Mixed Hemadsorption Assay Research Articles

Abstract 4756: Anti-melanoma-specific humoral immune responses in vitro in patients vaccinated with DC-rIL-2-VMO

Abstract Introduction: DC-rIL-2-VMO, an allogeneic cellular melanoma vaccine consisting of five melanoma cell lines, a recombinant vaccinia virus encoding for IL-2, as well as dendritic cells, has previously been shown to generate a potent cellular response in murine tumor models. We now demonstrate that this vaccine also has the capacity to generate a sustained humoral response in vitro. Methods: To detect anti-tumor antibodies in pre- and post-vaccinated melanoma patients’ sera, a mixed hemadsorption assay (MHA) was used. The binding of human antibody from diluted patients’ sera (1:05, 1:15 and 1:45) to cultured melanoma cells (Mel-2, Mel V, GLM-2 and KFM cells) was determined as indicator cells, sheep red blood cells (SRBC) coated sequentially with mouse anti-SRBC sera, goat sera against mouse sera, and mouse anti-human IgG sera to detect binding of human anti-melanoma antibodies. Results: Out of four patients evaluated with Stage IIIA-C and Stage IV resected with no evaluable disease (NED), three patients’ post-vaccination sera showed antibodies specifically binding to melanoma cell lines. Antibodies from two of the patients’ sera showed moderate to strong binding to three different melanoma cell lines (Mel 2, Mel V and GLM-2). Additionally, antibodies from patients’ sera and sera from two normal healthy donors did not show any binding to colorectal carcinoma (SW1116) cells (CRC), a negative control. Conclusions: Treatment of patients with Stage IIIA-C and Stage IV melanoma resected to be NED with DC-rIL-2-VMO induced a tumor-specific antibody response. The presence of these anti-melanoma antibodies suggests that this vaccine may be effective in slowing the progression of melanoma in some patients via cellular and humoral immune mechanisms. DC-rIL-2-VMO has been FDA-approved for Phase I trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4756.

Production and characterization of an active single-chain variable fragment antibody recognizing CD25

The α subunit of the interleukin-2 receptor (IL-2Rα, CD25) is a potential target in therapeutic approaches for hematolopoietic malignancies expressing CD25 on their cell surface, such as adult T cell leukemia/lymphomas. Recent reports have demonstrated that depletion of CD4 +CD25 + regulatory T cells with anti-CD25 antibodies may enhance host tumor immunity. We previously raised a mouse monoclonal antibody (mAb), Ta60b mAb (IgG1 κ), specifically recognizing CD25, and an attempt was made here to produce a single chain Fv fragment (scFv) from this mAb as an initial step to development of scFv-based therapeutics. cDNA fragments encoding for the variable regions of the light and heavy chains of the Ta60b mAb were thus isolated by polymerase chain reaction-mediated cloning, and, an expression vector constructed to express Ta60b scFv fused with the maltose binding protein (MBP) in the periplasm of Escherichia coli. The soluble form of MBP-Ta60b fused scFv could be extracted and affinity-purified with an amylose/agarose column, allowing its immunoreactivity to be analyzed by enzyme-linked immunosorbent assay (ELISA), mixed hemadsorption assay, and fluorescence activated cell sorting. In addition, binding activity was studied by competitive ELISA and surface plasmon resonance. The results showed that Ta60b scFv obtained from periplasm retains good reactivity, although its KD value was 4-fold lower than that of the whole Ta60b antibody, suggesting possible clinical use for treatment of patients with CD25-expressing tumors and also for enhancing anti-tumor immunity.

A monoclonal antibody recognizing human cancers with amplification/overexpression of the human epidermal growth factor receptor.

Epidermal growth factor receptor (EGFR) has attracted considerable attention as a target for cancer therapy. Wild-type (wt)EGFR is amplified/overexpressed in a number of tumor types, and several mutant forms of the coding gene have been found, with DeltaEGFR, a deletion mutation lacking exons 2-7 of the external domain, being the most common and particularly associated with glioblastoma. We generated monoclonal antibodies (mAbs) against NR6(DeltaEGFR) (mouse fibroblast line NR6 transfected with DeltaEGFR). mAb 806 with selective reactivity for NR6(DeltaEGFR) in mixed hemadsorption assays, fluorescence-activated cell sorting, Western blot, and immunohistochemistry was analyzed in detail and compared with mAbs 528 (anti-wtEGFR) and DH8.3 (anti-DeltaEGFR). In xenograft tumors and molecularly pretyped glioblastomas, the reactivity pattern was as follows: 528 reactive with amplified and nonamplified wtEGFR; DH8.3 reactive with DeltaEGFR; and 806 reactive with amplified/overexpressed wtEGFR (with or without DeltaEGFR). In normal tissues, 528 but not DH8.3 or 806 was widely reactive with many organs, e.g., liver expressing high EGFR levels. In glioblastoma and non-CNS tumor panels, 806 was reactive with a high proportion of glioblastomas and a substantial number of epithelial cancers of lung and of head and neck. DH8.3 reactivity was restricted to DeltaEGFR-positive glioblastoma. Thus, 806 represents a category of mAbs that recognizes tumors with EGFR amplification/overexpression but not normal tissues or tumors with normal EGFR levels. Our study also indicates that DeltaEGFR is restricted to glioblastoma, in contrast to other reports that this mutation is found in tumors outside the brain.

A mixed hemadsorption assay for detection of cell surface binding anti-tumor antibodies in human sera

A practical mixed hemadsorption assay (MHA) was developed for the detection of cell surface binding anti-tumor antigen antibodies (anti-TA Ab) in the human sera. The assay was compared to enzyme-linked immunosorbent assay (ELISA) and dot ELISA in the detection of the antibodies developed against GA733-2 antigen. SW1116 cell line and recombinant GA733-2E protein were used as targets in the MHA and solid phase immunoassays (SPIA), respectively. Sera were obtained from healthy donors and vaccinated-colon carcinoma patients. The sensitivity level of the MHA was very high for detection of anti-TA Ab in sera.

Induction of antibodies reactive with GM2 ganglioside after immunization with lipopolysaccharides from Camplyobacter jejuni.

Ganglioside GM2, expressed on the surface of some human cancers, is a promising target for immune therapy, since GM2 antibodies are cytotoxic, can be induced in humans by vaccination, and the presence of GM2 antibodies is associated with a better prognosis in melanoma patients. In our efforts to induce long-lived, cytotoxic GM2 antibodies, we investigated lipopolysaccharides (LPS) containing "GM2-like" oligosaccharides. LPS were prepared from Campylobacter jejuni serotypes O:1, O:23, or O:36 (all sharing the oligosaccharide structure GalNAcbeta1-4Gal(113NeuAc)-Hex with ganglioside GM2), and tested for their ability to induce GM2-reactive antibodies. Immunization of NZW rabbits (2 animals per vaccine) with LPS from C. jejuni serotype O:1 in Freund's adjuvant resulted in production of high-titer IgG antibodies reactive with purified bovine brain GM2 in ELISA, dot-blot immune strains and immune thin-layer chromatography, and with GM2 derived from various human tumors by immune thin-layer chromatography. These rabbit antibodies bound to cancer cell lines expressing GM2 on their cell surface, as determined by mixed hemadsorption assays, mediating strong antibody-dependent cellular cytotoxicity (ADCC) with tumor cells expressing cell-surface GM2. Antibodies induced by vaccination with C. jejuni serotype O:1 were higher-titer (IgG ELISA titer > 1:60,000) than antibodies induced by immunization with purified GM2 (IgG ELISA titer > 1:200). Immunization with LPS from C. jejuni serotype O:36 resulted in production of moderately high-titer IgM and low-titer IgG GM2 antibodies. Immunization with LPS from C. jejuni serotype O:23 did not elicit GM2-reactive antibodies. No clinical symptoms were observed in animals immunized with these LPS preparations, with purified GM2 ganglioside, or with LPS derived from C. jejuni serotype O:19 (containing a GM1-like oligosaccharide). Our results indicate that lipopolysaccharides sharing carbohydrate epitopes with gangliosides may be useful immunogens for inducing antibodies to ganglioside antigens.

Reactivities of Mouse Monoclonal Antibody K2.7 to Renal Cancers in Complement Dependent Cytotoxicity and Antibody Dependent Cell-Mediated Cytotoxicity

Immunohistochemical analysis by indirect immunoperoxidase staining demonstrated that monoclonal antibody (mAb) K2.7, derived from a mouse immunized with a renal cell carcinoma (RCC) cell line OS-RC-2, reacted with 89 of 95 renal cancer tissues (94 percent). Only 1 gastric and uterine cancer tissue showed positive staining among 87 cancer specimens from 9 different organs. Among normal human tissues, the renal tubule, testis, epithelium of the uterine endometrial gland and Fallopian tube, grey matter of cerebrum and cerebellum, and foreskin showed positive staining. Serological analysis by protein A mixed hemadsorption (PA) assay demonstrated that mAb K2.7 reacted with 25 of 31 RCC cell lines (81 percent), but with only 2 of 50 other cell lines from different organs. The specific antitumor activities of mAb K2.7 against RCC cell lines were investigated in vitro by complement dependent cytotoxicity (CDC) and antibody dependent cell-mediated cytotoxicity (ADCC) assays. In the CDC assay, all of the 9 RCC cell lines reactive serologically with mAb K2.7 were killed by mAb K2.7 with normal human serum, and the killing activity of mAb K2.7 correlated well with the reactivity of mAb K2.7 in the PA assay. mAb K2.7 showed the same killing activity against each of 3 RCC cell lines with sera from 9 patients with low and high stage renal cancers, as well as with normal human serum. In the ADCC assay, mAb K2.7 with peripheral blood leukocytes (PBLs) from 4 healthy donors showed cytotoxic activity against RCC cell lines. Peripheral blood leukocytes from the same 9 renal cancer patients also showed significant killing activity against the 3 RCC cell lines. These findings suggest the potential utility of mAb K2.7 for specific immunotherapy of renal cancer.

Characterization of Mouse Monoclonal Antibody B1.4 Reactive with Human Invasive Bladder Cancer and Some Other Malignant Tumors but Not with Normal Urinary Epithelium

Immunohistochemical analysis by indirect immunoperoxidase staining demonstrated that monoclonal antibody (mAb) B1.4 derived from a mouse immunized with a bladder cancer cell line EJ‐1 was reactive with a high proportion of high‐grade and invasive bladder tumors, but not with the majority of low‐grade and superficial bladder tumors, or normal urinary epithelium. Among 71 primary bladder tumors classified by pathological grading, positive stainings were observed in 1 of 34 tumors (3%) of grade 1, 8 of 20 tumors (40%) of grade 2 and 14 of 17 tumors (82%) of grade 3. When the tumors were classified by pathological staging, positive stainings were observed in only 8 of 54 (15%) superficial tumors of stages Ta and T1, but in 15 of 17 (88%) invasive tumors of stages T2 and T3. mAb B1.4 showed restricted positive stainings with normal tissues including renal glomerulus, vascular endothelium, squamous epithelium of esophagus, glandular epithelium of prostate, and epithelium of pancreatic acinar gland and minute duct, while positive stainings were observed in a range of tumor tissues other than bladder tumor. Mixed hemadsorption assays with a panel of cell cultures showed also that the antigen recognized by mAb B1.4 was expressed on a range of tumor cell lines. These findings suggest that the antigen recognized by mAb B1.4 may appear after malignant transformation, and be an indicator of malignant potential of bladder cancer.

Induction of Anti-Idiotypic (ab2) and Anti-Anti-Idiotypic (ab3) Antibodies in Patients Treated with the Mouse Monoclonal Antibody 17-1A (ab1). Relation to the Clinical Outcome — An Important Antitumoral Effector Function?

Forty-three patients with metastatic colorectal carcinoma (CRC) were treated with the unconjugated mouse monoclonal antibody (MAb) 17-1A (ab1) only. The presence of antiidiotypic antibodies (ab2) and anti-antiidiotypic antibodies (ab3) were analyzed using an ELISA technique and a mixed hemadsorption assay respectively. Ninety-five percent (41/43) of the patients developed ab2 both of the IgM and the IgG classes. Forty-seven percent (20/43) of the patients had detectable ab3 after therapy, two of them also before administration of MAb 17-1A. Binding in vitro of ab3 (ab1) to CRC cells could be specifically inhibited by ab1. Ab3 bound to human monoclonal antiidiotypic antibodies and to a goat antiidiotypic antibody (ab2). Both these ab2 were directed against MAb 17-1A (ab1). There was a strong correlation between the presence of ab3 and the clinical outcome. Ab3+ patients survived significantly longer than those who did not develop ab3 antibodies, 80 weeks vs 38 weeks (p less than 0.001). A statistically significant correlation was found between the presence of ab3 and the anti-tumor response (CR + PR + MR + SD) (p = 0.01). Thus, induction of an antiidiotypic cascade seems to be an important antitumor effector function of MAb in the treatment of cancer patients.

Molecular cloning of cDNA for the human tumor-associated antigen CO-029 and identification of related transmembrane antigens.

The human tumor-associated antigen CO-029 is a monoclonal antibody-defined cell surface glycoprotein of 27-34 kDa. By using the high-efficiency COS cell expression system, a full-length cDNA clone for CO-029 was isolated. When transiently expressed in COS cells, the cDNA clone directed the synthesis of an antigen reactive to monoclonal antibody CO-029 in mixed hemadsorption and immunoblot assays. Sequence analysis revealed that CO-029 belongs to a family of cell surface antigens that includes the melanoma-associated antigen ME491, the leukocyte cell surface antigen CD37, and the Sm23 antigen of the parasitic helminth Schistosoma mansoni. CO-029 and ME491 antigen expression and the effect of their corresponding monoclonal antibodies on cell growth were compared in human tumor cell lines of various histologic origins.

Fibronectin Distribution in Normal and Malignant Urothelium

Fibronectin is a glycoprotein that mediates the attachment of BCG to the murine bladder. To assess the potential role of fibronectin on bladder cancer cells as a specific substrate for BCG binding in man, a semi-quantitative method was employed to evaluate the presence of fibronectin on normal urothelium and bladder cancer. Monoclonal anti-fibronectin binding to normal and malignant urothelial tissues was evaluated by an immunoperoxidase assay. Human tumor cell lines were evaluated with mixed hemadsorption and immunoperoxidase assays. In both systems, immunoreactive fibronectin had low expression on unfixed normal and malignant urothelium. With fixation, immunoreactive fibronectin decreased on supporting stroma and increased in normal and malignant urothelium. Fibronectin distribution did not show tumor specificity either with fixed or unfixed specimens. (J. Urol., 143: 418–420, 1990)

Specific detection of antibodies in cancer patients following immunotherapy with anti-idiotype

Assays were compared for specificity and sensitivity in detecting in cancer patients' sera antibodies (Ab) raised during the course of immunotherapy with goat anti-idiotypic antibodies (Ab2) bearing the internal image of a colon carcinoma-associated antigen defined by monoclonal antibody (MAb) CO17-1A (Ab1). The human Ab were tested for binding to tumor cells, isolated tumor antigen (Ag), and Ab2, and for the capacity to inhibit binding of Ab2 to Ab1. Chimeric (human/mouse) MAb CO17-1A was used as a positive control in all assays. Of the four different cell binding assays used, the mixed hemadsorption assay (MHA) showed the highest specificity and sensitivity. For detection of Ag-binding human Ab, the enzyme-linked immunosorbent assay (ELISA) with Ag as target and peroxidase (PO)-labeled anti-human IgG antibodies as tracer for detection of human Ab binding to the target, showed higher specificity and sensitivity as compared to radioimmunoassay (RIA). For detection of human Ab binding specifically to Ab2, three different ELISAs and three RIAs were used. Best results were obtained in the ELISA with anti-human IgG antibodies as target and biotinylated Ab2 as tracer for detection of human Ab binding to the target. Of four different inhibition assays used, the ELISA which measures inhibition of binding of biotinylated Ab2 to Ab1 by human Ab or chimeric antibody at 37°C was the most sensitive and specific. These assays have general applicability for the characterization of human Ab responses in Ab2 vaccination approaches to various tumors and pathogens and therefore provide the basis for the establishment of a correlation between Ab responses and clinical outcome of the disease.

A novel neuronal antigen identified by sera from patients with systemic lupus erythematosus

Using human neuroblastoma cell lines (IMR-6, NMB-7, SK-N-Mc, SK-N-SH) as sources, we characterized surface neuronal antigens as an initial step in determining the pathogenic role and clinical significance of neuronal antibodies in systemic lupus erythematosus (SLE) patients. SLE sera were screened for the presence of surface neuronal antibodies using a mixed hemadsorption assay. Thirty SLE sera were further tested by Western blotting and immunoprecipitation of lysed IMR-6 cells. Western blotting revealed binding to predominantly intracellular antigens, none of which was restricted to neuroblastoma cells. In contrast, immunoprecipitation experiments demonstrated binding to a 97K antigen, which appeared to be of surface origin, by 3 SLE sera. This was not present on non-neuronal cells and was not precipitated by sera from healthy or disease controls. This 97K antigen was also precipitated from the neuroblastoma cell line NMB-7, but was not present on SK-N-Mc or SK-N-SH cells. Precipitation was depleted by preabsorption with viable IMR-6 and NMB-7 cells, but not with non-neuronal cells. Thus, some SLE sera recognize a 97K neuronal antigen on select neuroblastoma cells.

Cell-surface glycoproteins of human sarcomas: differential expression in normal and malignant tissues and cultured cells.

Normal differentiation and malignant transformation of human cells are characterized by specific changes in surface antigen phenotype. In the present study, we have defined six cell-surface antigens of human sarcomas and normal mesenchymal cells, by using mixed hemadsorption assays and immunochemical methods for the analysis of cultured cells and immunohistochemical staining for the analysis of normal tissues and greater than 200 tumor specimens. Differential patterns of F19 (Mr, 120,000/95,000 glycoprotein), F24 (Mr, 95,000 glycoprotein), G171 (Mr, 75,000 glycoprotein), G253 (Mr, 90,000 glycoprotein), S5 (Mr, 120,000 glycoprotein), and Thy-1 (Mr, 25,000 glycoprotein) antigen expression were found to characterize (i) subsets of cultured sarcoma cell lines, (ii) cultured fibroblasts derived from various organs, (iii) normal resting and activated mesenchymal tissues, and (iv) sarcoma and nonmesenchymal tumor tissues. These results provide a basic surface antigenic map for cultured mesenchymal cells and mesenchymal tissues and permit the classification of human sarcomas according to their antigenic phenotypes.

Definition of selectable cell surface markers for human chromosomes and chromosome segments in rodent-human hybrids.

A panel of 22 monoclonal antibodies (MAbs) recognizing 21 distinct human cell surface antigens was tested by mixed hemadsorption assays for reactivity with a large number of rodent-human somatic cell hybrids containing different subsets of the human chromosome complement. The serological typing results permit the assignment of six gene loci determining cell surface antigens to human chromosomes 3, 6, 11, 19, 20, and 22. In addition, analysis of hybrids retaining deleted copies (but no normal homologs) of specific human chromosomes provides regional assignments for 18 gene loci, located on seven different chromosomes. These findings extend and refine the genetic map for human cell surface antigens and identify new selectable markers for defined chromosome segments.

Identification of a human osteosarcoma-associated glycoprotein with monoclonal antibodies: relationship with alkaline phosphatase.

The molecular nature of an osteosarcoma-associated antigen was investigated with the three monoclonal antibodies Ost6 (immunoglobulin (IgG1), Ost7 (IgG1), and Ost15 (IgG2a), which selectively react with frozen sections of osteosarcoma and chondrosarcoma tissues. When tested with a panel of 41 human cell lines in the mixed hemadsorption assay, the antibodies reacted similarly with three of six osteosarcomas, one choriocarcinoma, one teratoma, and one osteoblast-like culture, but failed to react with 32 lines of normal and other tumor cell types. Immunoprecipitation plus sodium dodecyl sulfate (SDS)--polyacrylamide gel electrophoresis and sequential immunoprecipitation studies revealed that in [35S]methionine- or [14C]glucosamine-labelled osteosarcoma cells the three antibodies detected a single glycoprotein, with an apparent molecular mass of 86 kilodaltons (kDa), which was not affected by reducing conditions. Tunicamycin treatment and pulse-chase experiments showed glycosylation of this molecule to be N-linked; it arose from a 54-kDa polypeptide precursor. Alkaline phosphatase activity was detected in the material rich in 86-kDa molecules that was immunoprecipitated from serologically reactive cell lines with each antibody. These antibodies also cross-reacted with two isoenzymes of alkaline phosphatase (strongly with the liver and bone, and moderately with the placental isoenzyme), but not with the intestinal form.

Microelectrochemical radioiodination of monoclonal antibody: A preliminary study

AbstractThe optimal reaction conditions for the microelectrochemical iodination of immunoglobulins were determined with non‐specific human serum immunoglobulins. These conditions were used for the efficient radioiodination of a monoclonal antibody, 140.240, in submilligram quantities. An approximately five‐fold decrease in the titre of the antibody against melanoma cells, as determined by the miniaturized mixed hemadsorption assay, was observed after iodination with an average of 0.85 atoms of iodine per molecule of antibody.

Differential expression of the human Thy-1 gene in rodent-human somatic cell hybrids [corrected

Thy-1 is a cell surface differentiation marker which shows distinct patterns of tissue-specific expression in different species. In man, the Thy-1 antigen is encoded by chromosome 11. We have examined the regulatory signals determining human Thy-1 expression through serologic analysis of rodent-human somatic cell hybrids retaining human chromosome 11 in which the fusion partners belong to distinct differentiation lineages. Cell surface expression of human Thy-1 was determined by mixed hemadsorption assays with two monoclonal antibodies (mAb), K117 and L127, shown to detect authentic human Thy-1 through analysis of COS-7 monkey kidney cells transfected with a cloned human Thy-1 gene. Three different patterns of human Thy-1 expression were observed when hybrid cells, constructed with different human and rodent cell types, were tested with mAb K117 and L127. Hybrids formed between Thy-1+ human neuroblastoma cells and Thy-1- mouse neuroblastoma cells, or hybrids between Thy-1+ human fibroblasts and the Thy-1- mouse kidney carcinoma, RAG, retain human Thy-1 expression. In contrast, hybrids formed between either Thy-1+ human neuroblastoma cells or Thy-1+ human fibroblasts and Thy-1- mouse L cells lose expression of human Thy-1 even though chromosome 11 is retained. Finally, hybrids formed between Thy-1- human peripheral lymphocytes or a Thy-1- lymphoblastoid B cell line and Thy-1- Chinese hamster fibroblasts begin to express human Thy-1. These studies suggest that both positive and negative trans-acting signals may play a role in the tissue-specific regulation of the human Thy-1 gene.

Primary Melanoma Cells of the Vertical Growth Phase: Similarities to Metastatic Cells<xref ref-type="fn" rid="FN2">2</xref>

Three primary and 16 metastatic melanoma cell lines were established from primary and metastatic lesions of 4 patients with malignant melanoma. Comparison of metastatic melanoma cells with cells of the vertical growth phase (VGP) or late primary melanoma from the same individual revealed, generally, a shorter population-doubling time, growth to a higher cell density, higher tyrosinase activity, and more pigmentation in metastatic cells. Conversely, primary and metastatic melanoma cells had similar morphology, plating efficiency, and tumorigenicity in nude mice. Karyotypic analysis revealed clonality and nonrandom abnormalities in chromosomes 1, 6, and 7 in cells of the primary and metastatic lesions of the 3 patients studied. Few differences were found in the expression of melanoma-associated antigens on short-term and long-term cultured cells by tests with monoclonal antibodies in mixed hemadsorption assays, flow cytometry, and radioimmunoassays. Our results indicate that cells cultured from the VGP but not from the radial growth phase of primary melanoma are similar to metastatic melanoma cells.

Radioimmunodetection of a transplantable human bladder carcinoma in a nude mouse.

The results of an in vitro mixed hemadsorption (MHA) assay predicted the success of in vivo tumor localization using a radioiodinated, monoclonal, IgG1 antibody (A2) with reactivity to the human bladder carcinoma cell line RT4. In vitro, murine monoclonal antibodies A2 and G6, demonstrated reactivity to RT4 with titers of 1/1024 and 1/4 by MHA assay, respectively. In vivo results obtained with RT4 xenografts in athymic nude, Balb/c, mice indicated tumor uptakes of 1.10% dose/gram with A2 and 0.29% dose/gram with G6 at seven days after radiotracer injection. Successful scintigraphic imaging of tumor xenografts was achieved with A2 but not with G6 or radioiodinated mouse serum albumin.

Comparison of approaches for augmenting the serologic response to the individually specific methylcholanthrene-induced sarcoma-Meth A: pretreatment with cyclophosphamide is most effective.

We have previously reported the production of antisera against a highly restricted antigen expressed on the BALB/c sarcoma Meth A. Because induction of the antibody required many vaccinations with irradiated and unirradiated Meth A cells over a prolonged period, we have investigated methods of improving the efficiency of producing Meth A antibody in BALB/c mice. Three general approaches were used: immunizing with irradiated Meth A cells mixed with adjuvants, immunizing with irradiated Meth A cells modified by treatment with chemicals, or administration of tumor cell vaccines in conjunction with low dose cyclophosphamide. These vaccine preparations were injected subcutaneously into groups of BALB/c mice five to six times at 2-wk intervals. Sera from all mice were screened for reactivity to Meth A by complement-dependent cytotoxicity, protein A, and mixed hemadsorption assays. Twenty-three vaccine preparations containing adjuvants or modified cells were tested in individual groups of mice. Mice in some groups receiving adjuvants and in all groups receiving modified cells produced antibody, but only after four to six vaccinations. In contrast, nine of the 14 mice immunized with irradiated Meth A cells (unmodified and without adjuvant) in conjunction with 10 or 100 mg/kg body weight of cyclophosphamide, and all nine of the mice receiving 25 mg/kg cyclophosphamide made Meth A antibodies after only one or two vaccinations. The titer of antibodies produced after one treatment with cyclophosphamide and irradiated Meth A cells was at least as high as that achieved after five or six vaccinations in our other trials. The specificity of these antibodies for the Meth A antigen was established by absorption analysis. We conclude that treatment with cyclophosphamide before vaccination is highly effective in augmenting the humoral immune response to the Meth A antigen.