Adel L. Barsoum

Identification of HLA-A*0201-Presented T Cell Epitopes Derived from the Oncofetal Antigen-Immature Laminin Receptor Protein in Patients with Hematological Malignancies

The oncofetal Ag immature laminin receptor (OFA-iLR) is a potential target molecule for immunotherapeutic studies in several tumor entities, including hematological malignancies. In the present study, we characterize two HLA-A*0201-presented epitopes eliciting strong OFA-iLR peptide-specific human cytotoxic T cell (CTLs) responses in vitro. Both allogeneic HLA-A*0201-matched and autologous CTLs recognized and killed endogenously OFA-iLR-expressing tumor cell lines and primary malignant cells from patients with hemopoietic malignancies in an MHC-restricted fashion but spared nonmalignant hemopoietic cells. Spontaneous OFA-iLR peptide-specific T cell reactivity was detectable in a significant proportion of leukemia patients. Interestingly, in patients with chronic lymphocytic leukemia and multiple myeloma but not in those with acute myeloid leukemia, significant frequencies of OFA peptide-specific CTLs could be detected in an early stage of disease but disappeared in patients with progressive disease. The identification of OFA-iLR-derived peptide epitopes provides a basis for tumor immunological studies and therapeutic vaccination strategies in patients with OFA-iLR-expressing malignancies.

EXPRESSION AND IMMUNOGENICITY OF ONCOFETAL ANTIGEN-IMMATURE LAMININ RECEPTOR IN HUMAN RENAL CELL CARCINOMA

PURPOSE The 32 to 44 kDa. oncofetal antigen-immature laminin receptor (OFA-iLR) is a multifunctional protein expressed by various tumors, including breast, lung, ovary and prostate carcinoma as well as lymphoma. OFA-iLR has been implicated in tumor invasiveness, metastasis and growth. Interferon-gamma producing effector T cells and interleukin (IL)-10 producing suppressor T cells specific for OFA-iLR have been described. MATERIALS AND METHODS The 43515 IgG2a anti-OFA-iLR monoclonal antibody was used to detect OFA-iLR expression in human renal cell carcinoma tissue by flow cytometry and immunoblotting. Spontaneous or therapy induced immune responses against OFA-iLR were determined in patients with metastatic renal cell carcinoma. Proliferative and cytokine (interferon-gamma and IL-10) responses of peripheral blood mononuclear cells from patients with renal cell carcinoma against recombinant OFA-iLR were assessed. RESULTS Using flow cytometry OFA-iLR was detected in all 13 tumors tested. Immunoblotting revealed differences in OFA-iLR expression in renal cell carcinoma and normal kidney tissue. OFA-iLR specific proliferative and cytokine responses of mononuclear cells were detected in all 6 patients tested. Importantly evidence was also obtained that treating metastatic renal cell carcinoma with tumor lysate pulsed dendritic cells would enhance OFA-iLR specific immunity. CONCLUSIONS This study demonstrates that OFA-iLR is an immunogenic tumor associated antigen in human renal cell carcinoma. OFA-iLR specific effector T cells producing interferon-gamma may have a role in the control of tumor growth, whereas suppressor T cells producing IL-10 may promote tumor tolerance and, thus, tumor progression.

Tumors express both unique TSTA and crossprotective 44 kDa oncofetal antigen.

Abstract Tumors of inbred rodents and humans generally express a shared tumor-associated transplantation antigen (TATA)-like oncofetal antigen (OFA) and tumor-specific transplantation antigen (TSTA), which stimulate cytotoxic T lymphocyte (CTL) tumor rejection responses. Immunoregulatory CD8 + OFA-specific suppressor T cells are also stimulated, which promote tumor development via interleukin 10 secretion, inhibiting CTL function. Here, Joseph Coggin and colleagues argue that future investigations must distinguish responses to OFA and TSTA.

Humoral Immune Responses against the Immature Laminin Receptor Protein Show Prognostic Significance in Patients with Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is characterized by a highly variable clinical course. The role of an autologous tumor-specific immune control contributing to the variable length of survival in CLL is poorly understood. We investigated whether humoral immunity specific for the CLL-associated Ag oncofetal Ag/immature laminin receptor (OFA/iLR) has a prognostic value in CLL. Among sera of 67 untreated patients with CLL, 23 (34.3%) had detectable OFA/iLR Abs that were reactive for at least one specific OFA/iLR epitope. Patients with humoral responses compared with patients with nonreactive sera had a longer progression-free survival (p = 0.029). IgG subclass analyses showed a predominant IgG1 and IgG3 response. OFA/iLR Abs were capable of recognizing and selectively killing OFA/iLR-expressing CLL cells in complement-mediated and Ab-dependent cellular cytotoxi cityassays. In the analysis of 11 CLL patients after allogeneic hematopoetic stem cell transplantation, 8 showed high values for OFA/iLR Abs that specifically recognized the extracellular domain of the protein, suggesting a potential role of anti-OFA/iLR-directed immune responses to the graft-vs-leukemia effect in CLL. Our data suggest that spontaneous tumor-specific humoral immune responses against OFA/iLR exist in a significant proportion of CLL patients and that superior progression-free survival in those patients could reflect autologous immune control.

Identification of Oncofetal Antigen/Immature Laminin Receptor Protein Epitopes That Activate BALB/c Mouse OFA/iLRP-Specific Effector and Regulatory T Cell Clones

During tumor development in mice and humans, oncofetal Ag/immature laminin receptor (OFA/iLRP)-specific Th1, CTL, and IL-10-secreting T (Ts) cells are induced. The presence of too many Ts or too few effector T cells appears to predict a poor prognosis. We established clones of OFA/iLRP-specific splenic Th1, CTL, and Ts cells from the OFA/iLRP+ MCA1315 fibrosarcoma-bearing BALB/c mice or from BALB/c mice vaccinated with 1 or 10 μg of rOFA/iLRP. The MCA1315 tumor cell-reactive T cell clones were characterized as to surface Ag phenotype, cytokine secretion profile, and specificity for OFA/iLRP presented by syngeneic splenic APC. OFA/iLRP-specific Th1 and Ts clones were established from all mice. OFA/iLRP-specific CTL could be established from all mice except for mice immunized with 10 μg of rOFA/iLRP. Analysis of the proliferation profile of the OFA/iLRP-specific clones to overlapping OFA/iLRP 12-mer peptides that spanned the OFA/iLRP protein sequence defined the epitopes to which the T cell clones responded. There was a similar spatial distribution of the epitopes to which the two types of CD8 T cell clones responded. The nonapeptide epitopes of the Ts clones were located between aa 36 and 147 of OFA/iLRP, while the epitopes of the CTL clones were located between aa 52 and 163. Even though the CTL and Ts epitopes shared part of the protein, all of the CD8 CTL epitopes were distinct and separable from those of CD8 Ts cells.

High expression of the immature laminin receptor protein correlates with mutated IGVH status and predicts a favorable prognosis in chronic lymphocytic leukemia

The immature laminin receptor (iLR) is a tumor-associated antigen. We analyzed the expression of iLR on malignant B cells of 134 unselected patient samples with CLL and hypothesized that iLR expression would have prognostic significance due to a differential expression pattern. High ILR expression (cut-off value 30%) was correlated with mutated IGVH status (p<0.0001). Patients with high iLR-expression had a significantly longer time to progression (p=0.039). Combination of CD38, ZAP-70, and iLR by flow cytometry can be used to construct a diagnostic score identifying patients with a median progression free survival of 80 months, if no adverse marker is present.

Production, safety and antitumor efficacy of recombinant Oncofetal Antigen/immature laminin receptor protein.

We describe here for the first time an efficient high yield production method for clinical grade recombinant human Oncofetal Antigen/immature laminin receptor protein (OFA/iLRP). We also demonstrate significant antitumor activity for this protein when administered in liposomal delivery form in a murine model of syngeneic fibrosarcoma. OFA/iLRP is a therapeutically very promising universal tumor antigen that is expressed in all mammalian solid tumors tested so far. We have cloned the human OFA/iLRP cDNA in a bacterial expression plasmid which incorporates a 6x HIS-tag. Large scale cultures of the plasmid transformed Escherichia coli were performed and the crude HIS-tagged OFA/iLRP was isolated as inclusion bodies and solubilized in guanidine chloride. The protein was then purified by successive passage through three column chromatography steps of immobilized metal affinity, anion exchange, and gel filtration. The resulting protein was 94% pure and practically devoid of endotoxin and host cell protein. The purified OFA/iLRP was tested in mice for safety and efficacy in tumor rejection with satisfactory results. This protein will be used for loading onto autologous dendritic cells in an FDA approved phase I/II human cancer vaccine trial in OFA/iLRP-positive breast cancer patients.

True Immunogenicity of Oncofetal Antigen/Immature Laminin Receptor Protein

Su et al . [(1)][1] described the detection of RNA present in renal cell carcinoma (RCC) that achieved expression of oncofetal antigen (OFA) in RCC patient’s monocytes and gave rise to OFA-expressing dendritic cells ex vivo . When these OFA-expressing dendritic cells were infused back into

Requirement of TPO/c-mpl for IL-17A-induced granulopoiesis and megakaryopoiesis

IL‐17A is a critical, proinflammatory cytokine essential to host defense and is induced in response to microbial invasion. It stimulates granulopoiesis, leading to neutrophilia, neutrophil activation, and mobilization. TPO synergizes with other cytokines in stimulating and expanding hematopoietic progenitors, also leading to granulopoiesis and megakryopoiesis, and is required for thrombocytopoiesis. We investigated the effects of in vivo expression of IL‐17A on granulopoiesis and megakaryopoisis in TPO receptor c‐mpl−/− mice. IL‐17A expression expanded megakaryocytes by 2.5‐fold in normal mice but had no such effect in c‐mpl−/− mice. The megakaryocyte expansion did not result in increased peripheral platelet counts. IL‐17A expression did not impact bone marrow precursors in c‐mpl−/− mice; however, it expanded splenic precursors, although to a lesser extent compared with normal controls (CFU‐HPP). No peripheral neutrophil expansion was observed in c‐mpl−/− mice. Moreover, in c‐mpl−/− mice, release of IL‐17A downstream cytokines was reduced significantly (KC, MIP‐2, GM‐CSF). The data suggest that IL‐17A requires the presence of functional TPO/c‐mpl to exert its effects on granulopoiesis and megakaryopoiesis. Furthermore, IL‐17A and its downstream cytokines are important regulators and synergistic factors for the physiologic function of TPO/c‐mpl on hematopoiesis.

Monoclonal antibodies specific for oncofetal antigen – immature laminin receptor protein: Effects on tumor growth and spread in two murine models

The oncofetal antigen – immature laminin receptor protein (OFA/iLRP) has been linked to metastatic tumor spread for several years. The present study, in which 2 highly-specific, high-affinity OFA/iLRP-reactive mouse monoclonal antibodies were examined for ability to suppress tumor cell growth and metastatic spread in the A20 B-cell leukemia model and the B16 melanoma model, provides the first direct evidence that targeting OFA/iLRP with exogenous antibodies can have therapeutic benefit. While the antibodies were modestly effective at preventing tumor growth at the primary injection site, both antibodies strongly suppressed end-organ tumor formation following intravenous tumor cell injection. Capacity of anti-OFA/iLRP antibodies to suppress tumor spread through the blood in the leukemia model suggests their use as a therapy for individuals with leukemic disease (either for patients in remission or even as part of an induction therapy). The results also suggest use against metastatic spread with solid tumors.