immunotherapy of the syngeneic Friend virus-induced murine leukemia FBL-3 was mediated by a proliferative MHC-restricted, tumor-specific CTL clone in combination with recombinant human IL 2. This clone was previously shown to express the L3T4-, Lyt-1+, Lyt-2+ surface phenotype. Activation of the clone for 48 hr in vitro with irradiated tumor cells induced the expression of IL 2 receptors and markedly increased clonal proliferation in response to recombinant IL 2. Intravenous injection of 2 X 10(7) 48 hr in vitro-activated cloned cells, followed by 6 days of systemic (i.p.) administration of IL 2 resulted in the complete regression of tumors and the cure of 50% of the treated mice. IL 2 alone had no effect on tumor growth, whereas the injection of nonactivated (resting) clone plus IL 2 or activated clone without IL 2 had small but insignificant effects on tumor growth and survival. These results indicated that the in vivo effector functions of cloned T cells may be markedly enhanced by the concurrent systemic administration of recombinant IL 2 and by the induction of optimal IL 2 receptor expression on the cloned T cells at the time of cell administration.