Biomedical subjects

M A Ghetie

Publications and source records attributed to M A Ghetie.

At least 19 recordsLinked to original sources

PubMed1996

Combination immunotoxin treatment and chemotherapy in SCID mice with advanced, disseminated Daudi lymphoma.

We describe the use of an immunotoxin (IT) cocktail (anti-CD22- and anti-CD19-ricin A chain) and any 1 of 3 chemotherapeutic drugs (doxorubicin, cytoxan or camptothecin) to treat advanced disseminated Daudi lymphoma in SCID mice (SCID/Daudi). In a previous report, we demonstrated that this regimen was curative when given the day following tumor cell inoculation. Here, we show that combination therapy in mice with advanced tumor significantly increased their survival, although it was not curative. Importantly, the outcome of therapy was dependent upon the temporal order in which IT and chemotherapy were administered. Thus, the best anti-tumor effect was achieved when an IT cocktail was given before or at the same time as chemotherapy. When the IT was given after chemotherapy, there was no additional therapeutic benefit. Our results confirm the rationale of using combination therapy in the treatment of advanced B-cell neoplasia and suggest that ITs should be administered prior to or during chemotherapy.

Animals↗

PubMed1996

Effect of sublethal irradiation of SCID mice on growth of B-cell lymphoma xenografts and on efficacy of chemotherapy and/or immunotoxin therapy.

Severe combined immune deficiency (SCID) mice obtained from different vendors vary in their capacity to accept human tumor xenografts, and in some instances mice must first be irradiated. It has been reported that SCID mice are particularly sensitive to irradiation which, in addition, can partially restore their immune system so that they are no longer totally immunologically incompetent. For the past several years we have used nonirradiated SCID mice to grow human B-cell lymphomas. When we changed vendors, we found it necessary to irradiate the mice before xenografting. Sublethal irradiation of at least one source of SCID mice before tumor cell inoculation improved tumor take and dissemination, but irradiation changed the response of these mice to chemotherapy and immunotoxins. Thus the irradiated mice did not respond to chemotherapy, but the two immunotoxins used for therapy became more effective in extending survival of the mice. It then appears that irradiation affects the immune system of SCID mice in such a way as to change their response to the therapeutic regimens used here.

Animals↗

PubMed1994

Eradication of minimal disease in severe combined immunodeficient mice with disseminated Daudi lymphoma using chemotherapy and an immunotoxin cocktail.

Severe combined immunodeficient (SCID) mice injected intravenously with a human Burkitt's lymphoma cell line (Daudi) develop disseminated lymphoma (SCID/Daudi), which is fatal in 100% of the mice. Early treatment of these mice with either an immunotoxin (IT) cocktail (consisting of anti-CD19-ricin A chain plus anti-CD22-ricin A chain) or chemotherapy significantly prolonged survival but was not curative. Combination therapy with the IT cocktail and any one of three chemotherapeutic drugs (doxorubicin, cytoxan, or camptothecin) cured the mice. Cure was demonstrated by both histopathologic examination of treated mice and, more importantly, by adoptive transfer of cells from organs of the cured mice to naive SCID mice where 100 tumor cells would have caused disease in the recipients. These results provide a strong rationale for combining IT therapy with conventional chemotherapy in the treatment of B-cell neoplasia.

Animals↗

PubMed1994

Anti-CD19 inhibits the growth of human B-cell tumor lines in vitro and of Daudi cells in SCID mice by inducing cell cycle arrest.

In this report, we extend our previous findings that IgG or F(ab')2 fragments of HD37 anti-CD19 antibody (Ab) in combination with the immunotoxin (IT), RFB4-anti-CD22-deglycosylated ricin A chain (dgA) (but neither reagent alone), prolonged the survival of SCID mice with disseminated human Daudi lymphoma (SCID/Daudi mice) to 1 year at which time they still remained tumor-free. We explored the mechanisms by which the HD37 Ab exerts antitumor activity in vivo by studying its activity in vitro. We found that it has antiproliferative activity (IC50 = 5.2 - 9.8 x 10(-7) mol/L) on three CD19+ Burkitt's lymphoma cell lines (Daudi, Raji, and Namalwa) but not on a weakly CD19-positive (CD19lo) pre-B cell tumor (Nalm-6). The inhibitory effect was manifested by cell cycle arrest, but not apoptosis. Results using three additional anti-CD19 Abs, suggest that the affinity of the antibody and possibly the epitope which it recognizes may effect its capacity to transmit a signal that induces cell cycle arrest. Hence, therapeutically useful Abs may exert anti-tumor activity by a variety of mechanisms, each of which should be evaluated before undertaking clinical trials in humans.

Animals↗

PubMed1994

Recent developments in immunotoxin therapy.

Immunotoxin (IT) research has been ongoing for 15 years. During the past 2 years, work has focused on several areas: on improvements and developments in first- and second-generation ITs; the preparation of new immunotoxin constructs with anti-tumor activity; novel animal models for preclinical evaluation of immunotoxins; and clinical trials, which are now entering Phase II or III in humans.

Animals↗

PubMed1993

Ricin A-chain and ricin A-chain immunotoxins rapidly damage human endothelial cells: implications for vascular leak syndrome.

The results of Phase I/II clinical trials indicate that ricin A-chain-containing immunotoxins cause vascular leak syndrome, characterized by hypoalbuminemia with resultant weight gain and edema. Vascular leak syndrome may be a dose-limiting factor during treatment with ricin A-chain-containing immunotoxins. In this report, we determined the effect of ricin A-chain and ricin A-chain-containing immunotoxins on human umbilical vein endothelial cells with the aim of developing an in vitro model to study vascular leak syndrome. The major findings of our study are: (1) Human umbilical vein endothelial cells undergo rapid and dramatic changes in morphology after treatment with ricin A-chain and ricin A-chain-containing immunotoxins. These changes include rounding of the cells and, eventually, the formation of gaps between them. (2) The permeability of human umbilical vein endothelial cell monolayers to passage of molecules increases after exposure to ricin A-chain or ricin A-chain-containing immunotoxins and this is consistent with the morphologic changes. (3) Human umbilical vein endothelial cells bind 125I-rRTA in a dose-dependent manner but binding is not specific. (4) Human umbilical vein endothelial cells are moderately more sensitive to ricin A-chain-induced inhibition of protein synthesis and proliferation than simian virus-transformed mouse endothelial cells. (5) The morphologic changes are observed 1 h after exposure to the toxins, whereas inhibition of protein synthesis is not detectable until 4 h after a similar exposure. The in vitro model represents a first step in dissecting the complex events which occur in cancer patients who develop vascular leak syndrome after treatment with ricin A-chain-containing immunotoxins.

Animals↗

PubMed1992

The antitumor activity of an anti-CD22 immunotoxin in SCID mice with disseminated Daudi lymphoma is enhanced by either an anti-CD19 antibody or an anti-CD19 immunotoxin.

The antitumor activities of immunotoxins (ITs) constructed with deglycosylated ricin A chain (dgA) and either anti-CD19 (HD37) or anti-CD22 (RFB4) monoclonal antibodies were compared in SCID mice with disseminated human Daudi lymphoma (SCID/Daudi). As reported previously, after intravenous injection with Daudi cells, SCID mice develop disseminated lymphoma, which infiltrates the vertebral column and causes paralysis of the hind legs before death. The mean paralysis time (MPT) has been taken as an end point in this tumor model. We have previously reported that early treatment of SCID/Daudi mice with RFB4 coupled to dgA prolongs the MPT in a manner consistent with the killing of 4 logs of tumor cells. In the present study, we show that HD37-dgA kills 2 logs of tumor cells. The lower potency of the HD37-dgA is consistent with its lower IC50 on Daudi cells in vitro. We further show that the antitumor activity of a mixture of HD37-dgA and RFB4-dgA is significantly enhanced in SCID/Daudi mice and is consistent with the killing in excess of 5 logs of tumor cells. However, identical enhancement was observed when a mixture of the RFB4-dgA and the HD37 antibody was administered. In contrast, enhancement was not observed when mice were injected with a mixture of the RFB4 antibody and the HD37-dgA. The results indicate that a "cocktail" of HD37 antibody and RFB4-dgA immunotoxin can have significant antitumor activity in this mouse model of lymphoma and suggest that combinations of particular antibodies and ITs may have cooperative antitumor activity.

Animals↗

PubMed1991

Antitumor activity of Fab' and IgG-anti-CD22 immunotoxins in disseminated human B lymphoma grown in mice with severe combined immunodeficiency disease: effect on tumor cells in extranodal sites.

The antitumor effects of two anti-CD22 ricin A chain-containing immunotoxin (IT) constructs were compared in mice with severe combined immunodeficiency disease with human Daudi cell tumors (SCID-Daudi mice). SCID-Daudi mice develop disseminated lymphoma that clinically resembles African Burkitt's lymphoma, i.e., extranodal disease including infiltration of the vertebral column and spinal canal. In the absence of treatment, the mean survival time of SCID-Daudi mice was 45.9 +/- 4.3 days. The mice was given injections of a dose of IT equal to 40% of the 50% lethal dose. The ITs consisted of either IgG or Fab' fragments of mouse anti-CD22 antibody coupled to deglycosylated ricin A chain (dgA). Both ITs were potent and specific and inhibited protein synthesis in Daudi cells in vitro by 50% at concentrations of 1.2 x 10(-12) (IgG-dgA) and 1.3 x 10(-11) M (Fab'-dgA). When administered to mice beginning 1 day after inoculation with tumor cells, both ITs extended the mean survival time, to 87.2 +/- 18.9 days (IgG-dgA) or 57.9 +/- 3.8 days (Fab'-dgA). The latter represented the killing of 2 logs of Daudi cells, and the former 4 logs. IgG antibody alone killed 1 log of tumor cells. The IgG-dgA had an antitumor effect even when administered 20-23 days after tumor inoculation. Gross and histological examinations of IT-treated tumor-bearing mice showed a marked decrease in the number and size of neoplastic foci in both lymphoid organs and extranodal sites.

Animals↗

PubMed1991

The GLP large scale preparation of immunotoxins containing deglycosylated ricin A chain and a hindered disulfide bond.

The large scale preparation of two second generation immunotoxins containing murine monoclonal antibodies and deglycosylated ricin A chain is described. The procedure for the preparation of immunotoxins consists of the derivatization of antibody with SMPT and reduction of dgA with DTT followed by their reaction to establish a hindered interchain disulfide bond. The purification of the immunotoxin includes affinity chromatography on Blue-Sepharose to remove the free antibody and gel filtration on Sephacryl S-200HR to remove any high molecular weight material and free dgA. The two immunotoxins were prepared by GLP procedures and tested for yield, composition, purity, sterility and biological activity.

Antibodies, Monoclonal↗

PubMed1991

Covalent binding of human alpha 2-macroglobulin to deglycosylated ricin A chain and its immunotoxins.

In this report we demonstrated that human alpha 2-macroglobulin (alpha 2M) reacts with deglycosylated ricin A chain (dgA) and its immunotoxins to form high molecular weight complexes (molecular mass approximately 800 kDa). This interaction has a t1/2 at 37 degrees C of 5 h and reaches completion at 24 h. Complexes of alpha 2M-dgA cannot be dissociated by guanidine, sodium dodecyl sulfate, or low pH, but can be partially dissociated by reducing agents, such as 2-mercaptoethanol in the presence of sodium dodecyl sulfate. This indicates that dgA or dgA-containing immunotoxins are bound to alpha 2M by disulfide bonds. The dgA-binding site on alpha 2M and the mechanism underlying its interaction with dgA are different from those described for proteases or methylamine. alpha 2M complexes do not bind to Blue-Sepharose 4B or anti-A chain-Sepharose, suggesting that the sites on dgA which bind Cibacron Blue or polyclonal anti-A chain antibodies are sterically blocked or modified by interaction with alpha 2M. The interaction of alpha 2M with dgA or its immunotoxins results in a 2- to 3-fold decrease in the activity of the dgA in both cell-free assays and cytotoxic assays. However 12 h after injection into mice, only 11% of immunotoxin was bound to alpha 2M because of the slow kinetics of the interaction versus the more rapid t1/2 of the immunotoxin in the circulation.

Animals↗

PubMed1990

Disseminated or localized growth of a human B-cell tumor (Daudi) in SCID mice.

A human Burkitt lymphoma (Daudi) has been grown in the mutant mouse called C.B-17 SCID. Twenty-eight days after s.c. injection of Daudi cells, a palpable tumor grew only at the site of injection in all injected mice. In contrast, after intravenous (i.v.) or intraperitoneal (i.p.) injection, macroscopic, disseminated tumors developed. Following i.v. inoculation, tumors grew in the lungs, kidneys, ovaries and adipose tissue, and microscopic tumor infiltrates were observed in the spleen, bone marrow, spinal column and femur, whereas after i.p. injection, the tumors were localized in the abdomen, liver, spleen, ovaries and muscular tunics of the gut, but did not disseminate into the lung or bone marrow. The growth pattern and phenotype of the Daudi cells were similar whether the inoculated tumor cells were derived from the in vitro cell line or from in vivo passaged tumors. The survival time of the tumor-bearing animals was dependent on the dose of i.v.-administered Daudi cells; as few as 100 cells caused death. All mice injected i.v. showed paresis or paralysis of the hind legs just prior to death. This was associated with the presence of neoplastic nodules within the spinal canal. Two surface antigens on Daudi cells (CD19 and CD22) were stably expressed in all the neoplastic lesions. Radiolabelled anti-CD22 antibodies localized in organs infiltrated with tumor, but did not penetrate primary s.c. tumors. This model of disseminated vs. solid tumor should prove useful for evaluating the efficacy of different types and doses of therapeutic antibodies, immunoconjugates and immunotoxins prepared from anti-human B-cell antibodies.

Animals↗

PubMed1990

Large scale preparation of an immunoconjugate constructed with human recombinant CD4 and deglycosylated ricin A chain.

A method for the preparation and purification of large amounts (grams) of a conjugate containing recombinant CD4 antigen (rCD4) and chemically deglycosylated ricin A chain (dgA) is described. The cross-linking of rCD4 and dgA molecules was accomplished with N-succinimidyl-oxycarbonyl-alpha-methyl-(2-pyridyldithio)toluene (SMPT). The rCD4-dgA conjugate was purified by an automatic liquid chromatography system consisting of Blue-Sepharose CL-4B and Sephacryl S-200HR Pharmacia Bioprocess columns. The purified, endotoxin-free rCD4-dgA conjugate had a stable (hindered) disfulfide bond between rCD4 and dgA and was able to efficiently kill a human T cell line infected with HIV-1.

CD4 Antigens↗

PubMed1989

The epitope specificity and tissue reactivity of four murine monoclonal anti-CD22 antibodies.

The CD22 antigen is expressed on the surface of normal human B cells and some neoplastic B cell lines and tumors. Previous cross-blocking studies using a panel of monoclonal anti-CD22 antibodies have defined four epitope groups, termed A-D. In the present studies, we have further dissected the epitopes recognized by four monoclonal anti-CD22 antibodies using immunoprecipitation and cross-blocking techniques, immunofluorescence analyses with a variety of cell lines, and immunoperoxidase analyses of 36 normal human tissues. Two of the antibodies, HD6 and RFB4, have been described previously, and two, UV22-1 and UV22-2, are described in this report. Our studies indicate that the four monoclonal antibodies show unexpected complexities in their reactivity with CD22+ and CD22- cells and their reactivity with solubilized CD22 molecules. The four antibodies, which recognize epitopes defined previously as CD22-A and CD22-B, further subdivide these epitope clusters into four determinants, A1, A2, B1, and B2. Furthermore, only two of the antibodies, RFB4 and UV22-2, are B cell-specific. In summary, our data indicate that RFB4 and UV22-2 would be the antibodies of choice for constructing immunotoxins to treat B cell tumors.

Animals↗

PubMed1988

Evaluation of four CD22 antibodies as ricin A chain-containing immunotoxins for the in vivo therapy of human B-cell leukemias and lymphomas.

Ricin A chain-containing immunotoxins (IT-As) specific for the human B-cell antigen, CD22, were prepared from 4 monoclonal antibodies (MAbs) or their Fab' fragments: RFB4, HD6, UV22-I and UV22-2. The ITs were tested for their ability to kill cells from the Burkitt lymphoma line, Daudi, the pre-B-cell leukemia line, NALM-6, and the myeloma cell line, ARH-77. Daudi expresses high levels of CD22, whereas NALM-6 and ARH-77 express low levels of CD22. The IgG-RFB4-A was highly toxic to all 3 cell lines; it killed 50% of the Daudi cells at a concentration of 1.2 x 10(-12) M and 50% of NALM-6 and ARH-77 cells at concentrations of 1.5 to 2.1 x 10(-11) M. IgG-RFB4-A was 10-30 times more toxic to Daudi cells than were the IgG-As constructed from the other 3 CD22 MAbs and 10 times more toxic than ricin itself. IT-As constructed from the Fab' fragments of the 4 CD22 antibodies were 2 to 5 times less toxic to Daudi cells than their IgG-A counterparts. Fab'-RFB4-A was twice as toxic to Daudi cells as ricin, whereas the other Fab'-As were about 7 times less toxic than ricin. Scatchard analyses of the binding of the radio-iodinated antibodies to Daudi cells showed that the intact RFB4 antibody bound 3-10 times more strongly than the other antibodies, whereas the Fab'-RFB4 bound 1.2 to 3.5 times more strongly than the Fab' fragments prepared from the other antibodies. Thus, the potent cytotoxic activity of the RFB4-As appears to derive, in part, from their superior binding affinity. Prior studies have shown that UV22-I and HD6 cross-react with certain normal human tissues lacking cells of B-cell lineage, whereas UV22-2 and RFB4 are B-cell-specific. This fact, together with its superior potency as an IT-A, suggests that RFB4 is the antibody of choice for preparing Fab'-As or IgG-As for in vivo therapy of human B-cell leukemias and lymphomas.

Animals↗

PubMed1988

Large scale preparation of immunotoxins constructed with the Fab' fragment of IgG1 murine monoclonal antibodies and chemically deglycosylated ricin A chain.

In this report, we describe a method for the preparation of large amounts (grams) of immunotoxins (ITs) consisting of Fab' fragments of murine IgG1 monoclonal antibodies conjugated to chemically deglycosylated ricin A chain (dgA). The preparation of Fab' and dgA chain and the purification of the Fab'-dgA IT were accomplished by gel filtrations and affinity chromatography utilizing six Pharmacia Bioprocess columns (Sephadex G-25M, Sephacryl S-200HR and Blue Sepharose CL-4B) integrated into a semi-automatic chromatography system controlled by a Pharmacia C3-process controller. The final Fab'-dgA ITs were highly purified, potent, sterile and low in endotoxin concentration.

Antibodies, Monoclonal↗

PubMed1988

Evaluation of ricin A chain-containing immunotoxins directed against CD19 and CD22 antigens on normal and malignant human B-cells as potential reagents for in vivo therapy.

Ricin A chain-containing immunotoxins (IT-As) specific for the human B-cell antigens, CD22 and CD19, were constructed using the monoclonal antibodies, HD6 and HD37, respectively. IT-As were prepared by coupling intact antibodies, F(ab')2, or Fab' fragments to native or chemically deglycosylated ricin A chain. The IT-As were then evaluated for cytotoxicity to normal and neoplastic human B-cells in vitro with the major objective of appraising their suitability for in vivo therapy of human B-cell tumors. The IT-As prepared with both the HD6 and HD37 antibodies were specifically toxic to normal B-cells and to most of the neoplastic B-cell lines tested. However, the IT-As prepared from HD6 were generally more potent than those prepared from HD37. On Daudi cells, to which the two antibodies bound in similar numbers and with similar affinities, IT-As prepared with intact HD6 antibody or its Fab' fragment were 10-fold and 1.5- to 4-fold more potent, respectively, than the corresponding HD37 IT-As. The IT-As constructed from intact HD6 antibody and native or deglycosylated A chain reduced protein synthesis in Daudi cells by 50% at a concentration of 1.2 X 10(-11) M indicating that they were only 5-fold less toxic to the cells than ricin itself. Intact HD37 IT-As produced equivalent inhibition of protein synthesis at 1.5 X 10(-10) M. With both antibodies, IT-As constructed from the Fab' fragments were 10- to 20-fold less potent than their intact antibody counterparts. Different neoplastic B-cell lines varied in sensitivity to the IT-As. In most cases, their sensitivity correlated with the levels of CD19 and CD22 antigens expressed. Neither HD6 nor HD37 IT-As affected the ability of normal human bone marrow cells to form granulocyte-macrophage colony-forming units in soft agar, suggesting that both antigens are absent from these progenitor cells. Examination of sections of frozen human tissues using immunoperoxidase staining procedures indicated that the antibodies did not bind to a panel of normal tissues lacking B-lymphocytes. These results suggest that HD6 and HD37 IT-As are candidates for in vivo therapy in humans with certain B-cell tumors. However, HD6 IT-As are more potent, reduce protein synthesis more completely, and hence appear to be the ITs of choice for treating tumors expressing the CD22 antigen.

Antigens, CD↗

PubMed1988

Protein A vectorized toxins--II. Preparation and "in vitro" cytotoxic effect of protein A-ricin A chain conjugate on antibody coated human tumour cells.

Protein A of Staphylococcus aureus was covalently bound to reduced ricin A chain toxin by N-succinimidyl 3-(2-pyridyldithio)propionate. The conjugate consisting mainly of one molecule of protein A bound to two molecules of A chains (Mr 107,000) was purified by tandem affinity chromatography on ConA-Sepharose 4B and IgG-Sepharose 4B. The purified protein A-A chain conjugate was able to bind and kill human lymphoma cells coated either with monoclonal mouse IgG2a anti-kappa antibody or with polyclonal rabbit anti-kappa antibody. The cytotoxic activity of protein A-A chain conjugate in conjunction with either mouse or rabbit anti-kappa antibodies was 10 times higher than that of rabbit IgG anti-mouse IgG coupled with A chain on Daudi cells coated with mouse anti-kappa antibody and 100 times higher than that of rabbit anti-kappa antibody coupled with A chain on non-coated Daudi cells. The cytotoxic effect of protein A-A chain conjugate on antibody-coated Daudi cells (9 x 10(-12) M) was comparable with that of ricin toxin on non-coated Daudi cells (2 x 10(-12) M). The results recommend the use of protein A-ricin A chain toxin conjugate as a unique specific toxin for the "in vitro" killing of antibody-coated target cells.

Burkitt Lymphoma↗

PubMed1986

Interaction between rabbit IgG immune complexes and Ricinus agglutinin.

Ricinus communis agglutinin covalently bound to Sepharose 4B (RcA-Sepharose 4B) was able to selectively bind immune complexes consisting of antigen (bovine serum albumin) and rabbit IgG antibody which were rich in antibody (i.e., high molecular weight complexes of approximately 2000 000). No interaction was recorded between RcA-Sepharose 4B and immune complexes consisting of the antigen plus rabbit IgG antibody which were rich in antigen (i.e., complexes with a molecular weight of 300 000-500 000). These results indicate that binding to RcA is due to the increased density of galactose residues in the high molecular weight polymeric antibody component of the soluble immune complexes.

Animals↗