Immunologic and therapeutic evaluation of a synthetic peptide vaccine for the treatment of patients with metastatic melanoma.

The cloning of the genes encoding cancer antigens has opened new possibilities for the treatment of patients with cancer. In this study, immunodominant peptides from the gp100 melanoma-associated antigen were identified, and a synthetic peptide, designed to increase binding to HLA-A2 molecules, was used as a cancer vaccine to treat patients with metastatic melanoma. On the basis of immunologic assays, 91% of patients could be successfully immunized with this synthetic peptide, and 13 of 31 patients (42%) receiving the peptide vaccine plus IL-2 had objective cancer responses, and four additional patients had mixed or minor responses. Synthetic peptide vaccines based on the genes encoding cancer antigens hold promise for the development of novel cancer immunotherapies.

Targeting human T-lymphocytes with bispecific antibodies to react against human ovarian carcinoma cells growing in nu/nu mice.

In the present study we tested whether human T-cells from normal donors can be targeted against human ovarian carcinoma cells and block i.p. growth of an established tumor in immunodeficient mice. For targeting we used chemically cross-linked bispecific monoclonal antibodies (mAbs) reacting with CD3 on the T-cells and with cell-surface antigens selectively expressed by tumor cells. The tumor model consisted of mice given i.p. injections of a human ovarian carcinoma cell line, OVCAR-3, whose growth includes development of massive ascites. Peripheral blood lymphocytes from normal human donors were cultured overnight with 50-100 units/ml recombinant interleukin 2, coated with bispecific antibodies, and injected i.p. into mice 4-6 days after tumor inoculation, at which time tumor cells were established and growing in about 85% of the hosts. Tumor growth was assessed by the number of tumor cells, and in some tests by cell-free tumor antigen, recovered in peritoneal lavage fluid collected 15 days after tumor priming. Treatment with lymphocytes retargeted with bispecific mAbs, prepared with anti-CD3 and three different antitumor mAbs, 113F1, OVB-3, and MOv19, gave highly significant increases in percentages of mice without detectable tumor. Controls showed that the antitumor activity of retargeted lymphocytes did not result simply from antibody-dependent cellular cytotoxicity or from heteroconjugates reacting only with CD3 or with lymphocyte major histocompatibility complex determinants and tumor cells. These results show that targeted T-lymphocytes can significantly decrease the growth of an established tumor in a fashion specific for antigens expressed by the neoplastic cells.

Human T-lymphocytes targeted against an established human ovarian carcinoma with a bispecific F(ab')2 antibody prolong host survival in a murine xenograft model.

A bispecific F(ab')2 fragment with anti-CD3 and antitumor specificity was used to target activated human peripheral blood lymphocytes (PBL) against OVCAR-3 human ovarian carcinoma cells growing i.p. in athymic mice. Mice were given injections of OVCAR-3 cells on day 0 and treated with i.p. injections of activated PBL coated with the [anti-CD3 (TR66) x antitumor (MOv18)] bispecific F(ab')2 on day 4, using an approximate effector:target ratio of 1:1. Treatment was evaluated for the ability either to block tumor growth at 15 days or to prolong survival of tumor-bearing mice. After 15 days, the incidence of mice with tumor growth was 20% among those given PBL coated with bispecific F(ab')2, whereas the incidence among mice untreated or treated with PBL alone or PBL with either parental antibody ranged from 80 to 94%. The mean survival time of tumor-bearing mice treated with PBL and bispecific F(ab')2 was 104 days, which was 3.5 times that of untreated mice and twice that of mice given PBL alone or PBL with either parental antibody. These results provide support for the concept that treatment of ovarian cancer patients with targeted T-cells could prove beneficial.

Expansion and characterization of T cells transduced with a chimeric receptor against ovarian cancer.

Adoptive immunotherapy with genetically modified T lymphocytes is being utilized in clinical trials for the treatment of a broad range of diseases including cancer and HIV infection. To improve on these treatments, and to better understand their mechanisms of action, it is necessary to develop techniques to generate large numbers of cells and characterize the functional heterogeneity of the cells produced. In this study, patient peripheral blood lymphocytes were transduced with a chimeric antigen receptor (MOv-gamma) derived from a mouse monoclonal antibody against folate-binding protein, which is overexpressed on many ovarian cancers. Thus, irrespective of their original specificity, normal human T lymphocytes were redirected to react against ovarian cancer cells. Lymphocytes from five patients were transduced and grown to large numbers, with a median expansion of more than 7000-fold. When proliferation was inadequate, the cells were expanded by stimulation utilizing anti-CD3, IL-2, and irradiated allogeneic PBMCs. The cells maintained their functional ability to recognize ovarian cancer over several months. Cloning of transduced cells was undertaken to determine the level of gene expression and function of individual cells making up the bulk population. Transduced CD4(+) and CD8(+) cell clones were isolated from the bulk and demonstrated antitumor activity. These clones had a diverse repertoire with respect to secretion of cytokines, and individual clones maintained their cytokine profile on subsequent expansion. These studies establish the feasibility of consistently generating large numbers of gene-modified tumor-reactive lymphocytes, with a stable and diverse cytokine repertoire, that could be utilized for patient treatment.

Susceptibility of adherent versus suspension target cells derived from adherent tissue culture lines to cell-mediated cytotoxicity in rapid 51Cr-release assays.

Preparation of target cells from tissue culture lines which grow adherent to tissue culture vessels is often desirable for tests of cell-mediated cytotoxicity (CMC). In the present study we used cells derived from adherent tissue culture lines to compare the merits of suspension vs. adherent target cells in short-term 51Cr-release assays. Cytotoxic activity of murine spleen cells sensitized in vitro against allogeneic spleen cells or syngeneic sarcoma cells was tested with fibroblast or sarcoma target cells. In parallel tests, aliquots of tissue culture lines were detached and used as either suspension or adherent target cells in CMC assays, matching the concentrations of suspension and adherent target cells. In both allogeneic and syngeneic combinations adherent target cells released less 51Cr spontaneously and were more susceptible to CMC than their suspension counterparts.

Augmented antibody-dependent cell-mediated cytotoxicity following sensitization or nonspecific stimulation of human effector cells.

Human peripheral blood leukocytes were stimulated in vitro with mitogens (poke-weed mitogen, concanavalin A, and phytohemagglutinin) or allogeneic cells. Each form of stimulation augmented the cytotoxic effector cell activity of lymphoid cells in a 4-hr test for antibody-dependent cell-mediated cytotoxicity. This augmented activity did not involve release of detectable nonspecific toxins, nor did it require the presence of mitogen during the cytotoxicity test. Stimulated attacking cells appeared more cytotoxic either because of a more potent cytotoxic mechanism per individual cytotoxic cell or because of an increased percentage of cytotoxic cells.

Bacterial lipopolysaccharide acts synergistically with selected macromolecular polyanions to induce MHC-nonrestricted cytotoxic cells.

We examined whether bacterial lipopolysaccharide, at a dose range extending to less than 1.0 ng/ml, would work with cofactors to induce MHC-nonrestricted cytotoxic cells. To this end, normal mouse splenocytes were cultured for 5 days with LPS and potential cofactors, after which the cells were tested for cytotoxic activity in short-term 51Cr-release assays. We found that LPS can act synergistically with the macromolecular polyanions, dextran sulfate and polyinosinic acid. The effector cells induced by LPS and polyanions showed characteristics of activated NK cells in that they were (1) cytotoxic for widely differing sources of tumor cells, (2) not inhibited by an anti-T cell receptor antibody, and (3) not removed by depletion of CD4+ or CD8+ cells. LPS was active at picogram concentrations when dextran sulfate was included. Exposure of splenocytes to LPS was necessary during the early phase of the 5-day culture, but as little as 1 h of exposure was required, whereas exposure to the macromolecular polyanions during either the first or the last 2 days of a 5-day culture with LPS was effective. As expected with LPS activity, the cytotoxic cell response was prevented by polymyxin B or by the use of splenocytes from LPS non-responder C3H/HeJ mice. Screening of the S. minnesota R mutants and other partial LPS structures revealed that lipid A was closely associated with LPS activity in this assay system and that at least one partially detoxified structure, a deacylated LPS, could substitute for native LPS.

Targeting of anti-tumor responses with bispecific antibodies.

T cells can be induced to specifically lyse tumor cells with bispecific antibodies containing anti-T cell receptor mAbs crosslinked to anti-tumor mAbs. Such "targeted cytolysis" requires that the target cell be bound directly to the cytotoxic cell. In addition, targeted T cells mediate a second activity, the secretion of factors that can block the growth of both tumor target cells and bystander tumor cells. When given to nude mice bearing intraperitoneal human ovarian carcinoma, targeted human T cells cause the rapid removal of most tumor cells from the peritoneum, and markedly prolong the times of survival of treated mice. The efficacy of targeted T cells for treating human cancer is currently being tested in clinical trials.

Suppressor cells in tumor-bearing mice capable of nonspecific blocking of in vitro immunization against transplant antigens.

Spleen cells from mice with progressively-growing methyl-cholanthrene-induced tumors, when immunized in vitro against transplant alloantigens, developed less cytotoxic activity against these antigens as measured by a short-term chromium-release assay than did spleen cells from normal mice. The hyporesponsiveness of spleen cells from the tumor-bearing mice seemed to be due to the presence of suppressor cells which could be removed by nylon-column passage but not by anti-theta treatment and which, in mixture experiments, could inhibit the response of normal spleen cells. The suppression appeared to occur at the sensitization stage and not at the effector stage of the in vitro tests. No evidence was found for mediation of the suppression by soluble factors. These observations emphasize the growing importance of suppressive mechanisms in tumor immune systems.

The cellular nature of concanavalin A-stimulated antibody-dependent cell-mediated cytotoxicity.

Enhanced antibody-dependent cytotoxic cell (ADCC) activity, generated in vitro by concanavalin A (Con A) stimulation of human peripheral blood lymphocytes, was analyzed to identify the nature of the killer (K) cell. Stimulated and unstimulated 2-day cultured cells were separated into sheep erythrocyte rosetting (E+) and non-rosetting (E-) subpopulations, and ADCC activity was determined by using the slopes of linear dose-response plots as a measure of K cell activity. Quantitative comparisons were made by examining activity on a per cell basis ("specific" activity) and total K cell activity recovered per subpopulation. This analysis revealed that although the E- cells were enriched for specific ADCC activity, the E+ subpopulation contained the major proportion of the total recovered activity and nearly all of the Con A-induced increase in K cell activity.

Identification of macrophage-like characteristics in a cultured murine tumor line.

A mouse tumor line P388D1 passaged in tissue culture for many years has been characterized morphologically and functionally as a macrophage like cell. P388D1 cells phagocytize latex particles and firmly adhere to glass and plastics. In addition they have been shown to carry cellbound receptors for immunoglobulin (Fc) and complement (C3). They fail to stain with fluorescent anti-mouse Ig or heterologous anti-mouse. Functionally, these cells exhibited high effector activity in an antibody-dependent, cell-mediated cytotoxic system. The accessibility of well characterized and pure macrophage-like cell lines, such as P388D1, will facilitate studies where cell purity is essential.

T cell memory for the cytotoxic response to hapten-modified target cells.

This study describes the development of memory and cytotoxic murine T cells against syngeneic haptne N equals[N-(3-nitro-4-hydroxy-5-iodophenyl-acetyl)-Beta-alanylglycylglycyl] associated antigen. Memory activity in this system had the following characteristics. a) In vitro challenged cells primed in vivo resulted in an augmented cytotoxic response compared to cells primed in vitro. b) The augmented cytotoxic response in vitro was antigen-specific for both target cells in the lytic reaction and stimulator cells in the secondary response. c) Memory activity was long lasting (at least 2 months). d) Memory cells were not cytotoxic. e) Memory activity as well as the cytotoxic cells generated in a secondary response in vitro were T cell dependent, These findings are consistent with the results of others who have investigated T cell dependent memory in other cell-mediated reactions.

Changes in mouse MHC expression during the cell cycle of Con A-treated spleen cells.

This study was directed at correlating the expression of class I MHC determinants with different segments of the cell cycle by using dual laser flow microfluorometry to measure levels of both DNA and cell surface H-2Kd or H-2Dd determinants for single cells. Con A-treated mouse spleen cells were identified as being in the G2/M or G0/G1 phases of the cell cycle on the basis of propidium iodide or Hoechst 33342 dye bound to DNA. Monoclonal anti-H-2 antibodies, indirectly fluoresceinated with goat anti-mouse IgG, were used to detect MHC determinants. The average level of both Kd and Dd determinants expressed by G2/M cells was about 1.6-fold higher than that expressed by G0/G1 cells. These observations indicate that the average-size G0/G1 and G2/M cells have the same apparent surface density of Kd and Dd determinants, insofar as we estimate that these cells differ in surface area by a factor of about 1.5. We also analyzed the expression of Kd and Dd determinants by measuring how they changed as a function of the intensity of forward light scatter from cells. For both G2/M and G0/G1 cells, changes in light scatter intensity were associated with parallel changes in levels of Dd and Kd determinants, indicating a common mechanism(s) that controls their cell surface expression.