Different approaches for obtaining antibodies from human B cells.

Antibodies have emerged as powerful therapeutics effective for treating a number of human conditions and diseases. While early successes utilized small animals to generate therapeutic antibodies, human antibodies are now preferred in order to limit anti-antibody immune responses. Antibodies with human amino acid sequences can be generated in a number of ways, such as humanizing antibodies from other species or expressing human antibodies in transgenic animals. This review focuses on methods for obtaining antibodies directly from human B cells. These methods use both antigen exposed and non-exposed ("naïve") humans as B cell sources, and apply various technologies to isolate desired antibodies; including cell line generation, single cell isolation, display technologies, and B cell library generation.

Specific inhibition of the classical complement pathway with an engineered single-chain Fv to C1q globular heads decreases complement activation by apoptotic cells.

Apoptotic cells are potent complement activators; and proposed mechanisms include IgM-mediated classical pathway activation, C-reactive protein (CRP)-mediated classical pathway activation, and IgM-mediated lectin pathway activation. While complement activation is beneficial in clearing apoptotic cells, the resulting complement-mediated inflammation may extend damage to the surrounding cells and tissues, as observed in ischemia/reperfusion injury. We previously engineered and characterized a single-chain Fv against C1q globular heads (scFv(QuVHVL)) that blocked C1q binding to immobilized IgG and to IgG-sensitized cells, and thereby inhibited IgG-mediated classical pathway activation [Hwang H.Y., Duvall M.R., Tomlinson S., Boackle R.J., 2008. Highly specific inhibition of C1q globular-head binding to human IgG: a novel approach to control and regulate the classical complement pathway using an engineered single-chain antibody variable fragment. Molecular Immunology 45, 2570-2580]. In the present study, this scFv(QuVHVL) was examined for its ability to restrict complement deposition on apoptotic cells in the presence of fresh normal human serum (NHS). Interestingly, the addition of scFv(QuVHVL) to NHS decreased C1-mediated C4b deposition on apoptotic cells by 60% as compared to appropriate buffer-treated control serum. By inhibiting initiation of the early complement components, the subsequent C3b and membrane attack complex depositions were inhibited by 70%. Apoptotic cells may acquire serum CRP, a known classical complement pathway activator. It was observed that scFv(QuVHVL) blocked C1 binding to CRP and blocked CRP-mediated classical pathway activation using an ELISA format. However, under the experimental conditions used, the addition of exogenous CRP to apoptotic cells did not further increase the levels of C4b, C3b, or MAC deposition significantly, suggesting predominance by other activation mechanisms, such as antibody-C1-mediated complement activation. In summary, the results indicated that C1-mediated classical pathway activation was a highly significant mechanism for complement activation by apoptotic cells. In the future, specific inhibition of classical complement pathway activation by a humanized form of scFv(QuVHVL) may be useful in reducing inadvertent damage to healthy bystander tissue in a variety of acute, complement-mediated inflammatory conditions, including ischemia/reperfusion injury.

Back-burning to cure HIV: temporary depletion of all CD4+ cells and elimination of the extracellular reservoir with HIV immunotoxin therapy.

Temporary elimination of all host cells for the human immunodeficiency virus (HIV) combined with dislodging HIV from its extracellular reservoir could cure acquired immunodeficiency syndrome (AIDS). This combination would be effective because the virus is dependent on host cell integration or on the membrane protection of B cells or of follicular dendritic cells (FDCs) for its survival and because the CD4(+) host cells are leukocytes that are naturally renewable through hematopoiesis. By treating HIV patients with a combination of humanized antibodies it should be possible to achieve both goals. To deplete HIV host cells, a humanized antibody against CD4 should be fused to an apoptosis-inducing toxin; and to void the extracellular reservoir, a fragment of a humanized antibody against CD21 should be used. Because only CD4(+) cells would be destroyed, hematopoietic stem cells would be spared, and would spontaneously replace the depleted cells. We call this hypothetical new HIV treatment "HIV Immunotoxin Therapy (HIT)". Once the HIV viral load reaches zero, the HIT would be withdrawn and IL-2 or luteinizing hormone releasing hormone analogues (LHRH-A) might be administered to accelerate the natural replacement of the CD4(+) T(H) cells and macrophages. Killing all HIV host cells may seem counterintuitive at first, because it requires the purposeful destruction of the very cells that we ultimately hope to preserve for AIDS patients, but just as controlled back-burning purposefully creates a trap to stop a wildfire from burning out of control, this method could provide a mechanism to extinguish HIV.

Highly specific inhibition of C1q globular-head binding to human IgG: a novel approach to control and regulate the classical complement pathway using an engineered single chain antibody variable fragment.

We sought to specifically regulate the binding of human C1q, and thus the activation of the first complement component, via the construction of a single chain antibody variable binding region fragment (scFv) targeting the C1q globular heads. Here we describe details of the construction, expression and evaluation of this scFv, which was derived from a high-affinity hybridoma (Qu) specific for the C1q globular heads. The scFv was comprised of the Qu variable heavy chain domain (VH) linked to the Qu variable light chain domain (VL) and was termed scFv-QuVHVL. When mixed with either purified C1q or with human serum as a source of C1, scFv-QuVHVL bound to C1q and competitively restricted the interaction of C1q or C1 with immobilized IgG or with IgG1 antibody-coated cells, and prevented the activation of native C1 in human serum as determined by analyses of C1-mediated C4 deposition and fluid-phase C4 conversion. However scFv-QuVHVL could be manipulated to become a C1 activator when it was irreversibly immobilized onto microtiter ELISA plates, prior to contact with human serum complement. This functional dichotomy can be a useful tool in selectively elucidating, differentiating, inducing or inhibiting specific roles of human C1q and the classical complement pathway in complement-mediated physiological processes. We project that once fully humanized, fluid-phase scFv-QuVHVL could become a useful therapeutic in limiting inadvertent host tissue damage elicited by the classical complement pathway.