Julie A. Titus

Correct disulfide pairing and efficient refolding of detergent-solubilized single-chain Fv proteins from bacterial inclusion bodies

In vitro folding of denatured proteins has remained an inefficient and empirical process that has limited the use of bacterially expressed recombinant proteins. In this paper we show that in vitro folding of recombinant single-chain Fv (sFv) proteins is markedly facilitated when disulfide bonds are formed in detergent solution. sFv proteins from three different antibodies were expressed as bacterial cytoplasmic inclusion bodies and solubilized in the weak ionic detergent, sodium lauroylsarcosine (SLS). Upon oxidation in air in the presence of metal ion catalysts, all three sFvs quantitatively formed intrachain disulfide bonds which ran as a single band in SDS-polyacrylamide gel electrophoresis under non-reducing conditions. By contrast, oxidation from 6 M urea gave large amounts of disulfide linked aggregates, and three closely spaced bands of monomeric protein. Detergent was removed from the oxidized sFvs by addition of 6 M urea and absorption with an ion exchange resin. After dialysis and gel filtration in non-denaturing solution, moderate to high yields of monomeric sFv were obtained, depending upon the sFv. All three sFvs gave single bands on isoelectric focussing and SDS-PAGE gels and had similar or identical binding specificities and affinities as the parental Fabs, implying that the final products contained correctly paired disulfide bonds. The correct disulfide pairing suggests that the disulfide loops within the detergent-solubilized sFvs adopt a native-like structure.

The role of non-immune IgG in controlling IgG-mediated effector functions.

The majority of evidence supports the conclusion that IgG-dependent effectors respond to antibodies which have been polymerized artificially or by polyvalent antigens, but not to monomeric IgG antibodies. Effectors can distinguish polymerized IgG antibodies from monomeric IgG because they contain multiple receptor units and can interact multivalently with polymerized IgG. However, monomeric IgG is present at very high concns in plasma and interstitial fluids and will inhibit multivalent interactions in vivo between polymerized antibody and effectors. Such inhibition raises the question of how IgG-mediated effector responses could function in vivo. In this review we present a mathematical model which quantitatively predicts how polyvalent ligands interact multivalently with receptors in the presence of excess monovalent ligand. We then show that results from experiments in vitro using such diverse systems as the binding and endocytosis of immune complexes by macrophages, complement-mediated lysis of antibody-coated target cells, and ADCC can be explained qualitatively by the model. We conclude that monomeric IgG does not totally inhibit IgG-mediated effector functions but, rather, raises the threshold of antibody binding which is required to elicit a response. We then consider how non-immune IgG may serve as a homeostatic regulator of IgG-dependent responses, in vivo, perhaps for the purpose of inhibiting responses to low levels of cell-bound IgG autoantibodies.

A single-chain bispecific Fv2 molecule produced in mammalian cells redirects lysis by activated CTL

Single-chain Fv (sFv) molecules consist of the two variable domains of an antibody (Ab) connected by a polypeptide spacer and contain the binding activities of their parental antibodies (Abs). In this paper we have attached the C-terminus of 2C11-sFv (anti-mouse CD3 epsilon-chain) to the N-terminus of OKT9-sFv (anti-human transferrin receptor [TfR]) through a 23 amino acid inter-sFv linker consisting primarily of CH1 region residues from 2C11, to form a single-chain bispecific Fv2 [bs(sFv)2] molecule. The bs(sFv)2 was expressed in COS-7 cells, and was secreted at the same rate as the two parental sFvs. The secreted protein had both anti-CD3 and anti-TfR binding activities. Essentially all of the secreted bs(sFv)2 molecules bound TfR and the binding affinity of the bs(sFv)2 was comparable to that of OKT9 sFv and Fab. Thus, the attachment of the inter-sFv linker to the N-terminus of OKT9-sFv did not impair its binding function. The bs(sFv)2 retained both binding specificities after long-term storage at 4 degrees C or overnight incubation at 37 degrees C. It redirected activated mouse CTL to specifically lyse human TfR+ target cells at low (ng/ml) concentrations and was much more active than a chemically cross-linked heteroconjugate prepared from the same parental mAbs. Because bs(sFv)2 molecules secreted by mammalian cells are homogeneous proteins containing two binding sites in a single polypeptide chain, they hold great promise as an easily obtainable, economic source of a bispecific molecule suitable for in vivo use.

Targeted cytotoxic cells as a novel form of cancer immunotherapy.

Cellular cytotoxicity is mediated by receptors on the surfaces of cytotoxic cells (lysis promoting receptors) which are specific for cell surface components on target cells. Such receptors mediate the formation of conjugates between effector and target cells, and transduce signals which cause the cytotoxic ceils to deliver “lethal hits” to the bound target cells. Subsequently, the two cells separate, leaving moribund target cells and cytotoxic cells which are free to lyse other target cells (Martz, 1977; Henkart, 1985). This process can be artificially manipulated by using heterocross-linked antibodies which contain an antibody against the lysis promoting receptor linked to an antibody against a surface component on a perspective target cell (Karpovsky et al., 1984; Staerz et al., 1985; Perez et al., 1985; Segal and Wunderlich, 1987). Such antibody heteroconjugates can bind a target cell which would not normally be lysed to the lysis promoting receptor on the cytotoxic cell and signal the cytotoxic cell to deliver a lethal hit. Thus, heteroconjugates can change target specificities of cytotoxic cells. Because monoclonal antibodies (MAb) against pathogenic cells could be incorporated into heteroconjugates, they could have important medical applications. In particular, the authors and others (Perez ef al., 1986; Titus et al., 1987a; Jung et ul., 1986; Lanzavecchia and Scheidegger, 1987; Staerz and Bevan, 1986; Liu et al., 1985) have been interested in using heteroconjugates containing anti-tumor antibodies to direct cellular cytotoxic responses against human cancers. Several classes of cytotoxic cells have been targeted using heteroconjugated antibodies. Antibodies against components of the T-cell receptor complex (either T, or CD3) (Perez et ul., 1985; Staerz et al., 1985) or against CD2 (in man) (Siliciano et al., 1985) and Ly6 (in mouse) (Leo et al., 1987) redirect the specificity of cytotoxic T-cells. Other types of cells have been targeted through PC, receptors: human K-cells through the Fc,RIII (Titus et al., 1987b), also known as CD16, and monocytes and neutrophils through Fc,RI and II (Shen et al., 1986, 1987; Graziano and Fanger. 1987). Lysis mediated by targeted Tand K-cells is enhanced by incubating the effector cells with IL-2 (Titus er al., 1987b), whereas killing by monocytes and neutrophils is boosted by interferon-y (Graziano and Fanger, 1987). In this paper we will summarize our recent studies using targeted Tand K-cells to kill tumor cells, both in the and in vim.

Quantitation of Fc receptors and surface immunoglobulin is affected by cell isolation procedures using plasmagel and Ficoll-Hypaque

When mononuclear leukocytes are isolated directly from whole human blood using Ficoll-Hypaque or Plasmagel, cytophilic immunoglobulin is detected on cell surfaces. Upon incubation at 37 degrees C, this cell-associated immunoglobulin is shed slowly into the medium. However, when cells are prewashed in phosphate-buffered saline prior to isolation, they appear to be free of cytophilic immunoglobulin. Compared to prewashed cells, populations retaining cytophilic immunoglobulin on their surfaces demonstrate a decreased binding of soluble immune complexes and radiolabelled trimeric rabbit IgG. The data suggest that Ficoll-Hypaque and Plasmagel cause serum IgG to bind with abnormally high affinity to human mononuclear leukocytes, probably via Fc receptors. This artifact of preparation can lead to erroneous estimates of the numbers of cells bearing Fc receptors or intrinsic membrane immunoglobulin within a given population of cells and to an inaccurate assessment of the average number of Fc receptors per cell.

Bispecific antibodies retarget murine T cell cytotoxicity against syngeneic breast cancer in vitro and in vivo

Bispecific antibodies with specificity for CD3 and a tumor antigen can redirect cytolytic T cells to kill tumor targets, regardless of their natural specificity. To assess the clinical potential of bispecific antibodies for treatment of human cancers we have, in the present study, adapted a totally syngeneic mouse model to the targeting of mouse T cells against mouse tumors in immunocompetent mice. We show that gp52 of the mouse mammary tumor virus (MTV) can serve as a tumor-specific antigen for redirected cellular cytotoxicity. Chemically crosslinked and genetically engineered bispecific antibodies with specificities for gp52 and murine CD3 ε-chain induced activated mouse T cells to specifically lyse mouse mammary tumor cells from cultured lines and primary tumors from C3H-MTV+ mice. Retargeted T cells also blocked the growth of mammary tumors in vitro as well as their growth in syngeneic mice. These findings identify murine MTV-induced mammary adenocarcinomas as a solid-tumor, animal model for retargetin T cells with bispecific antibodies against syngeneic breast cancer.

Fluorescence flow cytometry in the study of lymphoid cell receptors.

Publisher Summary This chapter describes the way measurements can be made, using cell surface receptors for the Fc portion of IgG (FcR) as an example. In homogeneous cell populations, radioactive assays for receptor number and affinity give meaningful results. However, in heterogeneous cell populations, for example human peripheral blood leukocytcs and murine splenocytcs, average numbers of receptors per cell or average affinities of receptors have little meaning. Direct measures of receptor number and affinity on only those subsets of cells that express the receptor are required. These can be provided by fluorescence assays, especially when the fluorescence is measured quantitatively on a large number of cells by flow cytometry. By using multicolor flow cytometric analyses, receptors can be quantitated on individual subsets of cells, and those subsets can be further identified by virtue of their staining with a second marker, in a single measurement.

Targeting of cytotoxic cells with cross-linked antibody heteroaggregates

Most forms of cellular cytotoxicity are mediated by leukocytes which express specific receptors on their surfaces for target cell surface components. By forming multiple receptor-ligand interactions, target cells are specifically bound to the leukocyte effector cells in effector-target conjugates. Subsequently the target cells are Iysed and released from the effector cells, which are then free to lyse other target cells. The best understood interactions between effector and target cells occur in antibody-dependent cellular cytotoxicity (ADCC)t (Lovchik and Hong, 1977) and in lysis mediated by cytotoxic T cells (T,) (Mar&, 1977; Green and Henney, 1981; Bonavida et al., 1983). In ADCC, the cytotoxic cells bear receptors on their surfaces specific for the Fc portion of IgG (Dickler, 1976; Zuckerman and Douglas 1979; Unkeless et al., 1981). When these cells encounter target cells which are coated with IgG antibodies, lytic conjugates are produced by multiple interactions between FcR on the cytotoxic cells and the Fc portions of the targetbound antibodies (Segal and Stephany, 1984). Tc, by contrast, express a different type of receptor (“T-celi receptor”) by which they recognize MHC molecules (class I or class II) on target cells, either alone or in conjunction with a cell surface target cell antigen, such as a virus (Meuer et al., 1984; Haskins et al., 1984; Reinherz et al.. 1983). The T-cell receptor consists of at least two subcomponents, an antigen specific component, Ti. and a second invariant molecule. T3. In both ADCC and Tc-mediated cytotoxicity, the normal mode of effector-target interaction can be replaced by using appropriate hetero-cross-linked antibody preparations. as shown schematically in

Dialysis and Concentration of Protein Solutions

Conventional dialysis separates small molecules from large molecules by allowing diffusion of only the small molecules through selectively permeable membranes. Dialysis is usually used to change the salt (small‐molecule) composition of a macromolecule‐containing solution. The solution to be dialyzed is placed in a sealed dialysis membrane and immersed in a selected buffer; small solute molecules then equilibrate between the sample and the dialysate. Concomitant with the movement of small solutes across the membrane, however, is the movement of solvent in the opposite direction. There are several simple and relatively inexpensive methods for concentrating protein solutions. Dialysis against Aquacide 11A (Calbiochem), which removes water through the dialysis tubing, may be used. After concentration, the solution must be redialyzed into the appropriate buffer. Another method is to use Immersible‐CX Ultrafilters (Millipore) which, when connected to a vacuum, remove everything below their molecular weight cutoff (MWCO). Alternatively, centrifugal concentrators, which are operated with the aid of ordinary laboratory centrifuges may be used.

Purification of Immunoglobulin M and Immunoglobulin D

This unit describes two classical protocols for the purification of IgM - dialysis of ascites fluid, tissue culture medium, or bioreactor supernatants against distilled water to precipitate pure IgM, and ammonium sulfate precipitation. Both protocols can be followed by size-exclusion chromatography to obtain a highly purified product. Recently an affinity method for purification of IgM has been developed using mannan binding protein, and is described here. The third approach presented is a one-step IgD purification method, designed specifically for murine derived samples, that uses Sepharose coupled to lectin derived from the seeds of Griffonia simplicifolia-1. This represents a simple, rapid, and gentle approach to isolating this highly labile immunoglobulin from IgD-containing ascites or hybridoma sources.