Gary M. Stuhlmiller

In vivo tumor localization using tumor-specific monkey xenoantibody, alloantibody, and murine monoclonal xenoantibody.

Specific in vivo localization of antibodies reactive with human melanoma cell membrane tumor associated antigens (TAA) has been attempted using congenitally athymic nude mice bearing subcutaneous human melanoma tumor xenografts as the experimental model. IgG fractions were prepared from each of several immune and control sera. Antimelanoma antibody sources included human alloantibody obtained from melanoma patients immunized against allogeneic melanoma cells, a monkey antiserum raised by immunization against a single human melanoma cell line, and a murine monoclonal antimelanoma antibody-secreting hybridoma cell line. Localization of these radiolabeled antibodies and of control IgG preparations to tumor tissue was determined by whole body scintigraphy and by differential tissue counting. Compared with the different control IgG preparations, each of the antimelanoma IgG preparations exhibited significant specific accumulation within the melanoma tissue. However, variation existed in the ability of each antimelanoma IgG to tumor preparation to localize despite attempts to control model parameters such as tumor source, vivo passage number and mass. This variation appears to reflect basic biologic differences between tumors in different animals and possibly differences in the antigen-binding capacities of each IgG preparation following radioiodination. This technique for tumor localization is very promising and has obvious potential for clinical application

Allotransplantation of insulinoma into the testis of diabetic rats.

We have previously shown that many of the metabolic abnormalities of the streptozotocin-induced diabetic state in KX (Ag-B7) rats can be corrected by a transplantable syngeneic insulin-secreting insulinoma. In this study, we have evaluated the transplantability of this KX insulinoma into allogeneic Sprague-Dawley (SD) rats (Ag-B6). Graft rejection was evident at 7 days post-transplant of the tumor when engraftment was made in a subcutaneous site. Prolonged graft survival was observed in 1 of 16 normal SD rats and in 3 of 19 streptozotocin-induced diabetic SD rats when the tumor was grafted into the testis. Mean survival times for these successful allografts was 27 ± 4 days. Blood glucose levels in these animals returned to normal limits. Failure of the other intratesticular (IT) allografts was attributable to technical considerations rather than to specific antigraft immune reactions. Sensitization of SD rats with KX skin grafts or by s.c. injection of the KX insulinoma resulted in second-set survival times for subsequently applied KX skin grafts. Sera from animals sensitized in this manner contained antibody cytotoxic for KX lymphocytes, thus demonstrating the immunogenicity of the insulinoma. In contrast, SD rats sensitized by IT injection of the KX insulinoma rejected subsequent KX skin grafts with a primary antigraft immune reaction. Sera taken from SD rats which had unsuccessful IT engraftment of the insulinoma did not contain anti-KX antibody. These studies thus suggest that, although the KX insulinoma does express KX histocompatibility antigens, expposure of SD rats to these antigens via IT transplant fails to provoke an anti-KX immune response. Successfully engrafted insulinoma retains its metabolic activity as evidenced by its alleviation of the hyperglycemie state of the recipient diabetic animals.

The use of non-human primates for production of antisera to human tumor-associated antigens

High-titered antisera against human melanoma- and leukemia-associated antigens have been produced in non-human primates. The data from this and from other studies suggest that this model may prove a valuable source of highly specific antisera against a variety of human tumor-associated antigens or alloantigens. Our methods for production and characterization of these antisera are reported.

Immune response of chimpanzee to purified melanoma 250 kilodalton tumor-associated antigen

SummaryMelanoma high molecular weight tumor-associated antigen (TAA), having a molecular weight of 250 kilodaltons (Kd), was purified from a crude cell membrane extract through a combination of lectin affinity, immunoadsorption, and high performance liquid molecular filtration chromatography. Compared to the starting extract, purified TAA was 600-fold higher in TAA activity per microgram of protein. Purified TAA was used to immunize a chimpanzee and the resulting antiTAA immune response was evaluated. Postimmune chimpanzee serum reacted in solid phase radioimmunoassay against purified TAA with a titer in excess of 100,000. In contrast, preimmune serum had a titer of <100 in the same assay. By immunoprecipitation analysis, we were able to demonstrate reactivity of the chimpanzee immune serum with a 250 Kd TAA in spent culture medium from melanoma cells metabolically labeled with 35S-methionine and with iodinated purified 250 Kd TAA. Reactivity of the chimpanzee antiserum for the 250 Kd TAA was confirmed in blocking and reciprocal immunodepletion studies using murine monoclonal antibody 9.2.27. These studies suggest that the 250 Kd TAA defined by murine monoclonal antibodies may prove to be immunogenic in man and that manipulation of the immune response to this TAA might be used to the clinical benefit of the patient.

Serological response of non-human primates to human melanoma disialoganglioside GD3.

SummaryThe immunogenicity of the disialoganglioside, GD3, a melanoma-tumor-associated antigen, has been evaluated in non-human primates. Sera from four chimpanzees and two monkeys were evaluated for anti-GD3 antibody activity by solid-phase radioimmunoassay using GD3 and control gangliosides as targets. Serum from one monkey, immunized with cells from a melanoma cell line, was strongly reactive with GD3, having a titer of >2500. In contrast, serum from this animal was non-reactive with several other gangliosides including the structurally similar GM3. Anti-GD3 reactivity was also demonstrable, albeit in low titer, in the sera of an additional monkey and a chimpanzee. Each of these animals had likewise been immunized using cells from melanoma cell lines. On the basis of these observations, suggestive of a primate anti-GD3 antibody response, we initiated a series of immunizations of chimpanzee using purified GD3 bound to Salmonella minnesota, R595. IgG reactive with melanoma cells in the cell-binding assay was first detected in sera collected after 4 immunizations and increased in titer against each reactive melanoma cell line during the immunizations. Reactivity of this serum with melanoma cell lines demonstrated a direct correlation with the expression of GD3 by the respective cell line. Anti-GD3 reactivity was evident in solid-phase radioimmunoassay against purified GD3 beginning with serum collected after 11 immunizations. By comparison with its binding to the control ganglioside panel, this serum demonstrated strong specificity for GD3 (titer=640) while having only marginal reactivity with GM3 (titer=40). Immune serum from this animal was also able specifically to block subsequent binding of a murine IgM anti-GD3 antibody (DMab7) to target GD3 in solid-phase radioimmunoassay. Together, these observations suggest that GD3, in the form of a purified molecule bound to a bacterial matrix or as part of the intact melanoma cell membrane, can be immunogenic in non-human primates, and is able to elicit an antibody response of appropriate specificity.