The development of recombinant techniques for the rapid cloning, expression, and characterization of cDNAs encoding antibody (Ab) subunits has revolutionized the field of antibody engineering. By fusion to heterologous protein domains, chain shuffling, and inclusion of self-assembly motifs, novel molecules such as bispecific Abs can now be generated which possess the subset of functional properties designed to fit the intended application. Rapid technological developments in phage display of peptides and proteins have led to a plethora of applications directed towards immunology and antibody engineering. Many of the problems associated with the therapeutic use of Abs are being addressed by the application of these new techniques.

Single chain antibody variable region fragments (sFv), by virtue of their size and method of construction are potentially useful as therapeutic reagents and as tools for exploring cell surface receptor function. sFv offer several advantages over the intact immunoglobulin molecule. For instance, they are expressed from a single transcript and can be molecularly linked to other proteins to generate bispecific sFv molecules or single-chain immunotoxins. The relatively small size of sFv is an advantage in allowing for easier penetrance into tissue spaces, and their clearance rate is exceedingly rapid. sFv are useful for gene therapy since they can be directed to a specific cellular localization and can be fused to retroviral env genes to control viral host range. To prepare sFv to murine and human leukocyte CD antigens, we devised a method for rapid cloning and expression that can yield functional protein within 2-3 weeks of RNA isolation from hybridoma cells. The variable regions were cloned by poly-G tailing the first strand cDNA followed by anchor PCR with a forward poly-C anchor primer and a reverse primer specific for constant region sequence. Both primers contain flanking restriction sites for insertion into PUC19. Sets of PCR primers for isolation of murine, hamster and rat VL and VH genes were generated. Following determination of consensus sequences for a specific VL and VH pair, the VL and VH genes were linked by DNA encoding an intervening peptide linker [usually (Gly4Ser)3] and the VL-link-VH gene cassettes were transferred into the pCDM8 mammalian expression vector. The constructs were transfected into COS cells and sFvs were recovered from spent culture supernatant. We have used this method to generate functional sFv to human CD2, CD3, CD4, CD8, CD28, CD40, CD45 and to murine CD3 and gp39, from hybridomas producing murine, rat, or hamster antibodies. Initially, the sFvs were expressed as fusion proteins with the hinge-CH2-CH3 domains of human IgG1 to facilitate rapid characterization and purification using goat anti-human IgG reagents or protein A. We also found that active sFv could be expressed with a small peptide > or = tag > or = or in a tail-less form. Expression of CD3 (G19-4) sFv tail-less or Ig tailed forms demonstrated increased cellular signalling activity and suggested that sFv have potential for activating receptors.

Cross‐linking of B cell surface immunoglobulins (sIg) results in activation of mature B cells and stimulates a molecular signaling mechanism for antigen‐specific B cell expansion and differentiation. This signaling pathway is dependent on tyrosine (Tyr) phosphorylation and results in the activation of sIg‐associated src family kinases and p72SYK. Rapid Tyr phosphorylation occurs on multiple protein substrates. Here we show that activation of B cells by cross‐linking sIg results in an increase in Tyr phosphorylation of the lineage‐restricted B cell surface antigen CD19, and show that it is a major substrate of activated Tyr kinase following sIg stimulation. Lower levels of constitutive CD19 Tyr phosphorylation occurred in most sIg+ mature B cell lines examined and in normal dense tonsillar B cells. We also find that when CD19 is Tyr‐phosphorylated it becomes competent to interact with SH2 domains suggesting a mechanism whereby, following B cell activation, CD19 could be linked to intracellular signaling pathways. In sIg‐ pre‐B cell lines, CD19 was expressed but was not constitutively phosphorylated on tyrosine. Upon CD19 cross‐linking, Tyr phosphorylation of CD19 was induced in sIg‐ pre‐B cell lines. CD19 cross‐linking also directly induced Tyr phosphorylation of CD19 and other substrates in mature B cells. The ability of CD19 to signal in the absence of sIg expression may provide important stimulation in pre‐B cell development.

UV radiation is known to induce lymphocyte nonresponsiveness both in vitro and in vivo. We have found that UV radiation rapidly induced tyrosine phosphorylation and calcium signaling in normal human peripheral blood lymphocytes. In the leukemic T cell line Jurkat and the Burkitt's lymphoma cell line Ramos, UV rapidly induced tyrosine phosphorylation in a wavelength-dependent manner, giving strong signals after UVB and UVC, but not UVA, irradiation. Similarly, in Jurkat cells UV-induced calcium signals were dependent on the dose of UVB or UVC irradiation over a range of 150-1200 J/m2, but only a small signal was observed for UVA at a dose of 1200 J/m2. The UV-induced calcium signals were blocked by the tyrosine kinase inhibitor herbimycin A, indicating that they were dependent on tyrosine phosphorylation. Phospholipase C (PLC) gamma 1 was tyrosine phosphorylated in response to UV irradiation but to a lesser extent than observed after CD3 cross-linking. However, PLC gamma 1-associated proteins demonstrated to bind to the PLC gamma 1 SH2 domain were tyrosine phosphorylated strongly after UV irradiation. A similar dose response was observed for the inhibition by herbimycin A of UV-induced calcium signals and UV-induced tyrosine phosphorylation of PLC gamma 1 and associated proteins. We propose that in contrast to CD3/Ti stimulation, UV aberrantly triggers lymphocyte signal transduction pathways by a mechanism that bypasses normal receptor control.

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