Modifying interleukin-2 concentrations during culture improves function of T cells for adoptive immunotherapy.

BACKGROUND: Adoptive immunotherapy with cytotoxic T cells has shown promising clinical results in patients with metastatic melanoma and post-transplant-associated viral infections. Cell transfer therapies often require the ex vivo expansion of large numbers of reactive lymphocytes. Therefore interleukin-2 (IL-2), a potent T-cell mitogenic cytokine that critically affects the features and effectiveness of T cells, is frequently added to cell culture media. METHODS: We examined the influence of various IL-2 concentrations on cell growth, cytotoxicity, cytokine release and surface marker expression of tumor-infiltrating lymphocytes (TIL) during a standard 14-day rapid expansion phase. The study was conducted under good manufacturing practice (GMP) conditions, using approved reagents in a class 10000 laboratory. RESULTS: T-cell cultures grown in very high IL-2 concentrations (600-6000 IU/mL) expanded massively and maximally secreted interferon (IFN)-gamma in response to antigenic stimulation, but exhibited only low direct cytotoxicity. On the other hand, TIL cultures grown in low concentrations of IL-2 throughout the rapid expansion phase expanded to a lower extent and barely secreted IFN-gamma but displayed high cytotoxic activity. A combined approach of starting with 10-120 IU/mL IL-2 during the first week, followed by increasing the IL-2 concentration to 6000 IU/mL during the second week, results in T cells that expand well, maximally produce IFN-gamma and are highly cytotoxic against tumor cells. DISCUSSION: Fine tuning of the IL-2 concentration during ex vivo expansion of T cells can yield high numbers of T cells with optimal features for clinical use.

Novel antibodies as anticancer agents.

In recent years antibodies, whether generated by traditional hybridoma technology or by recombinant DNA strategies, have evolved from Paul Ehrlich's 'magic bullets' to a modern age 'guided missile'. In the recent years of immunologic research, we are witnessing development in the fields of antigen screening and protein engineering in order to create specific anticancer remedies. The developments in the field of recombinant DNA, protein engineering and cancer biology have let us gain insight into many cancer-related mechanisms. Moreover, novel techniques have facilitated tools allowing unique distinction between malignantly transformed cells, and regular ones. This understanding has paved the way for the rational design of a new age of pharmaceuticals: monoclonal antibodies and their fragments. Antibodies can select antigens on both a specific and a high-affinity account, and further implementation of these qualities is used to target cancer cells by specifically identifying exogenous antigens of cancer cell populations. The structure of the antibody provides plasticity resonating from its functional sites. This review will screen some of the many novel antibodies and antibody-based approaches that are being currently developed for clinical applications as the new generation of anticancer agents.

Engineering antibody Fv fragments for cancer detection and therapy: disulfide-stabilized Fv fragments.

Disulfide-stabilized Fv fragments of antibodies (dsFv) are molecules in which the VH-VL heterodimer is stabilized by an interchain disulfide bond engineered between structurally conserved framework positions distant from complementarity-determining regions (CDRs). This method of stabilization is applicable for the stabilization of many antibody Fvs and has also been applied to a T-cell receptor Fv. A summary of the design strategy, and the construction and production of various dsFvs and dsFv-fusion proteins is presented. Included in the discussion are the biochemical features of dsFvs in comparison with scFvs, the effect of disulfide stabilization on Fv binding and activity, and various applications of dsFvs and dsFv-immunotoxins for tumor imaging and the treatment of solid tumors in animal models.

Antitumor activity and pharmacokinetics in mice of a recombinant immunotoxin containing a disulfide-stabilized Fv fragment.

Disulfide-stabilized Fvs (dsFv) are recombinant Fv fragments of antibodies in which the inherently unstable VH-VL heterodimer is stabilized by a disulfide bond engineered between structurally conserved framework positions of VH and VL. We have recently described a recombinant immunotoxin, B3(dsFv)-PE38KDEL, that is composed of such a dsFv connected to a truncated form of Pseudomonas exotoxin (PE38KDEL). This disulfide-stabilized immunotoxin is indistinguishable in activity and specificity from its single-chain immunotoxin counterpart (Brinkmann et al., Proc. Natl. Acad. Sci. USA, 90: 7538-7542, 1993). We have now constructed and evaluated the stability, pharmacokinetics, and antitumor effect of a very similar disulfide-stabilized immunotoxin B3(dsFv)-PE38. This immunotoxin is specifically cytotoxic to human cancer cell lines such as A431 that express the B3 antigen on their surface. In addition, the dsFv-immunotoxin is more stable at 37 degrees C in human serum than the corresponding single-chain immunotoxin B3(Fv)-PE38. The survival of the disulfide-stabilized immunotoxin in the circulation of mice was determined by a bioassay on cultured A431 cells after administering the immunotoxin i.v. The half-life in blood was 23 min. To determine the therapeutic effects of the disulfide-stabilized immunotoxin, it was given i.v. to immunodeficient mice bearing s.c. human epidermoid carcinomas. The dsFv-immunotoxin caused complete regression of tumors with no toxic effect on mice. The antitumor effect was similar to that reported for the single-chain Fv-immunotoxin. Our data show that dsFv-immunotoxins retain full in vitro as well as in vivo activity when compared to scFv-immunotoxins. Because dsFv-immunotoxins have full activity, are more stable, and can be produced with significantly improved yields compared to scFv-immunotoxins, the dsFv-immunotoxins may be more useful for therapeutic applications than scFv-immunotoxins.

Recombinant immunotoxins in targeted cancer cell therapy.

Targeted cancer therapy in general and immunotherapy in particular combines rational drug design with the progress in understanding cancer biology. This approach takes advantage of our recent knowledge of the mechanisms by which normal cells are transformed into cancer cells, thus using the special properties of cancer cells to device novel therapeutic strategies. Recombinant immunotoxins are excellent examples of such processes, combining the knowledge of antigen expression by cancer cells with the enormous developments in recombinant DNA technology and antibody engineering. Recombinant immunotoxins are composed of a very potent protein toxin fused to a targeting moiety such as a recombinant antibody fragment or growth factor. These molecules bind to surface antigens specific for cancer cells and kill the target cells by catalytic inhibition of protein synthesis. Recombinant immunotoxins are developed for solid tumors and hematological malignancies and have been characterized intensively for their biological activity in vitro on cultured tumor cell lines as well as in vivo in animal models of human tumor xenografts. The excellent in vitro and in vivo activities of recombinant immunotoxins have lead to their preclinical development and to the initiation of clinical trail protocols. Recent trail results have demonstrated potent clinical efficacy in patients with malignant diseases that are refractory to traditional modalities of cancer treatment: surgery, radiation therapy, and chemotherapy. The results demonstrate that such strategies can be developed into a separate modality of cancer treatment with the basic rationale of specifically targeting cancer cells on the basis of their unique surface markers. Efforts are now being made to improve the current molecules and to develop new agents with better clinical efficacy. This can be achieved by development of novel targeting moieties with improved specificity that will reduce toxicity to normal tissues. In this review, the design, construction, characterization, and applications of recombinant immunotoxins are described. Results of recent clinical trails are presented, and future directions for development of recombinant immunotoxins as a new modality for cancer treatment are discussed.

An antibody single-domain phage display library of a native heavy chain variable region: isolation of functional single-domain VH molecules with a unique interface.

To develop very small antibody-derived recognition units for experimental, medical, and drug design purposes, a heavy chain variable region (VH) single-domain phage-display library was designed and constructed. The scaffold that was used for library construction was a native sequence of a monoclonal antibody with a unique VH/VL interface. There was no need to modify any residues in the VL interface to avoid non-specific binding of VH domain. The library repertoire, consisting of 4x10(8)independent clones, was generated by the randomization of nine amino acid residues in complementary determining region 3. The library was screened by binding to protein antigens, and individual clones were isolated. The VH genes encoding for specific binding clones were rescued and large amounts of soluble and stable single-domain VH protein were made from insoluble inclusion bodies by in vitro refolding and purification. Biochemical and biophysical characterization of the VH protein revealed a highly specific, correctly folded, and stable monomeric molecule. Binding studies demonstrated an affinity of 20 nM. The properties of these molecules make them attractive for clinical, industrial, and research applications, as well as a step toward improvement in the design of small molecules that are based on the hypervariable loops of antibodies.

Antibody engineering of recombinant Fv immunotoxins for improved targeting of cancer: disulfide-stabilized Fv immunotoxins.

Recombinant immunotoxins are chimeric proteins in which a truncated toxin is fused to a recombinant antigen-binding domain such as a recombinant Fv or Fab. Recombinant immunotoxins target cell surface receptors and other antigens on tumor cells. The antigen-binding and -targeting domains in recombinant immunotoxins are usually single-chain Fvs (scFv), which are the antibody variable regions connected by a flexible peptide linker and fused directly to a bacterial toxin. However, Fabs have also been used. Recombinant immunotoxins have very good activity in vitro on cultured human tumor cell lines and have produced complete regressions and cures of established tumor xenografts in nude mouse models. Problems with the stability and binding of some scFv immunotoxins as well as scFvs not linked to toxin led to the development of a new type of recombinant Fv immunotoxin in which the targeting variable domains of the Fv are stabilized by an interchain disulfide bond located in structurally conserved framework positions of the VH and VL domains. These are termed disulfide-stabilized Fvs (dsFv) or dsFv immunotoxins. dsFvs and dsFv immunotoxins have several advantages over scFv immunotoxins. This review summarizes the design, construction, activities in vitro and in vivo, and biochemical characteristics of dsFv immunotoxins and compares them with scFv immunotoxins.

Complement membrane attack complexes induce in human leukemic cells rapid expression of large proteins (L-CIP).

The effect of sublytic doses of the complement membrane attack complexes (MAC) on protein synthesis in human leukemic cells was examined. As shown herein, rapid protein synthesis is evident in K562 erythroleukemic cells upon exposure to sublytic complement doses. Analysis of cell extracts by SDS-PAGE revealed high molecular weight proteins which appeared in the cells already after 15 min treatment with complement at 37 degrees C, reaching a maximal level after 40-50 min. These large complement-induced proteins (L-CIP) were clearly observed in gels stained by Coomassie blue and in autoradiograms following [35S]-Met or [3H]-Leu incorporation. Rabbit antibodies prepared against L-CIP were reactive in immunoassays with extracts of MAC-treated cells but not of non treated cells. They also bound to the surface of intact K562 cells (as determined by immunofluorescence), but only after treatment of the cells with complement. Both heterologous (rabbit and guinea pig) and homologous (human) sera induced L-CIP synthesis. The induction of L-CIP was indeed mediated by the complement MAC since L-CIP could not be detected in K562 cells exposed to heat-inactivated human serum or C6-deficient rabbit serum. Similarly, C7- or C8-deficient human sera could not induce L-CIP production unless they were reconstituted with purified human C7 or C8, respectively. The synthesis of L-CIP was largely inhibited by the protein synthesis inhibitors cycloheximide and puromycin and partially inhibited by the RNA synthesis inhibitor actinomycin D. L-CIP was similarly induced in two other human leukemic cell lines, U937 and HL-60, but not in K562/S, a subline of K562 which is highly sensitive to complement damage. These results are discussed with respect to the resistance of leukemic cells, and nucleated cells in general, to complement-mediated immune damage.

Preparation and characterization of a disulfide-stabilized Fv fragment of the anti-Tac antibody: comparison with its single-chain analog.

Recombinant DNA techniques now allow the production of "mini-antibodies" called Fv fragments. These have been produced either as the independent variable domains of the heavy and light chains non-covalently associated in one-to-one stoichiometry or as single-chain gene products with the two domains linked by an intervening peptide sequence. Although Fv fragments can have excellent binding properties, they are often difficult to produce in good yield and lack the characteristic stability of whole antibodies. To improve the stability of the Fv molecule, we have introduced a cysteine residue into conserved framework regions of both the heavy and light variable domains from the anti-Tac antibody at positions compatible with the formation of an interdomain disulfide linkage (i.e. VH-44 and VL-99). The mutant subunits form a disulfide-bonded Fv molecule, which binds to the alpha-subunit of the IL2 receptor (IL2R alpha) with an affinity identical to that of humanized anti-Tac IgG. This disulfide-stabilized Fv (dsFv) proved to be substantially more resistant to denaturation by heat or urea treatment than the single-chain Fv (scFv). Furthermore, the yield of dsFv is -four-fold higher than that of the single-chain analog.

Recombinant Fv immunotoxins and Fv fragments as novel agents for cancer therapy and diagnosis.

Recombinant immunotoxins are new agents being developed for cancer therapy. They are composed of Fv fragments of antibodies that bind to cancer cells fused to a truncated form of a very potent bacterial toxin. The antibody moiety directs the toxin to cancer cells, which are killed, while normal cells are not recognized and thus survive. The excellent preclinical results in vitro and in vivo have led to the initiation of several clinical trials.

Antibody engineering for targeted therapy of cancer: recombinant Fv-immunotoxins.

Recombinant Fv-immunotoxins are a new class of biologic anticancer agents composed of a recombinant antibody fragment linked to a very potent bacterial toxin. These potent molecules are designed to specifically bind and kill cancer cells that express a specific target antigen on their cell surface. Recombinant Fv-immunotoxins are an excellent example for the concept of rational drug design. They combine the progress in understanding cancer biology, -the recent knowledge on the mechanisms of malignant transformation and the special properties of cancer cells, -with the enormous developments in recombinant DNA technology and antibody engineering. Recombinant Fv immunotoxins were developed for solid tumors and hematological malignancies and have been characterized intensively for their biological activity in vitro and in vivo in animal models. The excellent in vitro and in vivo activities of recombinant Fv-immunotoxins have lead to their pre-clinical development and to the initiation of clinical trial protocols. Recent trials have demonstrated potent clinical efficacy in patients with malignant diseases that are refractory to traditional modalities of cancer treatment. It is thus suggested that this strategy can be developed into a separate modality of cancer treatment with the basic rationale of specifically targeting cancer cells on the basis of their unique surface markers combined with potent effective biological toxic agents that directly kill the cancer cell. Efforts are now being made to improve the current molecules and to develop new agents with better clinical efficacy. In this review, we will describe the rationale in designing Fv-immunotoxins and will review current progress made in using these agents for cancer treatment.

Resistance to NK cell-mediated cytotoxicity (in K-562 cells) does not correlate with class I MHC antigen levels.

Natural Killer (NK) cells probably function as an early line of defense against virus-infected cells and tumor cells. In all cases, the killing by NK cell-mediated cytotoxicity (NK-CMC) is not MHC-restricted and the factors which determine the sensitivity to NK-CMC have not yet been identified. A positive correlation between resistance to NK-CMC and the level of class I MHC antigen (MHC I) expression on target cells has been reported in many studies, and in some cases a functional linkage between the two has been claimed. Several other studies have shown that there is no such correlation. By employing several experimental systems, we demonstrate here a lack of correlation between the level of MHC I and the sensitivity of K-562 cells to NK-CMC. Transfer of MHC I to MHC I-negative cells via vesicles had no effect on their resistance to NK-CMC. In addition, a decrease in resistance to NK-CMC and increase of MHC I levels was observed following target-cell membrane modulation by both application of cholesterol and hydrostatic pressure. Finally, no correlation between sensitivity to NK-CMC and MHC I expression was found in three sublines of K-562 cells. Since NK-CMC is a multistage process, it is concluded that components other than class I MHC antigens have a more prominent role in modulating the sensitivity of target cells to NK-CMC.

Killing of human tumor cells by antibody C3b conjugates and human complement.

To potentiate the lytic action of complement on tumor cells, we have constructed heteroconjugates composed of monoclonal antibody and of the human C3b component of complement. The conjugates were formed efficiently using the heterobifunctional cross-linking reagent SPDP. The monoclonal antibody-C3b conjugate promoted the killing of K562 tumor cells by normal human serum. Treatment of the tumor cells with the monoclonal antibody and normal human serum resulted in 10-15% lysis. However, following pretreatment of the cells with antibody-C3b conjugates, their lysis by normal human serum increased to 70%. The conjugate activated selectively the alternative pathway of complement and the C3b component in the conjugate was highly resistant to cleavage and inactivation by the complement regulatory proteins Factors H and I. These results suggest that the coupling of C3b molecules to monoclonal antibodies anti-unique tumor antigens produces a potent complement-activating reagent which may act specifically on tumor cells and promote cancer therapy.

Targeting of complement to tumor cells by heteroconjugates composed of antibodies and of the complement component C3b.

Tumor cells have adapted several strategies which permit them to grow in an immunologically hostile environment. The C system can potentially destroy these cells; however, its action needs to be specifically potentiated on the surface of the tumor cells. To this end, a heteroconjugate composed of a mouse mAb and of the human C3b C component has been generated by using the heterobifunctional reagent N-succinimidyl-3-(2-pyridyldithio)propionate. The two mAb which were used in this study are V1-10 and TIB219 which bind to the human and mouse transferrin receptors, respectively. The mAb-C3b conjugates were purified by gel filtration and were each composed of one mAb and one C3b. They bound to the human K562 and HL60 or mouse ALB1 cell lines and amplified the killing of these cells by C from 10 to 15% to 70 to 100%. Fresh normal human or mouse sera were used as a source of C. The mAb-C3b conjugates activated primarily the alternative pathway of C since only C3 and factor B but not C4 were cleaved in the sera. After disulfide-linking to the mAb, the C3b became highly resistant to inactivation by factors H and I, probably due to its reduced factor H binding capacity. On the other hand, the conjugated C3b bound factor B better than free C3b and produced more C3 convertases which expressed increased stability. These results suggest that mAb-C3b conjugates may serve as an effective tool for the specific activation of the cytolytic C system on selected cells. As such, they may be used in vitro or in vivo to target the autologous C to tumor cells or to lymphocytes and may promote tumor immunotherapy.

Sublytic complement attack protects tumor cells from lytic doses of antibody and complement.

Sublytic doses of the membrane attack complex (MAC) of complement are known to exert multiple stimulatory effects on metabolically active cells. Results presented herewith demonstrate that pretreatment of the human leukemic cells K562 and HL-60 with sublytic doses of antibody and normal human serum protects them from lytic complement concentrations, a phenomenon proposed to be called "complement-induced protection". C7- and C8-deficient human sera are ineffective in inducing resistance unless they are reconstituted with purified human C7 and C8, respectively. The complement-induced protection is inhibitable by actinomycin D and cycloheximide indicating that the increased complement resistance depends on RNA and protein synthesis triggered by the sublytic complement doses. Free extracellular Ca2+ is also required to achieve maximal protection, indicating a role for Ca2+ ions in the cell stimulatory events which culminate in increased complement resistance. Quantitative analysis of bound complement components indicated that similar amounts of C3 and C9 molecules are deposited on "protected" and control cells during complement activation. The "protected" K562 and HL-60 cells regain sensitivity to lytic MAC doses after about 8 or 3 h, respectively, of culture in growth medium, in the absence or presence of actinomycin D and cycloheximide. The "induced protection" is not species restricted and protection from human complement can be induced in K562 cells by treatment with sublytic doses of antibody and rabbit or guinea pig sera.

Cytotoxic and antitumor activity of a recombinant immunotoxin composed of disulfide-stabilized anti-Tac Fv fragment and truncated Pseudomonas exotoxin.

Disulfide-stabilized Fv (dsFv)-immunotoxins are recombinant immunotoxins in which the inherently unstable Fv moiety, composed of the VH-VL heterodimer, is stabilized by a disulfide bond engineered between structurally conserved framework positions of VH and VL. Anti-Tac(dsFv)-PE38KDEL is composed of such a dsFv, directed to the alpha subunit of the IL2 receptor (IL2R), and containing a truncated form of Pseudomonas exotoxin (PE38KDEL). We have found this new type of immunotoxin to be indistinguishable in its in vitro activity and specificity from its single-chain immunotoxin counterpart, anti-Tac(Fv)-PE38KDEL. We have now examined the therapeutically relevant factors, including stability, pharmacokinetics, and antitumor activity of this new disulfide-stabilized Fv-immunotoxin. We found that anti-Tac(dsFv)-PE38KDEL was specifically cytotoxic to human activated T-lymphocytes in addition to IL2R bearing cell lines. Anti-Tac(dsFv)-PE38KDEL was considerably more stable at 37 degrees C in human serum and in buffered saline than the single-chain immunotoxin, anti-Tac(Fv)-PE38KDEL. The half-life in blood was similar for both immunotoxins (approx. 20 min). The therapeutic potential of the disulfide-stabilized immunotoxin was evaluated using an animal model of immunodeficient mice bearing subcutaneous tumor xenografts of human IL2R-bearing cells. Anti-Tac(dsFv)-PE38KDEL caused complete regression of tumors with no toxic effects in mice. Because dsFv-immunotoxins are more stable and can be produced with significantly improved yields compared to scFv-immunotoxins, dsFv-immunotoxin may be more useful for therapeutic applications.

Administration of disulfide-stabilized Fv-immunotoxins B1(dsFv)-PE38 and B3(dsFv)-PE38 by continuous infusion increases their efficacy in curing large tumor xenografts in nude mice.

B1 (dsFv)-PE38 and B3(dsFv)-PE38 are recombinant immunotoxins in which the Fv fragments of MAbs B1 and B3, respectively, are stabilized by an engineered interchain disulfide bond and are fused at their C-termini to a modified Pseudomonas exotoxin from which the cell-binding domain has been deleted (PE38). Both immunotoxins have been shown to be specifically cytotoxic toward human cancer cell lines which express Le gamma-related carbohydrates on their surface, and when given i.v., eradicated 30- to 50-mm3 s.c. A431 tumors growing in nude mice. A major advantage of dsFv-immunotoxins is their stability at 37 degrees C compared with the relatively unstable single-chain Fvs. This allows them to be given continuously by osmotic pumps placed in the peritoneal cavity. In an attempt to increase the therapeutic index of the immunotoxins, we have now delivered them continuously for 6 days through mini-osmotic pumps placed in the peritoneal cavity of tumor-bearing nude mice. Using this mode of administration, we were able to maintain a constant level of immunotoxin in the serum which was non-toxic to the mice, but caused complete regressions of large 150- to 200-mm3 tumors which lasted for over a month at 1/11 of the LD50 with B1(dsFv)-PE38 and 1/6 of the LD50 with B3(dsFv)-PE38. Complete regression of tumors of similar size could also be achieved by i.v. bolus injections of these immunotoxins at 1/7 of the LD50 with B1(dsFv)-PE38) and 1/3 of the LD50 with B3(dsFv)-PE38. These results suggest that in patients it may be advantageous to administer dsFv-immunotoxins by continuous infusion, since a larger therapeutic index is achieved.

Critical role for CD8 in binding of MHC tetramers to TCR: CD8 antibodies block specific binding of human tumor-specific MHC-peptide tetramers to TCR.

There are conflicting opinions about the role that the T cell coreceptors CD4 and CD8 play in TCR binding and activation. Recent evidence from transgenic mouse models suggests that CD8 plays a critical role in TCR binding and activation by peptide-MHC complex multimers (tetramers). Here we show with a human CTL clone specific for a tumor-associated MHC-peptide complex that the binding of tetramers to the TCR on these cells is completely blocked by anti-human CD8 Abs. Moreover, the staining of CTLs with specific MHC-peptide tetramers simultaneously with anti-CD8 Abs was completely blocked with three different anti-CD8 Abs. This blockage was mediated by anti-CD8 Abs but not anti-CD3 Abs and was dose dependent. The blocking effect of the anti-CD8 Abs was attributable to directly inhibiting tetramer binding and was not attributable to Ab-mediated TCR-CD8 internalization and down-regulation. Our results have important implications in TCR binding to MHC-peptide tetramers. MHC-peptide tetramers are widely used today in combination with anti-CD8 Abs for the phenotypic analysis of T cell populations and in the study of T cell responses under various pathological conditions such as infectious diseases and cancer. Our results indicate that also in the human system CD8 plays a critical role in the interaction of MHC-peptide multimers with TCR.

Recombinant human single-chain MHC-peptide complexes made from E. coli By in vitro refolding: functional single-chain MHC-peptide complexes and tetramers with tumor associated antigens.

Soluble recombinant MHC-peptide complexes are valuable tools for molecular characterization of immune responses as well as for other functional and structural studies. In this study, soluble recombinant single-chain human MHC (scMHC)-peptide complexes were generated by in vitro refolding of inclusion bodies from bacterially expressed engineered HLA-A2 in the presence of tumor-associated or viral peptides. The scMHC molecule was composed of beta2-microglobulin connected to the first three domains of the HLA-A2 heavy chain through a 15-amino acid flexible linker. Highly purified scMHC-peptide complexes were obtained in high yield using several peptides derived from the melanoma antigens gp100 and MART-1 or a viral peptide derived from HTLV-1. The scMHC complexes were characterized in detail and were found to be correctly folded and able to specifically bind HLA-A2-restricted peptides. We also generated scMHC-peptide tetramers, which were biologically functional; they induced a peptide-specific CTL clone to be activated and secrete IFN-gamma, and were able to stain specifically CTL lines. Such recombinant soluble scMHC-peptide complexes and tetramers should prove of great value for characterization of immune responses involving CTL, for visualization of antigen-specific immune responses, for in vitro primary CTL induction, and for peptide binding assays and structural studies.

Engineering interchain disulfide bonds into conserved framework regions of Fv fragments: improved biochemical characteristics of recombinant immunotoxins containing disulfide-stabilized Fv.

Using molecular modeling technology, we have recently identified two positions in conserved framework regions of antibody Fv fragments (Fvs) that are distant from CDRs, and potentially can be used to make recombinant Fv fragments in which the unstable VH and VL heterodimer is stabilized by an interchain disulfide bond inserted between structurally conserved framework positions. A disulfide bond has been introduced at one of these positions, VH44-VL105, and shown to stabilize various Fvs that retain full binding and specificity. Recombinant immunotoxins, e.g. B3(dsFv)-PE38KDEL in which this disulfide-stabilized Fv moiety is connected to a truncated form of Pseudomonas exotoxin (PE; PE38KDEL) which contains the translocation and ADP ribosylation domains, are indistinguishable in binding and specificity from its single-chain immunotoxin counterparts. We have now analyzed the alternative position, (VH111-VL48), predicted by the modeling methodology, for disulfide stabilization of mAb B3(Fv) by producing a recombinant immunotoxin with such disulfide-stabilized (ds) Fv. This immunotoxin was also very active and retained full specificity to B3 antigen-positive cells. However, it was 2- to 3-fold less active than the VH44-VL105 dsFv-molecule. We also tested various biochemical features of VH44-VL105 and VH111-VL48 dsFv immunotoxins and compared them with the corresponding single-chain immunotoxin. We found the dsFv immunotoxins were more stable in human serum and more resistant to thermal and chemical denaturation than the single chain (sc) Fv immunotoxin. Because dsFv immunotoxins and dsFvs have full activity and specificity and improved stability, they may be more useful than scFv immunotoxins as therapeutic and diagnostic agents.