Immunotoxin Research Articles

Human glioma cell lines: tumour associated antigens distribution and sensitivity to antibody-toxin or ligand-toxin conjugates. A preliminary report.

We have investigated the phenotype of seven human glioma cell lines established in vitro from primary tumour explants. Indirect immunofluorescence and flow cytofluorimetry revealed a heterogeneous distribution of surface GE 2 and CG 12 Tumour Associated Antigens (TAA). In one group of cell lines TAA were detected both at the cell surface and in the cytosol, whereas in a second group of glioma cell lines TAA were found only in the cytosol. We have also investigated the sensitivity of glioma-derived cell lines to antibody-toxin and ligand-toxin conjugates (Immunotoxins). Monoclonal antibodies anti GE 2 antigen linked to ricin toxin A subunit (RTA) showed poor cytotoxicity, which increased about 50 fold when the whole toxin was linked to anti GE 2 monoclonals. Treatment with human recombinant interferon gamma (IFN-gamma) greatly augmented the percentage of HLA-DR+ cells and the amount of HLA-DR antigens per cell. IFN-gamma treatment resulted in a net increase of sensitivity to anti HLA-DR Immunotoxins (IT). Human diferric transferrin linked to RTA exhibited a potent cytotoxic effect against human glioma-derived cells when used in the presence of the lysosomotropic carboxylic ionophore monensin.

Clinical studies: solid tumors.

Immunotoxins (ITs) permit delivery of a therapy at the tumor site specifically. The use of ITs as therapy of patients with solid tumors presents problems which require unique solutions. These include stability in vivo, cellular heterogeneity, access to tumor, biodistribution, and the immune response to the immunoconjugate (Table 1). It is necessary to address some of these issues before contemplating entry into clinical trials, whereas others are more appropriately undertaken after clinical trials have been initiated, using the results of the clinical observations as a focus for planning improvements as part of a second generation effort. This involves both optimizing the administration of the currently available products and developing new, improved products.KeywordsPreclinical EvaluationBlood Group SubstanceMurine ImmunoglobulinCarbohydrate ReceptorAntibody ComponentThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Toxin selection and modification: utilization of the A chain of ricin.

The first immunotoxins (ITs) assembled by Moolten et al. in the seventies [1, 2] were composed of antibodies linked to whole toxins such as diphtheria toxin and ricin. In order to circumvent the nonspecific binding due to the B chains, ITs with the A chain subunit of the polypeptide toxin ricin were constructed [3–11].

Human immune response to immunotoxins.

The potential value of immunotoxins (ITs) in the treatment of human cancer may eventually prove to be limited by the development of host antibodies against the conjugate. The development of antimurine antibodies after administration of unconjugated monoclonal antibodies is well documented and has been associated with more rapid murine antibody clearance and decreased clinical effect [1, 2]. Similarly, antitoxin antibodies have been detected in both animals and humans treated with ricin and abrin [3, 4]. When given lethal doses of ricin, survival was prolonged in mice which had developed antiricin antibody levels in excess of 10–20 ng/ml [3].

RATIONALE FOR THE SELECTION OF RICIN A-CHAIN ANTI-T IMMUNOTOXINS FOR MATURE T CELL DEPLETION

A series of 25 different ricin A-chain immunotoxins (IT) were prepared with monoclonal antibodies reacting with several T cell antigens belonging to different clusters of differentiation (CD) to select the most appropriate immunotoxins (IT) for mature T cell depletion. Our screening procedure was performed in 2 steps. First, IT were evaluated using protein synthesis inhibition assay on clonogenic malignant cells in order to determine the most active IT for a given CD. Second, IT thus selected were evaluated for both mature T cell killing efficacy and tolerance on hematopoietic progenitor cells (HPC). This study showed that (1) different IT directed against the same CD antigen displayed a wide range of activity, suggesting the need for a large screening of IT to determine the most appropriate antibody for a given target antigen; (2) anti-CD3 and anti-CD5 ricin A-chain IT are the most active, displaying a cytoreduction of more than 2 logs on mature T cells; (3) the 3 different anti-CD2 ricin A-chain IT evaluated in this study induced poor mature T cell cytoreduction despite their relative efficacy on leukemic cells; (4) anti-CD8 ricin A-chain IT showed almost no efficacy on relevant target cells; (5) no toxicity on HPC was found for concentrations up to 10(-8) of A-chain whatever the IT used.

Elimination of T cells from human peripheral blood and bone marrow using a cocktail of three anti-T cell immunotoxins.

Four anti-T cell monoclonal antibodies were coupled to ricin-A and tested for their ability to kill T cells in peripheral blood and bone marrow using a clonogenic assay to quantify T cell survival. The immunotoxins (IT) prepared from RFT11 (CD2) and WT1 (CD7) antibodies were the most toxic to peripheral blood T cells. The immunotoxin prepared from RFT1 (CD5) was the next most efficient toxin and the immunotoxin prepared from RFT8 (CD8) was the least toxic. When these reagents were applied to peripheral blood cells at 3 x 10(-8) M the number of T cell colonies was reduced by an average of 95%, 94%, 84% and 50%, respectively. Peripheral blood T cells from different donors showed marked variability in their sensitivity to ITs. However, a cocktail of three ITs prepared from RFT11, WT1 and RFT1 gave superior and consistent killing (mean 99.9%; range 99.8-100%) of peripheral blood T cells from six donors. When this cocktail was applied to bone marrow cells from six donors, an average of 99.6% (range 99.5-99.8%) of the T cells were killed. Under the same conditions there was little or no reduction in the number of normal haematopoietic progenitors (CFU-GM, CFU-GEMM, CFU-Meg and BFU-E).

Human leukemia cell binding and killing by anti-CD5 radioimmunotoxins

We have synthesized a reagent for antibody directed cell targeting composed of the monoclonal antibody (MoAb) T101 linked to the potent toxin ricin. The immunotoxin (IT) was subsequently radiolabeled by a cyclic anhydride procedure with 90Yttrium ( 90Y) to construct a radioimmunotoxin (RIT) that may have potential for cancer therapy. We evaluated the reagent for selectivity in binding and protein synthesis inhibition (PSI) assays. The RIT selectively bound antigen positive leukemia T-cell lines, with minimal binding to antigen negative control lines. The IT inhibited 87% or greater protein synthesis activity at 1 μ g ml and exhibited an IC 50 (the dose inhibiting 50% activity) of 0.18 ± 0.08 μ g ml in the presence of lactose. RIT and nonlabeled IT showed comparable degrees of PSI at 1 μ g ml and 10 μ g ml , suggesting that labeling had little overall effect on the activity of the immunoconjugate. However, indirect evidence showed that the galactose binding site of ricin was inhibited 10-fold by its exposure to 90Y. Control RIT were minimally inhibitory. IT labeled with 131Iodine ( 131I) by an iodine monochloride technique also retained its capability to selectively inhibit protein synthesis. When RIT were tested for potency in a clonogenic assay against human leukemia T-cell lines, they inhibited 3.61 logs of tumor cell growth at μ g ml ml. This did not represent an improvement over the log elimination with radiolabeled antibody alone, which showed 4.19 log elimination of tumor cells. Our observation that the 90Y-labeled RIT and labeled antibody can selectively eliminate about four logs of tumor cells in an in vitro clonogenic assay is unique. The ability of RIT to kill several logs of tumor cells in vitro renders RIT interesting anti-tumor reagents.

Graft-versus-host disease prevention in allogeneic bone marrow transplantation from histocompatible siblings. A pilot study using immunotoxins for T cell depletion of donor bone marrow.

Seventeen patients, ages 7-53 years were transplanted with histocompatible bone marrow that had been depleted of T lymphocytes by ex vivo immunotoxin (IT) treatment. Twelve patients had high-risk acute leukemias, and five had chronic myelogenous leukemia. No other graft-vs.-host disease (GVHD) prophylaxis was used. A mixture of three anti-T-cell monoclonal antibodies conjugated to ricin were used in this study: TA-1, UCHT-1 (anti-CD3), and T101 (anti-CD5). The mean number of bone marrow cells infused was 1.5 X 10(8) mononuclear cells/kg recipient weight. Thirteen of the 17 patients demonstrated complete and sustained engraftment. Four patients experienced autologous marrow recovery and/or graft rejection. Compared with an historical group of leukemic patients who received GVHD prophylaxis with methotrexate alone or combinations of methotrexate, and prednisone plus antithymocyte globulin, (ATG) or OKT3, the IT patients with stable engraftment demonstrated shorter time to recovery of leukocytes greater than or equal to 1000mm3 for three consecutive days (median, 20 days vs. 26 days, P = .03). The recovery of total lymphocytes, B and T cell subsets, and T cell function by day 28 was highly variable, but similar, for patients in both the IT-treated group and historical controls. Four patients (ages 13, 18, 21, and 38) developed grade II skin GVHD, but none had severe GVHD. Eight of the 13 patients with durable engraftment have had posttransplant leukemic relapse. Currently only four patients remain alive; two have not relapsed posttransplant, while the other two achieved remission following posttransplant relapse. We conclude that severe GVHD was not observed in this small series with ex vivo T cell depletion for GVHD prophylaxis, and that favorable recovery of hematologic and lymphocytic function was demonstrated for cases where primary engraftment was sustained. A larger randomized controlled study will be needed to establish whether T cell depletion of donor bone marrow with IT can significantly reduce GVHD, and/or improve disease-free survival.

Whole ricin and recombinant ricin A chain idiotype-specific immunotoxins for therapy of the guinea pig L2C B cell leukemia.

The therapeutic efficacy of whole ricin, or recombinant ricin A chain, coupled to a monoclonal antibody that reacts with the idiotype of the surface IgM expressed on guinea pig L2C lymphoblasts, was assessed. In vitro studies were done to characterize the immunotoxins (IT) and to demonstrate their specificity before use in vivo. The concentration of whole ricin IT (M6-Ricin) that inhibited protein synthesis by 50% (IC50) in L2C cells was 1.4 X 10(-9) M, in a 5-hr assay, in the presence of lactose to block non-antibody-directed toxicity. M6-Ricin did not inhibit protein synthesis in two control guinea pig cell lines that did not express the idiotype, nor did a whole ricin IT prepared with an isotype-matched monoclonal antibody of irrelevant specificity inhibit protein synthesis in L2C cells. Two recombinant ricin A chain IT, which differed from one another by a factor of 2 to 3 in the number of A chains conjugated per antibody molecule, were less effective in vitro than M6-Ricin (IC50 of greater than 5 X 10(-8) M). For in vivo experiments, the IT were given by the i.p. route 24 hr after the i.p. inoculation of 1 X 10(5) L2C cells. The highest doses of M6-Ricin and M6-Ricin A chain IT tested, 30 micrograms/kg and 3000 micrograms/kg, respectively, were within fourfold to fivefold of their maximum tolerated doses; no deaths or ill effects due to ricin toxicity were noted. These doses increased the median survival time of L2C-bearing guinea pigs to 31 to 34 days, compared with 15 days for untreated animals. This magnitude of increase in survival indicates that 99.999% (5 logs) of injected tumor cells were eliminated, thus accounting for the 12% long-term survival rate obtained. Median survival times for guinea pigs treated with 30 micrograms/kg of the A chain IT were 18 and 21 days for the two conjugates tested, and the median survival for guinea pigs treated with 3000 micrograms/kg of unconjugated antibody was 18 days. Our data demonstrate that recombinant A chain IT are active in vivo and that the B chain of ricin can potentiate IT activity in vivo. Although the potency differs by 100-fold, the therapeutic index of the intact ricin IT is similar to that of the ricin A chain IT.

Comparison of inhibitory effect of galactose analogs on the binding and cytotoxicity of an anti‐globotriaosylceramide monoclonal antibody coupled or not coupled to pokeweed antiviral protein

The results described here provided an example of a human IgM monoclonal antibody against a tumor-associated glycolipid and of the unusual properties of its corresponding immunotoxin (IT). The monoclonal antibody referred to as 38-13 has been previously described and reacted with the globotriaosylceramide [Gb3:Gal(alpha 1----4)-Gal(beta 1----4)-Glc(beta 1----1)ceramide] specifically expressed on surface membrane of Burkitt's lymphoma (BL) cells. An immunotoxin (38-13 IT) combined with the pokeweed antiviral protein (PAP) toxin via S-S bridges showed paradoxically a lower cytotoxic effect in BL Ramos cells than in non-BL cells such as leukemic mouse L1210 cells, while these cells appeared not to be involved by flow cytometric analysis and complement-dependent cytotoxicity. Consequently, the inhibitory effect of selective galactose analogs on the binding and the cytotoxicity of 38-13 antibody conjugated or not to PAP toxin was compared on BL and non-BL cells. Only the galactose blocked in alpha configuration provided a fine inhibition of 38-13 binding on BL Ramos cells and both alpha and beta-galactose allowed us to establish a clear distinction between the pathway entry of 38-13 IT in BL and non-BL cells; in close correlation with the 38-13 binding specificity the 38-13 IT cytotoxic effect in Ramos BL cells could also be prevented by alpha-Gal only, suggesting that this toxic action is probably mediated through the IT binding to Gb3 antigenic sites. In contrast, on apparently irrelevant L1210 cells, 38-13 IT showed a cytotoxic effect which was inhibited preferentially by lactose (Gal in beta configuration). It was discussed that IT binding alone to either antigenic sites which are inhibited by the hapten alpha-Gal, or nonspecific sites which can compete with the hapten beta-Gal is unable to induce efficient killing of cells. But cooperation of both bindings might give an attractive explanation of IT cytotoxic effect. It was concluded that the unexpected activity of 38-13 IT in non-BL cells probably could be mediated through an active macromolecular transport process which could implicate a beta-galactoside-binding protein (lectin).

Antitumor activity against L1210 mouse leukemia of immunotoxins containing a monoclonal antibody to the glycolipid globotriaosylceramide

Since immunotoxin (IT) containing the anti-globotriaosylceramide monoclonal antibody was found to be cytotoxic in murine L1210 leukemia cells, its potential antitumor activity could be evaluated in animals using the L1210 model. In vitro, L1210 cells incubated IT before grafting in DBA/2 mice failed to induce leukemia. All tumor cells were neutralized by IT. In animals, a significant but limited therapeutic effect on leukemic mice was obtained when IT was injected i.p. shortly after L1210 cell grafting. In contrast, no toxic effect of IT was observed in non-leukemic mice at doses far above those used in our therapeutic treatment. The potentiation effect of chloroquine on IT was moderated when a cloning efficiency assay was used, but 70% of the mice grafted with in vitro chloroquine-treated L1210 cells were cured with IT treatment.

Effect of cyclophosphamide on the immunogenicity of monoclonal antimelanoma antibody-ricin A chain immunotoxin in rats.

This study was performed to determine the effect of treatment with cyclophosphamide (CY) on the formation of antimurine and anti-ricin A chain antibodies in rats treated with a murine monoclonal antimelanoma antibody-ricin A chain immunotoxin (IT). Animals received treatment with either IT alone or IT plus CY. IT treatment consisted of daily IV injections at a dose of 1 mg/kg/day on days 0, 1, 2, 3, 4. CY treatment consisted of a 25 mg/kg IP dose on day -1 followed by daily IP doses of 5 mg/kg/day on days 0, 1, 2, 3, 4. Antibody binding activities in treated animals were measured by enzyme-linked immunosorbent assay and reported as optical density values. Rats treated with IT plus CY had lower binding activity on day 7 (0.09 vs 0.6; p = .02), day 14 (0.42 vs 1.22; p = .001), and day 21 (0.11 vs 1.3; p = .001) compared to rats treated with IT alone. Lower levels of anti-ricin A chain binding activity were observed in CY treated rats on day 14 (0.35 vs 1.25; p = .001), but not on day 7 or day 21. These results indicate that treatment with CY can abrogate the immune response to murine antibody and partially abrogate the immune response to ricin A chain.

Marrow Purging in Autologous Bone Marrow Transplantation for T-Lineage Acute Lymphoblastic Leukemia: Efficacy of Ex Vivo Treatment With Immunotoxins and 4-Hydroperoxycyclophosphamide Against Fresh Leukemic Marrow Progenitor Cells

A lymphoblast progenitor cell assay was used to evaluate the antileukemic efficacy of marrow-purging protocols that employed intact ricin immunotoxins (IT) and 4-hydroperoxycyclophosphamide (4-HC) against clonogenic primary T-lineage marrow blasts freshly obtained from 12 T-lineage acute lymphoblastic leukemia (ALL) patients. Residual T-lineage blast colonies were observed after treatment with 1 micrograms/mL T101 (anti-CD5)-Ricin (R) + G3.7 (anti-CD7)-R in eight of 12 cases and after 100 micrograms/mL 4-HC in six of nine cases. By comparison, a combination of IT and 4-HC proved very effective against T-lineage leukemic progenitor cells, and no residual blast colonies were observed in any of the eight cases studied. We conclude that future trials should consider combined treatment protocols such as IT + 4-HC for more effective purging of autologous marrow grafts.

Marrow purging in autologous bone marrow transplantation for T-lineage acute lymphoblastic leukemia: efficacy of ex vivo treatment with immunotoxins and 4-hydroperoxycyclophosphamide against fresh leukemic marrow progenitor cells

A lymphoblast progenitor cell assay was used to evaluate the antileukemic efficacy of marrow-purging protocols that employed intact ricin immunotoxins (IT) and 4-hydroperoxycyclophosphamide (4-HC) against clonogenic primary T-lineage marrow blasts freshly obtained from 12 T-lineage acute lymphoblastic leukemia (ALL) patients. Residual T-lineage blast colonies were observed after treatment with 1 micrograms/mL T101 (anti-CD5)-Ricin (R) + G3.7 (anti-CD7)-R in eight of 12 cases and after 100 micrograms/mL 4-HC in six of nine cases. By comparison, a combination of IT and 4-HC proved very effective against T-lineage leukemic progenitor cells, and no residual blast colonies were observed in any of the eight cases studied. We conclude that future trials should consider combined treatment protocols such as IT + 4-HC for more effective purging of autologous marrow grafts.

Evaluation of ricin A-chain immunotoxins directed against human T cells

We have synthesized four immunotoxins (ITs) by covalently coupling the A chain of ricin to murine monoclonal antibodies that recognize surface antigens on human T cells. Treatment of human peripheral blood lymphocytes with either 10.2-A, directed against the CD5 (Tp67) antigen, or 64.1-A, directed against the CD3 (Tp19) antigen, abolished protein synthesis in cells subsequently cultured with phytohemagglutinin (PHA). In contrast, two other ITs (9.6-A and 35.1-A), both directed against the CD2 (Tp50) antigen, had minimal effects on protein synthesis in PHA-stimulated cells. The binding of each IT to T cells was shown by immunofluorescence with fluorescein-conjugated goat anti-mouse immunoglobulin (FITC-GAMIg) and fluorescein-conjugated rabbit anti-ricin A-chain (FITC-RAR) antibodies. Activity of the ricin A chain in each IT was demonstrated by its ability to inhibit protein synthesis in a cell-free reticulocyte lysate assay. Ultrastructural immunoperoxidase analysis of IT internalization showed that ineffective and effective ITs were endocytosed at the same rate (50% of cells had labeled endosomes after 15 min). However, ineffective IT 35.1-A was more rapidly delivered to lysosomes (15–30 min) than effective ITs (10.2-A and 64.1-A) (⩾30 min). The data support the hypothesis that there are several distinct pathways for internalization of ITs and that the ability of ricin A chain to reach and inactivate ribosomes may depend upon the specific membrane receptor involved in binding a given IT, its route of internalization, and the rate of entry of the IT into lysosomes.

Antigenic modulation by anti-CD5 immunotoxins.

We evaluated the modulation of T101 immunotoxins (IT) and free T101 antibody from the surface of normal and leukemic cells to determine whether the presence of toxin on antibody affected antigenic modulation. Reagents were made by conjugating T101, which binds to the T cell antigen CD5, to either intact ricin or purified ricin A chain. We found that T101-A chain modulated CD5 more efficiently than T101-ricin, which modulated CD5 more efficiently than T101 alone. Kinetic studies showed that maximal modulation of IT was reached within 3 hr. When toxicity of the reagents was tested in protein synthesis inhibition assays, T101-ricin in the presence of lactose inhibited 99% of the protein synthesis of CEM cells. T101-A chain was less toxic, inhibiting protein synthesis only 23 to 43%. The addition of the potentiating agent monensin nearly doubled the toxicity of T101-A chain, but did not affect T101-A chain modulation. To determine the fate of bound IT, T101 and T101-ricin were labeled with 125I. Cells were incubated under modulating conditions in the presence of radiolabeled reagents. T101 and T101-ricin were internalized into CEM cells. In contrast, T101, but not T101-ricin, appeared to be shed from peripheral blood mononuclear cells. Our findings show clearly that: 1) the presence of toxin on antibody does not inhibit--and may actually enhance--modulation; 2) T101-IT are internalized, not shed from the cell surface; 3) the lack of toxicity of T101-A chain is not attributed to inability to modulate; 4) there is no correlation between enhancement of T101-A chain toxicity by monensin and antigenic modulation by A chain reagents; and 5) modulation, which is undesirable in monoclonal antibody therapy, may be advantageous in the therapeutic use of IT.

Anti-CD2 (T, p50) intact ricin immunotoxins for GVHD-prophylaxis in allogeneic bone marrow transplantation

We evaluated the inhibitory effects of two immunotoxins (IT) synthesized by linking two different anti-CD2 (T, p50) murine monoclonal antibodies (MoAb) to intact ricin (R). Pretreatment with 1000 ng ml −1 35.1-R or OKT 11 a R inhibited PHA-induced T-cell proliferation by 93% and 86%, respectively. At this IT concentration generation of alloreactive cytotoxic T-cells (CTL) was inhibited by more than 99% by either IT. 35.1-R and OKT 11 a were minimally toxic to natural killer (NK) effectors or pluripotent bone marrow progenitor cells (CFU-GEMM). Blocking experiments suggested that 35.1-R and OKT 11 a-R might recognize different epitopes of the CD2 (T, p50) surface determinant. Our findings show that anti-CD2 IT may be useful for T-cell depletion in allogeneic bone marrow transplantation. We compared TU3, an equimolar mixture of T101 [anti-CD5]-R, UCHT-1 [anti-CD3]-R and 35.1 [anti-CD2]-R with the TUT-cocktail (a mixture of T101-R, UCHT-1-R and TA-1 [antiCDwl8]-R. TUT is currently under evaluation in Phase 1 clinical trials as a T-cell depletion regimen for GVHD prophylaxis. TU3 was as effective as TUT-cocktail in inhibition of PHA response and CTL generation but unlike TUT spared NK effectors. Cocktails of immunotoxins directed against subpopulations of lymphocytes may be useful (a) for more effective anti-GVHD strategies, and (b) to circumvent problems of graft failure/rejection associated with current purgation regimens.

Use of multiple T cell-directed intact ricin immunotoxins for autologous bone marrow transplantation

The monoclonal antibodies (MoAb) T101, G3.7, 35.1, and TA-1 were conjugated to intact ricin using a thioether linkage. These MoAb detect, respectively, the CD5[gp67], CD7[p41], CD2[p50], and [gp95, 170] determinants that are found in the vast majority of cases of T cell acute lymphocytic leukemia (T-ALL). The resulting immunotoxins (ITs) and an equimolar mixture of these ITs were evaluated as potential purgative reagents for autologous transplantation in T-ALL. Leukemic cell lines were used to compare the kinetics of protein synthesis inactivation mediated by each IT. The cells were treated with IT in the presence of lactose in order to block the native binding of ricin. The observed rates of protein synthesis inactivation correlated with target antigen expression detected by fluorescence-activated cell sorter analysis. Of the four ITs, T101-ricin (T101-R) exhibited the fastest rate of inactivation, followed in order by G3.7-ricin, TA-1-ricin, and 35.1-ricin. At concentrations greater than 300 ng/mL, a cocktail containing an equimolar amount of all four ITs (referred to as the four- IT cocktail) exhibited kinetics that were as fast or faster than those of T101-R. The long-term cytotoxic effects of individual ITs and the four-IT cocktail were evaluated using a sensitive clonogenic assay. Each IT was specifically cytotoxic and inhibited 1 to 4 logs of clonogenic leukemic cells at doses (300 to 600 ng/mL) that can be used clinically. The four-IT cocktail was highly cytotoxic; a concentration of 300 ng/mL inhibited greater than 4 logs of leukemic cells while sparing the majority of committed (CFU-GM, CFU-E) and pluripotent (CFU- GEMM) hematopoietic stem cells. The determination of both short-term kinetics of protein synthesis inactivation and longer-term inhibition of clonogenic growth allowed new insight into cell killing by IT. Our results suggest that ITs continue to act on clonogenic target cells for a period of three to five days. Interestingly, the four-IT cocktail was not as potent against clonogenic leukemic cells as T101-R alone, although it exhibited kinetics of protein synthesis inhibition that were as fast as those of T101-R alone. This finding suggests that internalized ITs may differ in the length of time they remain active within the cell. Our results also demonstrate the importance of using several different assays to evaluate IT reagents.

Use of Multiple T Cell-Directed Intact Ricin Immunotoxins for Autologous Bone Marrow Transplantation

AbstractThe monoclonal antibodies (MoAb) T101, G3.7, 35.1, and TA-1 were conjugated to intact ricin using a thioether linkage. These MoAb detect, respectively, the CD5[gp67], CD7[p41], CD2[p50], and [gp95,170] determinants that are found in the vast majority of cases of T cell acute lymphocytic leukemia (T-ALL). The resulting immunotoxins (ITs) and an equimolar mixture of these ITs were evaluated as potential purgative reagents for autologous transplantation in T-ALL. Leukemic cell lines were used to compare the kinetics of protein synthesis inactivation mediated by each IT. The cells were treated with IT in the presence of lactose in order to block the native binding of ricin. The observed rates of protein synthesis inactivation correlated with target antigen expression detected by fluorescence-activated cell sorter analysis. Of the four ITs, T101-ricin (T101-R) exhibited the fastest rate of inactivation, followed in order by G3.7-ricin, TA-1-ricin, and 35.1-ricin. At concentrations >300 ng/mL, a cocktail containing an equimolar amount of all four ITs (referred to as the four-IT cocktail) exhibited kinetics that were as fast or faster than those of T101-R. The long-term cytotoxic effects of individual ITs and the four-IT cocktail were evaluated using a sensitive clonogenic assay. Each IT was specifically cytotoxic and inhibited 1 to 4 logs of clonogenic leukemic cells at doses (300 to 600 ng/mL) that can be used clinically. The four-IT cocktail was highly cytotoxic; a concentration of 300 ng/mL inhibited >4 logs of leukemic cells while sparing the majority of committed (CFU-GM, CFU-E) and pluripotent (CFU–GEMM) hematopoietic stem cells. The determination of both short-term kinetics of protein synthesis inactivation and longer-term inhibition of clonogenic growth allowed new insight into cell killing by IT. Our results suggest that ITs continue to act on clonogenic target cells for a period of three to five days. Interestingly, the four-IT cocktail was not as potent against clonogenic leukemic cells as T101-R alone, although it exhibited kinetics of protein synthesis inhibition that were as fast as those of T101-R alone. This finding suggests that internalized ITs may differ in the length of time they remain active within the cell. Our results also demonstrate the importance of using several different assays to evaluate IT reagents.

Combined ex vivo treatment with immunotoxins and mafosfamid: a novel immunochemotherapeutic approach for elimination of neoplastic T cells from autologous marrow grafts.

We evaluated a novel ex vivo "purging" protocol for selective elimination of neoplastic T cells from human marrow by using a sensitive clonogenic assay. Immunotoxins (IT) were synthesized by conjugating ricin (R) to four different monoclonal antibodies (MoAb) directed against distinct markers of T cell lineage. Treatment with anti-p67-R produced effective elimination of leukemic T cells from human marrow. The cyclophosphamide congener mafosfamid (ASTA Z 7577) markedly enhanced the target cell cytotoxicity of IT and extended the final level of clonogenic kill 2 to 3 logs. Our data show that anti-p67-R in combination with mafosfamid resulted in a maximum elimination of 6.2 logs of neoplastic T cells with minimal toxicity to normal bone marrow progenitors. The efficiency of this protocol was not reduced in the presence of excess normal bone marrow cells. Similar findings were obtained by using a cocktail of four different anti-T cell IT. This approach is unique in combining both immunologic (IT) and chemical (mafosfamid) strategies for more effective ex vivo bone marrow purging in autologous bone marrow transplantation for T cell acute lymphoblastic leukemia/lymphoblastic lymphoma.