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Biomedical subjects

J W Uhr

Publications and source records attributed to J W Uhr.

At least 19 recordsLinked to original sources
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↗
PubMed1992

The toxicity of chemically deglycosylated ricin A-chain in mice.

Tumor-reactive antibodies coupled to ricin or its A-chain (immunotoxins) have been used in rodents and humans to treat a variety of neoplastic diseases. Side-effects of such treatment include hepatotoxicity, vascular leak syndrome, myalgia and low grade fever. At high doses, severe toxicities include liver damage, pulmonary edema, aphasia, rhabdomyolysis and kidney failure. There have been a limited number of toxicologic studies on uncoupled ricin or its A-chain and none on deglycosylated A-chain. Since the latter has been utilized in "second generation" immunotoxins, the current studies were carried out to evaluate the toxicities induced by deglycosylated ricin A-chain (dgA) in mice. The administration of dgA to normal BALB/c mice causes early (24 h) weight loss and late (10 day) accumulation of ascites. These effects could be partially altered by changing the route of injection of dgA from i.v. to i.p. Thus, i.p. administration caused weight loss but not ascites, whereas i.v. administration caused both. Weight loss was associated with reduced fluid intake by the treated mice, and was not associated with increased levels of serum TNF-alpha. SCID mice injected with the same dose of dgA as normal BALB/c mice developed ascites, but it was of lesser severity, suggesting that a functional immune system, differences in microbial flora, or strain differences may be involved in the development of ascites.

Animals↗
PubMed1992

A CD4-derived peptide carrier blocks acute HIV-1 infection in vitro and binds to gp120 in the presence of Walter-Reed stage 1-6 HIV+ sera.

A peptide containing amino acid residues 41-84 of the CD4 molecule was synthesized and coupled through a thioether bond to human serum albumin. This conjugate bound to gp120 with an affinity that was half that of CD4 and blocked the HIV infection in vitro with an efficacy tenfold lower than that of CD4. More importantly, the CD4 peptide-human serum albumin conjugate could bind to gp120 in the presence of HIV+ sera from 18 Walter Reed stage 1-6 patients.

Acquired Immunodeficiency Syndrome↗
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

Cancer dormancy: studies of the murine BCL1 lymphoma.

Dormancy in the murine BCL1 lymphoma can be induced by several strategies including cytoreductive therapy of mice with large tumor burdens and challenge of allogeneic chimeric mice or idiotype-immunized mice with BCL1 tumor. Dormant tumor cells were isolated from the spleens of the chimeric mice and the majority were shown to be noncycling. In idiotype-immunized mice that had lost dormancy, tumor growth occurred at a relatively rapid rate. A proportion of idiotype-immunized mice that had lost dormancy spontaneously regressed and then again relapsed; in these mice, the serum antiidiotypic levels were inversely related to the tumor burden.

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

CD4 peptide-protein conjugates, but not recombinant human CD4, bind to recombinant gp120 from the human immunodeficiency virus in the presence of serum from AIDS patients.

Sera from human immunodeficiency virus-positive (HIV+; Walter Reed stage 6) individuals inhibit the interaction between recombinant human CD4 and recombinant gp120 from HIV (rCD4 and rgp120, respectively), thereby interfering with the ability of soluble rCD4 to block infection with HIV or rCD4-toxin conjugates to kill HIV-infected cells. In this report we demonstrate that the inhibitory activity of such sera is caused primarily by anti-gp120 antibodies that do not recognize the CD4 interaction site on gp120. To circumvent the problem of inhibition, we have generated a construct containing a peptide of CD4 (residues 41-84) conjugated to ovalbumin (three to five peptides per molecule). This multivalent conjugate binds to rgp120 and binding is not inhibited by antibodies in HIV+ sera.

Acquired Immunodeficiency Syndrome↗
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↗
PubMed1991

Intracellular routing rather than cross-linking or rate of internalization determines the potency of immunotoxins directed against different epitopes of sIgD on murine B cells.

Several variables influence the potency of an immunotoxin (IT) prepared with a monoclonal antibody (mAb) and ricin A chain (IT-A). These include the affinity of the mAb, the nature and density of the target antigen (Ag), the epitope on the target Ag bound by the mAb, the type of cell target, and the rate of endocytosis and route of internalization of the bound IT-A. In a previous report, we demonstrated that anti-delta mAbs directed against epitopes which are putatively more proximal to the plasma membrane make more effective IT-As than those directed against epitopes that are putatively more distal from the plasma membrane. It is known that the latter mAbs cross-link sIgD less effectively than the former. Therefore, in the present study, we determined whether the differential cytotoxicity of IT-As directed against these epitopes is related to their ability to cross-link their specific surface antigen (sIgD). We further determined whether they were internalized at different rates by normal B cells. Our results show that neither cross-linking nor rate of internalization account for the different potencies of anti-Fc vs anti-Fd IT-As. However, when these IT-As were used in the presence of the lysosomotropic agent chloroquine, the less potent IT-A became 100-fold more potent and was as cytotoxic as the effective anti-Fc IT-A. Taken together with the results of other studies, these findings further support the hypothesis that the epitope specificity of a given mAb may be an important factor in determining the intracellular routing of an IT-A after internalization.

Animals↗
PubMed1990

Evaluation of ricin A chain-containing immunotoxins directed against different epitopes on the delta-chain of cell surface-associated IgD on murine B cells.

Over the past decade, immunotoxins (IT) composed of mAb covalently coupled to toxins or their subunits have been developed for the treatment of malignancies and autoimmune diseases. Despite specific binding to target cells, not every mAb makes a therapeutically potent ricin A chain-containing IT (IT-A). A number of variables influence the potency of a mAb as an IT-A, including the affinity of the mAb, the nature and density of the cell surface Ag, and the type of target cell used. The present report investigates the influence of the epitope specificity of a mAb on the effectiveness of that mAb as an IT-A. Seven mAb directed against different regions of the mouse delta H chain of surface IgD, were conjugated to deglycosylated ricin A chain, and tested for their ability to kill murine B cells. The panel of IT-A had similar A chain activities and similar binding avidities. However, the mAb directed against epitopes in the Fc portion of surface IgD made more effective IT-A than those directed against epitopes in the Fd region. Overall, the anti-Fc-A were approximately 60- to 150-fold more toxic than the anti-Fd-A. Taken together with previous studies, these findings suggest that the epitope on a target Ag recognized by a given mAb is an important variable in determining the potency of a mAb as an IT-A.

Antibodies, Monoclonal↗
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↗
PubMed1990

Immunoconjugates containing ricin A chain and either human anti-gp41 or CD4 kill H9 cells infected with different isolates of HIV, but do not inhibit normal T or B cell function.

We have previously reported that immunoconjugates composed of deglycosylated ricin A chain coupled to either recombinant (r) CD4 or two different monoclonal human anti-gp41 antibodies (rCD4-dgA and anti-gp41-dgA, respectively) are specifically toxic to HIV-infected lines of human T cells (H9) and monocytes (U937). In order to further evaluate these immunoconjugates as potential therapeutic reagents for killing HIV-infected cells, H9 cells infected with five different isolates of HIV were used as target cells in vitro. All three HIV-specific immunoconjugates were toxic to H9 cells infected with each HIV isolate, but were virtually nontoxic to uninfected cells. Chloroquine markedly potentiated the specific toxicity of all three conjugates, particularly the anti-gp41-dgAs. None of the conjugates affected the ability of normal peripheral blood B cells to respond to mitogen or the ability of normal T cells to respond to alloantigens.

Antibodies, Monoclonal↗
PubMed1989

IL-4 induces the specific rearrangement of gamma 1 genes on the expressed and unexpressed chromosomes of lipopolysaccharide-activated normal murine B cells.

Small, resting, surface IgM+/surface IgD+ murine B cells undergo an Ig class switch to IgG1 or IgE after stimulation with LPS and T cell supernatants containing IL-4. To firmly establish the role of IL-4 in the directed switch recombination observed in IgG1-secreting cells, we have 1) used highly purified native IL-4 instead of T cell supernatants, 2) used resting B cells from F1 mice in which the active IgH allele was determined before culture, 3) taken advantage of the allelic differences in the restriction fragment lengths of mu, gamma 1, gamma 2b, and gamma 3 loci to determine the status of the CH genes on both the expressed and unexpressed chromosomes, and 4) used different restriction enzymes to distinguish between deletion and rearrangement of a given CH gene. Our results indicate that LPS alone induces rearrangement of the gamma 3 genes on both chromosomes whereas stimulation with LPS plus IL-4 results in deletion of gamma 3 genes and rearrangement of gamma 1 genes on both chromosomes. The studies definitively establish the role of IL-4 in directed switch recombination to the gamma 1 locus in LPS-stimulated murine B cells.

Animals↗
PubMed1989

In vivo administration of Fab' fragments of anti-L3T4 (GK1.5) antibody inhibits the T helper cell function of murine lymph node cells.

The effect of intravenous injection of Fab' fragments of anti-L3T4 antibody (GK1.5 monoclonal antibody) into mice was studied. This treatment depleted L3T4+ cells from the popliteal lymph nodes of keyhole-limpet hemocyanin-primed mice. The T cells that remained were unable to provide help to antigen-specific B cells in vitro. The results obtained using Fab' fragments were comparable with those using intact anti-L3T4 antibody and demonstrate that either form of GK1.5 is a potentially useful immunosuppressive agent in mice.

Animals↗
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↗
PubMed1989

Cross-linking of interleukin 4 to surface molecules on murine T and B lymphocytes.

Interleukin 4 (IL-4) is a T-cell derived lymphokine with multiple activities on a variety of cells. An intriguing feature of the different IL-4-mediated activities is their requirements for markedly different concentrations of IL-4 even on the same cell population. To gain some insight into the phenomenon, we have analyzed the structure of IL-4-binding proteins on T and B cells by cross-linking different concentrations of 125I-labeled IL-4 (125I-IL-4) to cells. Cross-linking of 125I-IL-4 at relatively low concentrations in the presence of the cross-linking agent 3,3'-dithiobis(propionic acid hydroxysuccinimide ester) resulted in the detection of the previously described 60- to 75-kDa protein. At higher 125I-IL-4 concentrations, however, an additional molecule of 105 kDa was observed. Cross-linking of 125I-IL-4 to both molecules was specific; it was inhibited by the presence of a 50-fold excess of either unlabeled IL-4 or monoclonal anti-IL-4 antibody. Furthermore, considerable size heterogeneity of the IL-4-binding proteins was evident in different cell populations. These results suggest that IL-4 receptors might have a more complex structure than originally reported and/or that high concentrations of IL-4 might induce interactions of the previously described IL-4 receptor (60-75 kDa) with a 105-kDa molecule. Hence, it is possible that IL-4 might generate a different signal at high concentrations through interaction of its receptor with another membrane molecule.

Animals↗