ALBERT

Description: Relapse is the most common cause of treatment failure for advanced cancer, even those treated with autologous hematopoietic cell transplantation (HCT). Effective tumor-specific immunotherapy may decrease relapse, however, this will fail if the immune system is not able to respond appropriately. To test the integrity of the immune response, we vaccinated 27 normal volunteers, 14 patients after HCT, and 19 patients with advanced cancer with a single injection of KLH, a neo-antigen that humans have not been exposed to previously. KLH (Intracel, Rockville, MD or Biosyn Corp, Carlsbad, CA) was given as a single 1 mg subcutaneous injection. Immune response was measured by ELISA, proliferation or ELISPOT (validated for cryopreserved cells) assays on blood 1 month after vaccination compared to preimmunization. Intracel KLH (containing the intact KLH molecule, 6000–8000 kDA) induced a potent IgM, IgG1 and IgG2 response as well as a cellular response measured by proliferation assay in normal volunteers (n=17). Because of sporadic availability, Biosyn KLH, produced as 350/390 kDA subunits of KLH, was then tested. Biosyn KLH alone failed to induce either a humoral or cellular response. We hypothesized that lack of a response to a single Biosyn KLH immunization was due to the product purity and decreased complexity of size resulting in loss of adjuvant properties compared to the intact KLH product. Biosyn KLH was then mixed with the oil/surfactant Montanide ISA-51, which completely restored immunogenicity to a single 1 mg KLH test vaccine in normal subjects (n=10) similar to the Intracel intact KLH product. In marked contrast to the normal response, humoral responses were significantly impaired in 11 patients 58–144 [median 103] days after autologous HCT and in 1 as late as 16 months after HCT. This latter patient was the only one who mounted a cellular response to KLH after HCT. Patients with metastatic melanoma and renal cell carcinoma exhibited an intermediate response (Table). The decreased KLH-specific IgG2 response after HCT was partially explained by the decreased total IgG2 levels after transplant as compared to normal volunteers and solid tumor patients. Total IgG1 and IgM immunoglubulin levels were not significantly different between groups. The defect in isotype switching correlated with decreases in absolute CD4 counts between the normal (891±58/mm3), solid tumor (501±73/mm3) and HCT (290±119/mm3) cohorts. We conclude that patients with cancer and especially those patients after autologous HCT have immune defects significantly impairing neo-antigen responses. This has important implications for tumor vaccine strategies in these settings. KLH vaccines are safe, and 100% of normal volunteers require only a single injection to test cellular and humoral neo-antigen responses including isotype switching, an optimal platform for definitive testing of strategies to promote immune reconstitution after HCT. Response to KLH 1 month after vaccination. Readout Normal (n=27) Solid Tumor (n=19) HCT (n=14) * values are mean ± SEM in mcg/ml; # reported as spots/million cells IgM* 7.7 ± 1.3 7.6 ± 2.1 1.4 ± 0.28 IgG1* 234 ± 50 37.3 ± 13.2 7.9 ± 4.5 IgG2* 34 ± 5.1 12.7 ± 4.1 0.67 ± 0.23 ELISPOT# 311 ± 61 (n=10) Not done 14 ± 9 (n=7)

Description: Cytotoxic T lymphocytes (CTLs) specific for hematopoietic-restricted minor histocompatibility antigens (mHags) are important reagents for adoptive immunotherapy of relapsed leukemia after allogeneic stem cell transplantation. However, expansion of these CTLs to therapeutic numbers is often hampered by the limited supply of antigen-presenting cells (APCs). Therefore, we evaluated whether cell-sized latex beads coated with HLA/mHag complexes HLA-A2/HA-1 or HLA-A2/HA-2 and recombinant CD80 and CD54 molecules can replace professional APCs. The artificial antigen-presenting constructs (aAPCs) effectively stimulated HA-1– and HA-2–specific CTL clones as shown by ligand-specific expansion, cytokine production, and maintenance of cytotoxic activity, without alteration of CTL phenotype. Furthermore, HA-1–specific polyclonal CTL lines were enriched as efficiently by aAPCs as by autologous HA-1 peptide-pulsed dendritic cells. Thus, aAPCs coated with HLA/mHag complexes, CD80, and CD54 may serve as tools for in vitro enrichment of immunotherapeutic mHag-specific CTL lines.