Soluble Tumor-associated Antigens Research Articles

Transfer factor in vitro abrogation of blocking factors and amplification of immunoreactivity directed to soluble tumor associated antigens in the leukocyte adherence inhibition assay

26 breast cancer patients with recurrent disease were studied in order to evaluate whether transfer factor (TF) could abrogate serum blocking factors (SBF) and transfer or amplify immune reactivity directed to tumor-associated antigens (TAA) by using the leukocyte adherence inhibition assay (LAI). When 11 leukocyte samples obtained from patients reactive to cancer extract were preincubated with autologous blocking sera in the presence of PBS, nonspecific TF, or specific TF, leukocytes from these patients gained reactivity against breast cancer extract in none out of 11, one out of 11, and nine out of 11 experiments, respectively. Specific TF was obtained from donors with positive LAI breast cancer extract. Mean percentage LAI differences between control extract and breast cancer extract were −4.5±2.6, 0.5±1.7, and 15.5±2.4. The results of specific TF were significantly different from those of nonspecific TF (P<0.005). When nine leukocyte samples obtained from patients unreactive to cancer extract were preincubated with autologous blocking sera in the presence of PBS, nonspecific TF, or specific TF, leukocytes from these patients gained reactivity against breast cancer extract in none out of nine, one out of nine, and seven out of nine experiments, respectively. Mean percentage LAI differences between control extract and breast cancer extract were −4.6±2.0, 0.6±2.3, and 11.8±3.5. The results of specific TF were again significantly different from those of nonspecific TF (P<0.005). When allogeneic blocking sera were utilized, similar results were obtained. When specific TF was administered in two doses, 1 week apart subcutaneously to six breast cancer patients who were unreactive to breast cancer extracts, four patients gained reactivity against breast cancer extracts. Our in vitro experiments support the presence of a specific component in transfer factor extract which can transfer or amplify cell-mediated reactivity with abrogation of SBF directed at TAA.

Stimulation of Mouse Migration Inhibitory Factor (MIF) Production from MSV-Immune Lymphocytes by Soluble Tumor-Associated Antigen: Requirement for Histocompatible Macrophages

Abstract Spleen cells from C57BL/6 mice immunized with murine sarcoma virus (MSV) are capable of producing migration inhibition factor (MIF) in response to stimulation with a specific tumor-associated antigen prepared by solubilization with 3 M KCl. We have previously demonstrated that this response is T cell dependent. Further investigations into the effector cells involved in the production of MIF have revealed that spleen cells from mice immunized with MSV cannot produce MIF when stimulated with tumor extract if the population has been previously depleted of macrophages. However, the response can be restored by adding nonimmune syngeneic macrophages but not by allogeneic macrophages. The inability of allogeneic macrophages to provide this function was not due to their increased suppressor activity since in mixing experiments they did not interfere with the ability of immune spleen cells to produce MIF. Furthermore, they were not defective since they could supply this “cooperative function” to appropriate F1 mice. The results indicate that macrophages are required for stimulation of MIF by soluble tumor antigens and that for efficient interaction the macrophages and lymphocytes must share some genetic similarities.

An in Vitro Immuno-Enzymatic Assay of Tumor Antigens in the Mouse with β-Galactosidase

Abstract A method for detection of the primary binding of soluble tumor-associated antigens by antibodies has been developed by using an enzyme immunoassay (EIA). A heteroantiserum was produced by injecting tumor cells from a chemically induced murine sarcoma into rabbits, and antibodies reacting with most normal tissue components were removed by exhaustive in vivo absorption. A soluble preparation of tumor cells, obtained by 3 M KCl extraction, was conjugated to β-galactosidase from Escherichia coli. The antibody binding was measured by determining the enzyme activity that could be separated by anti-antibody coprecipitation. The reaction follows saturation kinetics, and nonlabeled antigen can be readily quantitated by inhibition. The present method detects determinants common to several MC-induced tumors on the same mouse strain but absent in normal cells and nonrelated tumors in addition to individual tumor-specific transplantation antigens. The sensitivity and simplicity of the new method compare favorably with a binding assay that utilizes radioactive iodine as a label. Thus, EIA becomes a flexible tool for the further characterization and purification of these antigens.

Isolation of a soluble tumor-associated antigen from human melanoma.

Human melanoma-associated antigen was solubilized from fresh surgical specimens by 3 M KC1 extraction. The antigenicity of this extract was demonstrated by delayed cutaneous hypersensitivity responses and inhibition of complement fixation. Twenty-one of 33 melanoma patients had delayed cutaneous hypersensitivity responses to melanoma antigen, while only four of 28 reacted to autologous muscle. Although KC1 extracts did not fix complement directly, they reacted with antibody, thus inhibiting complement fixation by the autologous melanoma antigen extracted from tissue culture supernatants. The soluble antigenic moiety was then purified by fractionation on a G-150 Sephedex column and polyacrylamide gels, and the antigenic activity was monitored by delayed cutaneous hypersensitivity responses in melanoma patients. A 20-fold purification was achieved. Solubilization of tumor antigens with 3 M KC1 provides tumor-associated antigen of high activity which is amenable to further biochemical purification.

Tumor-associated antigen (TAA) and anti-TAA antibodies in the serum of BALB-c mice with plasmacytomas.

Sera from BALB/c mice bearing plasmacytomas were studied for the possible presence of soluble tumor-associated antigen (TAA) and anti-TAA antibodies. The antibodies were detected by passive hemagglutination, sheep red blood cells being sensitized by a soluble tumor extract as a source of antigen. TAA was detected by inhibition of the hemagglutination of a syngeneic antitumor immune serum. Sera were hemagglutinating (antibody activity), inhibitory (antigen activity), or neutral. A neutral pool of serum was submitted to exclusion chromatography, and the fraction containing the Ig 7S split with acid into 2 fractions: The one of high molecular weight (A) was hemagglutinating (antibody?); the other of low molecular weight (B) was inhibitory (antigen ?). The mixture A+B was neither hemagglutinating nor inhibitory. The peak of lowest molecular weight (albumin fraction) contained free antige.

Multifaceted evaluation of human tumor immunity using a salt extracted colon carcinoma antigen.

Research during the past decade has indicated that most, if not all, human tumors possess tumor-associated antigens (1). This is based on in vivo and in vitro immunologic test systems which use intact tumor cells (2, 3), tumor cell homogenates (4) and subcellular fractions obtained by sonic disruption, enzyme digestion, and ultracentrifugation (5-7) as the source of antigen. There is also one report of a soluble tumor-associated antigen found in the fluid of a cystic melanoma (8).Ideally, the use of soluble tumor antigens will not only eliminate a number of technical difficulties entailed in preserving and storing tumor cells, but will also enable the investigator to study patients sequentially in relation to their response to treatment. This soluble antigen, if shown to be tumor specific and immunogenic, may also be useful for specific immunotherapy. Recent success in salt extraction of soluble HL-A antigens (9) has led investigators to apply similar methods to the extraction of tumor-associated antigen ...

Disappearance of a tumor-associated antigen in malignant melanoma after imidazole carboxamide therapy

Tumor fluid from a patient with cystic malignant melanoma contained a homogeneous protein, which induced in vitro stimulation of autologous lymphocyte DNA synthesis. The protein is believed to be a soluble tumor-associated antigen. Following therapy with dimethyl triazeno imidazole carboxamide (DTIC) the tumor antigen disappeared. Post-treatment lymphocytes of this patient were incubated with pre- and post-treatment tumor fluid. In the latter case, they were shown to be unaffected, and in the former to have a reduction of stimulation. In addition, homologous pretreatment lymphocytes from other melanoma patients showed cross reaction to pretreatment tumor fluid from the index patient, but had marked reduction in responsiveness to tumor fluid removed during treatment. These studies suggest that the anticancer drug DTIC may inhibit the synthesis of tumor-associated antigens, as well as lymphocyte mediated immunosensitivity to them.