A murine monoclonal antibody (OKT9) raised against human leukemic cells binds to a wide variety of leukemia and tumor cell lines and to a minority of leukemia cells taken directly from patients. Fetal thymus and liver are strongly reactive as are some normal, immature hemopoietic cells and activated lymphocytes. Reactivity with OKT9 appears to correlate with proliferation status in both normal and malignant populations. Biochemical analysis indicates that this structure is a approximately equal to 180,000-dalton glycoprotein with two disulfide-bonded subunits of approximately equal to 90,000-daltons. Isolation of the transferrin receptor from a T-cell line (MOLT-4) indicates that it also has a dimeric approximately equal to 180,000-dalton structure. Radio-labeled transferrin bound to its receptors can be specifically precipitated by the monoclonal OKT9, although the latter does not bind transferrin itself, indicating that the antigenic structure defined by this antibody is likely to be the transferrin receptor.

Human peripheral blood lymphocytes can be transduced to express antigen-dependent CD3zeta chimeric immune receptors (CIRs), which function independently of the T-cell receptor (TCR). Although the exact function of these domains is unclear, previous studies imply that an extracellular spacer region is required for optimal CIR activity. In this study, four scFvs (in the context of CIRs with or without extracellular spacer regions) were used to target the human tumor-associated antigens carcinoembryonic antigen (CEA), neural cell adhesion molecule (NCAM), the oncofetal antigen 5T4, and the B-cell antigen CD19. In all cases human T-cell populations expressing the CIRs were functionally active against their respective targets, but the anti-5T4 and anti-NCAM CIRs showed enhanced specific cytokine release and cytotoxicity only when possessing an extracellular spacer region. In contrast, the anti-CEA and anti-CD19 CIRs displayed optimal cytokine release activity only in the absence of an extracellular spacer. Interestingly, mapping of the scFv epitopes has revealed that the anti-CEA scFv binds close to the amino-terminal of CEA, which is easily accessible to the CIR. In contrast, CIRs enhanced by a spacer domain appear to bind to epitopes residing closer to the cell membrane, suggesting that a more flexible extracellular domain may be required to permit the efficient binding of such epitopes. These results show that a spacer is not necessary for optimal activity of CIRs but that the optimal design varies.

The L1 adhesion molecule is a member of the immunoglobulin (Ig) superfamily initially identified in the nervous system which contains six Ig-like domains. Besides the known L1-L1 homotypic interaction, L1 was recently shown to bind to very late antigen (VLA)-5 in the mouse and alpha v beta 3 in the human. The sixth Ig domain is critical for this function. We now demonstrate that human CD4+ peripheral blood T lymphocytes, monocytes and B lymphocytes, but not CD8+ T lymphocytes, express L1. When compared to the expression of CD31, another ligand for alpha v beta 3 on T lymphocytes, only a small proportion of cells were CD31+L1+ double positive. L1 was also detected on the surface of human monocytic and lymphoid tumor lines and was shown to have a molecular mass of approximately 220 kDa, similar to the molecule present on neuroblastoma cells. The function of the sixth Ig domain of human L1 as an integrin ligand was also investigated. Using an RGD-containing peptide derived from the sixth Ig domain as well as a fusion protein of the sixth Ig domain of L1 and the Fc portion of human IgG1 (6.L1-Fc), we demonstrated the binding of human MED-B1 (alpha v beta 3hi, alpha 5 beta 1lo) tumor cells and this binding was blocked by alpha v-specific mAb. In contrast, human Nalm-6 cells (alpha v beta 3lo, alpha 5 beta 1hi) did not bind to the 6.L1-Fc fusion protein. MED-B1 cells could also be stained with the 6.L1-Fc fusion protein. Our results suggest that human L1 binds predominantly to alpha v beta 3 and that its presence on leukocytes could be important for adhesion and migration.

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A novel in situ hybridization technique is described. This non-radioactive technique combines, for the first time, the high spacial resolution and rapid signal development of the non-isotopic approach with the previously unrivalled sensitivity of autoradiography. The procedure, which employs biotin labelled DNA probes and a streptavidin-alkaline phosphatase based detection system, is compatible with pre G-banding and can be performed on archival material. Unique sequences as small as 1 Kb are detectable. Using this technique, we have mapped the N-myc oncogene and the gene for beta-Nerve Growth Factor to 2p24 and 1p13 respectively.