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Biomedical subjects

A C Morgan

Publications and source records attributed to A C Morgan.

At least 37 records · Page 2Linked to original sources
PubMed1989

Murine monoclonal IgG3 to human colorectal tumor-associated antigens: enhancement of antibody-dependent cell-mediated cytotoxicity by interleukin 2.

Murine monoclonal antibodies (MAB) of the IgG3 subclass generated to colorectal tumor-associated glycolipids were assessed for enhancement of antibody-dependent cell-mediated cytotoxicity (ADCC) by interleukin 2 (IL-2). Mononuclear cell preparations containing large granular lymphocyte effectors required only low doses of IL-2 (0.1-10 units/ml) and short exposure (3 h) for maximal enhancement of ADCC. Exposure of mononuclear cells for longer periods (1-6 days) to higher levels of IL-2 (100-1000 units/ml) resulted in the generation of lymphokine-activated killer N cells which were lytically active without antibody. A non-ADCC MAB of the IgG1 subclass showed no ADCC even after stimulation of effector cells with IL-2. Effector cells pretreated with IL-2 showed an enhanced rate of cytolysis of target cells in the presence of antibody. Pretreatment of effector cells with IgG3 MAB resulted in lower ADCC, but pretreatment of target cells or simultaneous addition of MAB and effectors to target cells gave higher levels. These results indicated that ADCC with murine IgG3 is enhanced by levels of IL-2 achievable in current patient trials. The combination of antibody and IL-2-boosted effector cells gives comparable levels of killing to lymphokine-activated killer cells but should not suffer from similar toxicity. The NR-Co-04 antibody in combination with lymphokine may prove effective in treating colon cancer, a disease for which both chemotherapeutic and current biological therapies suggest a need for improved forms of therapies.

Animals↗
PubMed1989

Macrophage colony-stimulating factor enhances monocyte and macrophage antibody-dependent cell-mediated cytotoxicity.

In vitro culture of either human peripheral blood monocytes or murine peritoneal macrophages for 72 hr in the presence of macrophage colony-stimulating factor (M-CSF) dramatically increased their subsequent ability to mediate antibody-dependent cellular cytotoxicity (ADCC). The M-CSF-treated cells were more effective in ADCC at lower effector to target cell ratios and in the presence of lower concentrations of tumor-specific monoclonal antibody than the untreated control cells. Two other hematopoietic cytokines, granulocyte-macrophage colony-stimulating factor and interleukin-3, reported to enhance other macrophage effector functions were ineffective in promoting the development of ADCC by cultured human monocytes. All three hematopoietic growth factors were capable of enhancing the ability of the cultured monocytes to secrete TNF alpha; however, TNF alpha is unlikely to be an important cytotoxic factor in ADCC because neutralizing antibodies against TNF alpha had no affect on ADCC in vitro. Further, much higher concentrations of M-CSF were required to augment monocyte TNF alpha release (20-100 ng/ml) than ADCC capacity (1-10 ng/ml). These results suggest that M-CSF administration might prove effective in increasing the tumoricidal activities of tumor-specific monoclonal antibodies by enhancing the capacity of monocytes and macrophages to mediate ADCC.

Animals↗
PubMed1989

Characterization of a human-mouse chimeric antibody reactive with a human melanoma associated antigen.

The data presented demonstrate that a human-murine chimeric antibody has been generated that retains the immunoreactivity and has similar pharmacokinetic properties of its parent murine monoclonal antibody NRML-05. A competitive ELISA assay demonstrated that antigen reactivity of both parent and chimera were nearly identical. In a direct cell binding assay, NRML-05 and chimeric antibodies were immunoreactive, 81% and 83%, respectively. Scatchard Analysis of the antibodies indicate very similar affinities for NRML-05 (4.4 x 10(9) M-1) and chimera (2.7 x 10(9) M-1). Localization of the two antibodies to tumor xenografts in nude mice were very similar in biodistribution studies. The chimeric antibody is not as well recognized by antiglobulin from patients who have responded to non-idiotypic murine antibody determinants. Although this data does not predict how immunogenic a chimera would be in the clinical setting, it does suggest that patients without significant anti-idiotypic antiglobulin response could benefit from second and subsequent administrations with chimeric antibody. Even though anti-idiotypic responses may occur with the chimera, these may be reduced as a result of the presentation of these epitopes on the less immunogenic human constant domains.

Animals↗
PubMed1989

Successful imaging of malignant melanoma with technetium-99m-labeled monoclonal antibodies.

F(ab')2 and Fab fragments of murine monoclonal antibody 9.2.27, that recognizes the 250 kD melanoma-associated antigen, were labeled with 99mTc using the bifunctional chelate method of Fritzberg et al. Twenty-seven (27) patients received, intravenously, 10 mg of either F(ab')2 (8), or the Fab (27), labeled with up to 30 mCi of 99mTc. These doses were preceded by an infusion of cold irrelevant antibody. The average serum T1/2 of the F(ab')2 and the Fab were 11 hr and 2 hr, respectively. Twenty-two percent (22%) of the total injected F(ab')2 dose was excreted in the urine in 20 hr, compared to 55% for the Fab group. Imaging was optimal 6-9 hr postinjection for the Fab patients. No nonspecific uptake in liver, spleen, bone marrow, or lung was observed for either antibody form. Overall, (43/53) 81% of known metastases were seen with visualization of tumors as small as 250 mg and tumor localization as high as 0.03% injected dose/g. Immunoperoxidase staining of freshly-frozen tumor nodules removed 24 hr postinjection confirmed antibody deposition in the tumor. Thirty-six previously unknown ("occult") metastatic sites were detected. To date, 12/36 of these sites have been confirmed. We conclude that 99mTc-labeled antibody to melanoma produces high resolution images with a high sensitivity of detecting metastatic melanoma. The detection of previously unknown sites of disease has proven helpful in directing additional diagnostic studies (i.e., CT) as well as planning of therapeutic options.

Antibodies, Monoclonal↗
PubMed1989

Development of a stable radioiodinating reagent to label monoclonal antibodies for radiotherapy of cancer.

A method of radioiodinating monoclonal antibodies such that the labeled antibodies do not undergo in vivo deiodination has been studied. The method utilizes conjugation of succinimidyl para-iodobenzoate to the antibody. The iodobenzoate was radiolabeled by using an organometallic intermediate to facilitate the reaction. Thus, succinimidyl para-tri-n-butylstannylbenzoate was radiolabeled in 60-90% radiochemical yield and subsequently conjugated to the antibody in 80-90% yield. Animal biodistribution studies were carried out with two separate anti-melanoma antibodies (9.2.27 and NR-M1-05) labeled by this method, and examined in nude mice bearing human melanoma tumor xenografts. Very large differences in the localization of radioactivity were observed in the thyroids and stomachs of mice when the iodobenzoyl-labeled antibodies were compared with the same antibodies labeled using the chloramine-T method of radioiodination. Few other significant differences in the tissue distribution of the radioiodinated antibodies were seen.

Animals↗
PubMed1988

Specific and stable labeling of antibodies with technetium-99m with a diamide dithiolate chelating agent.

Technetium-99m labeling of antibodies has been suboptimal because of low affinity adventitious binding, nonspecific labeling, and loss of immunoreactivity. The diamide dithiolate ligand system (N2S2) forms highly stable, well-defined tetradentate complexes with Tc(V). Antibodies and their fragments have been labeled by conjugation of preformed 99mTc-4,5-bis(thioacetamido)pentanoate active ester to protein amine groups to give a chemically known 99mTc-N2S2 complex covalently linked to antibody. Evaluations of the 99mTc-N2S2-bound antibodies and their fragments have shown high stability and retained immunoreactivity.

Animals↗
PubMed1988

Effect of antibody dose on the imaging and biodistribution of indium-111 9.2.27 anti-melanoma monoclonal antibody.

Eleven patients with metastatic melanoma underwent serial gamma camera imaging and biodistribution measurements after i.v. injection of escalating doses of [111In]9.2.27, an antimelanoma murine monoclonal antibody. Patients received a fixed dose of 1 mg of [111In]9.2.27, with no additional 9.2.27 (five patients), or co-infused with 49 mg (five patients) or 99 mg (one patient) of unlabeled, unconjugated 9.2.27. Higher doses resulted in prolonged blood-pool retention, less uptake in spleen and bone marrow, and appeared to have a positive effect in improving tumor imaging. A dose of 1 mg of 9.2.27 permitted detection of tumors in two of five patients and two of ten lesions, while with greater than or equal to 50 mg, tumors were detected in all patients and in 24 of 32 lesions. Human gamma globulin injected prior to administration of [111In]9.2.27 failed to block the prominent liver, spleen, and bone marrow uptake. No toxicity was observed. These results indicate the feasibility of imaging metastatic melanoma with [111In]9.2.27 and suggest that antibody dose may be a critical determinant of biodistribution and tumor uptake.

Antibodies, Monoclonal↗
PubMed1987

Antibody conjugates with cobra venom factor. Synthesis and biochemical characterization.

Immunoconjugates are semi-synthetic hybrid proteins which bear great promise to become a new generation of anti-tumor agents. While many immunoconjugates have been shown to be selectively cytotoxic in in vitro model systems, dramatic in vivo anti-tumor effects have not been reported. To improve the activity of immunoconjugates, careful structure-function analyses have to be performed. We report here such an analysis for immunoconjugates consisting of a monoclonal anti-tumor antibody (MoAb) and cobra venom factor (CVF), the complement-activating glycoprotein from cobra venom, synthesized with the heterobifunctional crosslinking reagent N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP). It is shown that a reaction mixture after protein coupling contains free MoAb and CVF as well as hybrid proteins of different compositions (dimers (MoAb-CVF), trimers (MoAb2-CVF, MoAb-CVF2), tetramers (MoAb-CVF3, MoAb2-CVF2, MoAb3-CVF), and some higher oligomers). While free MoAb and CVF can be removed by size exclusion chromatography, separation of different oligomeric hybrid proteins is not possible by this method. From the biochemical characterization of the hybrid proteins, which included the determination of sedimentation coefficients, recording of circular dichroism spectra with subsequent determination of secondary structure, and ultrastructural analysis by transmission electron microscopy, it was concluded that the two proteins do not undergo major structural changes upon coupling, and that the coupling of the two proteins is random with no preferential relative orientation. The functional inactivation of CVF was substantial (approximately 70%) due to both derivatization with SPDP and subsequent conjugation to the MoAb, with conjugation being relatively more inactivating than derivatization. In contrast, the binding activity of the antibody was far less susceptible to inactivation. In conclusion, our data indicate that immunoconjugate synthesis with heterobifunctional crosslinking reagents results in a mixture of heterogeneous hybrid proteins and causes substantial functional inactivation. For successful in vivo anti-tumor activity of future immunoconjugates with CVF and other protein ligands better methods for immunoconjugate synthesis will have to be developed.

Antibodies, Neoplasm↗
PubMed1987

Analysis of rat natural killer cytotoxic factor (NKCF) produced by rat NK cell lines and the production of a murine monoclonal antibody that neutralizes NKCF.

Natural killer cytotoxic factor (NKCF) is produced as a result of the interaction of murine, rat, or human natural killer (NK) cells with NK-susceptible targets. This factor has been linked to the target cell lysis mediated by the NK effector cell. In the present results, culture supernatants from rat large granular lymphocyte (LGL) tumors exhibited NKCF activity which lysed the susceptible targets, MBL-2 and YAC-1. NKCF production from these rat tumor lines was spontaneous and was not significantly increased by co-incubation of the LGL tumors with target cells, target cell membranes, or by preincubation of the LGL tumor cells with interferon or interleukin 2. In addition to NKCF activity, the supernatants lysed L929, indicating the presence of tumor necrosis factor (TNF) in these preparations. The presence of this latter cytokine was verified using specific antibodies to recombinant murine TNF which neutralized the L929 activity while not affecting the NKCF activity against MBL-2 or YAC-1. Mouse monoclonal antibodies (mAb) A0287, A0462, and A0316) which significantly inhibit the NKCF cytolytic activity of these LGL-derived supernatants were also produced. These antibodies were shown to cross-react with human NKCF in a manner similar to that seen in the rat. Interestingly these same mAb demonstrated no inhibition of L929 cytotoxicity from either LGL-derived supernatants or by recombinant murine or human TNF. To examine further the specificity of these antibodies, they were chemically linked to Sepharose 4B and found to remove a significant proportion of the NKCF cytolytic activity from LGL supernatants, while not affecting the TNF reactivities in these preparations. In addition, these antibodies demonstrated significant inhibition of cell-mediated cytotoxicity by rat LGL against YAC-1 target cells. Biochemical analysis of labeled NKCF-containing supernatants indicated the major protein recognized by these anti-NKCF mAb to be approximately 12,000 m.w. The use of these mAb against NKCF should be very useful in further purification and biochemical characterization of NKCF and in studying its role in a variety of cell-mediated cytotoxicity assays.

Animals↗
PubMed1987

Immunotoxins to a human melanoma-associated antigen: comparison of gelonin with ricin and other A chain conjugates.

Gelonin, a ribosome-inactivating protein from the seeds of Gelonium multiflorum, has been conjugated to antibodies. Previous reports have indicated variable potency of such immunotoxins. The lack of toxicity of gelonin, however, makes it attractive for immunoconjugate production. The ribosome-inactivating protein was covalently linked (using N-succinimidyl-3-(2-pyridyldithio)propionate) to monoclonal antibody, 9.2.27, directed to a human melanoma-associated glycoprotein/proteoglycan. The immunoconjugate showed high selectivity with dose-dependent cytotoxic activity to cultured human melanoma cells (50% inhibitory dose; 1-3 X 10(-11) M versus antigen-positive cells; 1-3 X 10(-7) M versus antigen-negative cells). Specificity and immunoreactivity of the conjugate were similar to those of unconjugated antibody. Biodistribution studies with iodine trace-labeled conjugate in nude mice indicated that tumor localization of the gelonin conjugate was decreased compared to unconjugated antibody. However, a significant therapeutic effect of the conjugate was found with multiple but not single dose i.v. treatment in nude mice bearing established palpable melanoma. These in vivo experiments showed that gelonin conjugates are not toxic up to 2 mg total dose/mouse and significantly retarded the growth of established s.c. tumor. Comparison of gelonin conjugates in vitro and in vivo with other A-chain conjugates of 9.2.27 (abrin and ricin) indicated that gelonin had similar potency, better selectivity, better tumor localization, and more significant therapeutic effects.

Abrin↗
PubMed1987

Kinetic model for the biodistribution of an 111In-labeled monoclonal antibody in humans.

Using data from 12 patients, we have analyzed the pharmacokinetics of 111In-9.2.27, an antimelanoma monoclonal antibody, following i.v. infusion. Plasma data and scintillation camera images obtained from patients receiving either 1, 50, or 100 mg of monoclonal antibody indicated dose-dependent (i.e., saturable) kinetics. Based on these observations and known immunoglobulin kinetics, we developed a nonlinear compartmental model to describe the biodistribution of 111In-9.2.27 and the other coinjected 111In-associated compounds. The model included (a) three compartments representing intact 111In-9.2.27 ("plasma," "nonsaturable," and "saturable binding" compartments), (b) four compartments representing 111In-diethylenetriaminepentaacetic acid, and (c) one compartment representing 111In in an undetermined chemical form ("extravascular delay" compartment). Analysis of the rate of urinary excretion relative to plasma concentration indicated that the saturable binding compartment was a site for catabolism of monoclonal antibody. Further examination of the urinary data, together with previous studies of the site(s) of immunoglobulin catabolism, suggested that additional elimination took place from either the plasma or the nonsaturable compartment. The model indicated that to fill the saturable sites would require a dose of approximately 0.5 mg and suggested that greater than 3.5 mg would maintain saturation for 200 h. Computer integration of gamma camera counts over the spleen revealed a clear saturable component of uptake, whereas integration over the liver showed no such pattern. The proposed model was fitted to the liver and spleen imaging data by summing fractions of model simulations of each compartment. That analysis confirmed the suspected saturable uptake by the spleen (21% of the saturable binding compartment) and revealed a quantitatively important component of saturation in the liver (35% of the saturable binding compartment) that was not obvious from initial examination of the images. When the results were expressed on a concentration basis, the spleen accounted for 247% of the saturable compartment per kg, whereas the liver accounted for 25%/kg. The bone marrow also showed saturable uptake; hence, the saturable uptake may relate to the sinusoidal blood supply characteristic of liver, spleen, and marrow. The model predicts the dose levels required to overcome saturable background, suggests appropriate doses and schedules for cold loading strategies, and provides a format for explicit inclusion of tumor antigen.

Antibodies, Monoclonal↗
PubMed1987

Analysis of effector cells in human antibody-dependent cellular cytotoxicity with murine monoclonal antibodies.

We examined purified human large granular lymphocytes, peripheral monocytes, and T cells for their ability to mediate antibody-dependent cellular cytotoxicity (ADCC) with murine monoclonal antibodies. We also evaluated the effects of pretreatment of cells with interleukin 2 and interferon to augment ADCC activity. MB3.6, a murine monoclonal antibody directed against the GD3 ganglioside, induced high levels of ADCC. This ADCC was mediated predominantly, if not completely, by human killer cells (large granular lymphocytes) whereas other effector cell populations demonstrated no significant cytotoxic activity in 6- or 18-hr assays. The IgG2a an anti-melanoma antibody 9.2.27 generated low or no ADCC with most normal donors or melanoma patients. IL 2 was a very potent booster of ADCC activity. Interferon alpha also was effective, whereas interferon gamma did not augment but rather inhibited reactivity. We tested a large panel of antibodies of various isotype against colon carcinoma cells and found that gamma-3 isotype antibodies more frequently generated ADCC and produced higher levels of cytotoxic activity than did IgG1 or IgG2 antibodies. It appears that a variety of parameters can affect ADCC reactions, including the type of effector cell and its level of activation, the isotype of the antibody, and properties of the target cell line such as its susceptibility to lysis.

Animals↗
PubMed1987

Occult (non-surface expression) of T, B and monocyte markers in human large granular lymphocytes.

Human large granular lymphocytes were examined for non-surface expression with a panel of monoclonal antibodies to T cell, B-cell and monocyte markers. T101, antibody to the T65 antigen, showed binding to crude fractions containing intracellular membranes but not to immobilized whole cells. Non-surface expression of T65 was also demonstrated by flow cytometry using lysolecithin to transiently permeabilize cells. With the latter technique nonsurface expression was also demonstrated with monoclonal antibodies B2 and MO-2. T65 was shown to be synthesized by large granular lymphocytes by metabolic labeling, indirect immunoprecipitation and SDS-PAGE. T65 from large granular lymphocytes was the same mol. wt as antigen for T cells derived from the same donor. These results indicate that human large granular lymphocytes synthesize, but do not express on the surface, certain monoclonal antibody-derived markers heretofore considered specific for other cell lineages.

Antibodies, Monoclonal↗
PubMed1987

Immunotoxins to a human melanoma-associated antigen: resistance to pokeweed antiviral protein conjugates in vitro.

Pokeweed antiviral protein (PAP) from the summer leaves of Phytolacca americana was purified and conjugated via N-succinimidyl-3-(2-pyridyldithio)propionate to 9.2.27 anti-melanoma antibody to a glycoprotein-proteoglycan complex. The conjugate was highly potent (50% inhibition dose of 5 X 10(-11)-10(-13) M on antigen-positive melanoma) and highly selective (5 X 10(-8) on antigen-negative melanoma). Human melanoma cells were selected for resistance to in vitro killing of the PAP conjugate by cycling through a killing and recovery sequence. Resultant cultures were shown to be more than 2 logs less sensitive to the killing of the PAP conjugate than untreated cultures. Isolation of clones by limiting dilution and reanalysis indicated that the resistant polyclonal culture contained clones with a range of sensitivities. Resistant cultures were also resistant to other A-chain conjugates of 9.2.27, but not to intact toxins like ricin and abrin. Resistant cultures showed no change in antigen expression after selection with the PAP conjugate of 9.2.27. Thus, just as with many other chemotherapeutic agents, tumor cells can become resistant to agents inhibiting protein synthesis even when targeted with monoclonal antibody. The mechanisms of this resistance and modalities to minimize resistance are currently being explored.

Antibodies, Monoclonal↗
PubMed1987

Monoclonal antibody therapy in malignant melanoma: factors effecting in vivo localization.

Thirteen patients with metastatic malignant melanoma received intravenous therapy with the murine antimelanoma monoclonal antibody 9.2.27. Five patients were entered on a dose escalation protocol with twice weekly escalating doses of 10-500 mg, in an extension of a previously reported trial. These patients demonstrated near saturation of available antibody binding sites in vivo following the 500 mg dose, with minimal toxicity. The remaining patients were entered onto a dose schedule comparison study, with a 500 mg dose administered either in a single 2 h infusion or as five daily 2 h infusions of 100 mg to examine the effects of different dose schedules and of an interrupted schedule on subsequent therapy with the same antibody. Intratumor localization of the monoclonal antibody did not appear to vary with respect to the dose schedule; however, interruption in therapy for 4 weeks was accompanied by somewhat poorer localization of antibody. This effect appeared to be primarily attributable to development of human antimurine antiglobulin in 25-30% of patients with resultant decrease in intratumor localization of antibody and more rapid clearance of the 9.2.27 antibody from the circulation. Earlier reports with other antibodies notwithstanding, initial infusions of 500 mg of 9.2.27 did not induce tolerance to the murine immunoglobulin. This study confirms and extends the findings of our initial trial of the 9.2.27 antibody by demonstrating that, although clinical responses were not observed, the antibody can be safely administered at doses up to 500 mg, with good intratumor localization of antibody. The diminished localization of antibody associated with antiglobulin responses indicates the importance of monitoring antiglobulin levels during therapy, and the necessity of controlling or preventing this phenomenon when monoclonal antibodies are administered in multiple doses as drug, toxin, or radionuclide immunoconjugates.

Adult↗
PubMed1986

Evaluation of alternative methodologies for the generation of murine monoclonal anti-idiotypic antibodies to human B cell leukemias and lymphomas.

The standard immunization procedure for the development of monoclonal antibodies to human malignant B cell idiotype immunoglobulin in our laboratory consists of intraperitoneal immunization with 50-100 micrograms of purified immunoglobulin followed 7 days later by intravenous immunization with an equal quantity of protein. In order to shorten the process and decrease the amount of idiotypic immunoglobulin necessary for successful immunization, we have evaluated 2 alternative immunization procedures. When immunized by the standard method, the percentage of hybridomas demonstrating anti-idiotype activity was 0.3-1.0%. In order to increase the proportion of anti-idiotype hybrids tolerance to the constant regions of human IgM was established by intraperitoneal administration of disaggregated human gamma globulin 14 days prior to immunization of both male and female mice. The percentage of wells with anti-idiotype activity rose to 16-52% in tolerant male mice. The percentage of anti-idiotype hybrids generated was significantly lower in tolerant female mice. To accelerate the process of anti-idiotype development, a single intrasplenic immunization with soluble idiotype IgM was also evaluated. No anti-idiotype or anti-IgM secreting heterohybrids were formed out of nearly 1400 wells seeded in 3 separate fusions using soluble IgM. When the idiotype IgM was immobilized onto protein A-Sepharose, and then injected intrasplenically, approximately 1% of the wells seeded showed anti-idiotype activity. Thus, a single intrasplenic immunization with immobilized immunoglobulin resulted in significant time saving, while prior tolerization may greatly increase the percentage of anti-idiotype hybrids.

Antibodies, Anti-Idiotypic↗