A Phase I Trial of B7-Transfected or Parental Lethally Irradiated Allogeneic Melanoma Cell Lines to Induce Cell-Mediated Immunity Against Tumor-Associated Antigen Presented by HLA-A2 or HLA-A1 in Patients with Stage IV Melanoma. National Institutes of Health,Bethesda,Maryland

Abstract

Technical Abstract

A phase I trial of B7-transfected allogeneic melanoma cell lines to induce cell-mediated immunity against tumor-associated antigens presented by HLA-A2 or HLA-A1 in patients with stage IV melanoma.

BRMP 9401

NCI Protocol T93-0161

Biological Response Modifiers Program, DCT, NCI

The scientific basis for this protocol is derived from two main points. Recent data indicate that two stimuli are required to activate T cells to proliferate and secrete cytokines. Signal one is provided by the T cell receptor, and the other requires interaction of other T cell surface receptors with their ligants on antigen presenting cells. The second point, is that in the case of human melanoma, tumors from many patients express the same tumor antigens, and that peptides derived from these tumor antigens are often presented on tumor cell surfaces by MHC class I molecules HLA-A1 or A2. Thus, it may be possible to induce immunity against these shared melanoma antigens by vaccination with allogeneic tumor cell lines that express the tumor antigens and are HLA-A1 or HLA-A2 positive. In order to study these questions in a clinical trial of advanced stage melanoma patients, we introduced the gene encoding the T cell co-stimulatory molecule B7 (CD28 lignad) into 3 human melanoma cell lines. These tumor lines are HLA-A2⁺ (one is HLA-A1⁺), express significant levels of LFA-3 and ICAM-I (and in one case HLA-DR) on their surfaces, and produce RNAs encoding the shared melanoma antigens MAGE-1 and -3, and tyrosinases. We have examined the stimulatory capacity of parental tumor lines and B7-transfectants (DM150/B7-8, DM13/B7-7, and DM93/B7-4) in 7 day primary mixed lymphocyte cultures (MLC) with allogeneic human T cells obtained from PBL of normal donors or melanoma patients. In multiple experiments, parental tumor lines (which do not express detectable B7) fail to induce significant activation of allogeneic T cells as determined by lack of increased expression of HLA-DR or CD25 on T cell surfaces. In contrast, B7-transfected lines induce increased expression of HLA-DR and CD25 (in both CD4⁺ and CD8⁺ subsets), and a 5-10 fold increase in T cell number compared to cultures with parental cell lines. CTL induction in primary MLC was determined in this system by 7d co-culture of allogeneic T cells with the parental DM150 or DM150-B7 cell lines. While T cells cultured with DM150 exhibit only background levels of lytic activity, T cells cultured with DM150-B7 lyse this line, and also the unmodified parental line and the HLA-A2+ DM13 cell line. TIL lines that recognize the shared melanoma antigen(s) presented by HLA-A2 lyse all three parental lines, providing functional data that these lines present shared melanoma antigens via the endogenous MHC class I pathway. These data indicate: 1. B7 is expressed by all three lines and is biologically functional as assessed by the ability to activate resting human T cells. 2. The cell lines express the genes encoding the known shared melanoma antigens and are lysed by HLA-A2 specific TIL derived from melanoma patients, demonstrating functional expression of the shared melanoma antigens by these lines. These studies form the preclinical basis for a vaccine trial in which patients will be vaccinated with lethally irradiated allogeneic melanoma cell lines genetically engineered to express human B7. The three lines will be injected subcutaneously at two week intervals for three vaccinations, followed by 3 injections at monthly intervals. The cell lines will be given on a rotating basis. Cohorts of patients will receive excalating doses of 10⁷, 10⁸, or 10⁹ cells. This is a Phase I trial to determine the MTD of this therapy, but immunologic parameters such as generation of CTL precursors in peripheral blood and draining lymph nodes will also be monitored.

Non-Technical Abstract-BRMP 9401

A phase I trial of B7-transfected allogeneic melanoma cell lines to induce cell-mediated immunity against tumor-associated antigens presented by HLA-A2 or HLA-A1 in patients with stage IV melanoma.

BRMP 9401

NCI Protocol T93-0161

Biological Response Modifiers Program, DCT, NCI

It is possible for the body's immune system to recognize and reject some cancers. This probably occurs when blood immune cells called lymphocytes recognize an abnormal protein (called an antigen) on the tumor cell, attach to the tumor, and then either kill the tumor or call other immune cells to the site to help eliminate the cancer cells. Scientists have recently discovered that the tumors from some patients with melanoma contain distinct antigens that can be recognized by lymphocytes. However, for reasons that we still do not understand, in most patients with melanoma the immune system has somehow been paralyzed and cannot recognize or attack the tumor.

It may be possible to activate the immune system to recognize the anitgens on melanoma cells. We now understand that if lymphocytes see an antigen on a tumor cell but don't receive an improtant second signal (which is not normally provided by the melanoma cell), the lymphocyte will be turned off. We have grown tumor cell lines in the laboratory that contain some of the melanoma antigens. We have taken a gene (a gene is the DNA that directs the production of a protein in a cell) which codes for a moledule called B7 and placed it in the melanoma tumor cell lines we have in the laboratory. We believe that the B7 will provide a necessary second signal to activate lymphocytes to recognize the melanoma tumor antigens. Once activated, the lymphocytes can recognize and kill other melanoma cells even if they do not express the B7 signal.

In order to administer treatment, we match the patient to the tumor cell lines we have in the laboratory. We do this by determining the expression of specific proteins on the surface of patient lymphocytes that we obtain prior to the study. Only patients whose lymphocytes express the proteins HLA-A1 or HLA-A2 can receive treatment on this study.

For treatment, we plan to inject a large number of cells from the tumor cell lines containing B7 under the skin. The cells are radiated so there is little chance that a tumor might grow where we inject the cells. The tumor cells are injected every two weeks for 3 doses, then once a month for 3 doses. We rotate the sites of injection, usually starting in the leg, then the arm, then the opposite arm, then the opposite leg, and so on. HLA-A1 patients receive the same dose of the same cell line each time. HLA-A2 patients will alternately receive one of three cell lines. In either group, a maximum of 6 treatments is planned.

We will treat groups of patients with each group receiving a larger number of cells. The purpose of the study is to determine how many cells we can give safely and whether an immune response develops to the cells as we predict from the laboratory studies. In the first or second group of patients, 2 injections are given (about 2 inches apart) each treatment. In the third group, 10 injections are given each time. We do not know if the B7 actually improves the immune response to the cells. Therefore, the last group will receive the same tumor cell line(s) but not containing B7.