Treatment with tumor-reactive Fab-IL-2 and Fab-staphylococcal enterotoxin A fusion proteins leads to sustained T cell activation, and long-term survival of mice with established tumors.

C215Fab-IL-2 fusion protein, with full IL-2 and antigen binding activity, was produced in E. coli at high level (>50 mg/l). When co-administered with Fab-superantigen fusion protein (C215Fab-SEA) in mice strong and sustained T cell activation was observed. Combination treatment of mice carrying B16 melanoma transfected with C215 antigen was also more efficient than using C215Fab-SEA (p<0.01) or C215Fab-IL-2 alone (p<0.001). In a long-term survival experiment 5/12 mice having received combination treatment 5 days after i.v. inoculation of B16 cells survived >85 days. Improved therapeutic efficacy correlated with increased tumor infiltration by activated CD25+ T cells, indicating a T cell mediated mechanism.

Tumor-growth inhibition with bispecific antibody fragments in a syngeneic mouse melanoma model: the role of targeted T-cell co-stimulation via CD28.

The ability of bispecific antibodies with anti-tumor x anti-CD3 specificity to mediate the killing of tumor cells by activated T cells has been demonstrated in many in vitro experiments. Moreover, long-term survival of lymphoma-bearing mice has been observed after treatment with such reagents. The therapeutic effect of bispecific antibodies in solid-tumor models has been less impressive, in particular if fragmented antibodies were used to avoid systemic T-cell activation by bispecific constructs binding to Fc-receptor-positive cells. Here we report that bispecific anti-tumor x anti-CD3-fragments markedly inhibit intraperitoneal as well as pulmonary tumor growth in mice inoculated with B16 melanoma cells, resulting in the long-term survival of animals. Therapeutic success critically depends on the number of recruitable effector cells at the site of tumor growth. A second bispecific construct triggering the co-stimulatory CD28-molecule on the T-cell surface increased tumor-cell killing in vitro and in vivo, despite rather low avidity of this reagent to mouse T cells. Finally, long-term-surviving animals showed improved survival after i.v. rechallenge with tumor cells, indicating that bispecific antibodies are capable of inducing long-lasting protective immunity.

MHC class II-independent, Vbeta-specific activation of T cells by superantigen mutants fused to anti-tumor Fab fragments: implications for use in treatment of human colon carcinoma.

Genetically engineered fusion proteins of the super-antigen staphylococcal enterotoxin A (SEA) and tumor-reactive monoclonal antibodies, C215Fab-SEA and C242Fab-SEA, have been generated and shown to be effective in mediating superantigen-antibody directed cellular cytotoxicity against human carcinoma cells expressing the CA215 or CA242 antigens in an MHC class II-independent manner. In an attempt to reduce the in vivo toxicity of superantigen administration, alanine substitution mutations in SEA at residues F47 and D227 that affect SEA binding to class II molecules have been created and genetically linked to C215Fab or C242Fab. The purpose of this study was to determine whether these Fab-SEA mutant fusion proteins, that have low MHC class II binding affinities, were still able to stimulate human T cells in a Vbeta-specific manner in the presence or absence of MHC class II molecules. The SEA wt- and SEA-D227A-based fusion proteins shared the ability to activate V beta5. 2-, Vbeta6-, Vbeta7-, Vbeta9- and Vbeta18-bearing T cells, whereas Fab-SEA-F47A protein activated only Vbeta6- and Vbeta7-bearing T cells. The fusion of Fab fragments onto SEA wt, SEA-F47A or SEA-D227A had no effect on the Vbeta specificity of these superantigens. Fab fusion proteins containing either SEA wt or SEA mutants were presented, in the absence of class II molecules, by CHO cells transfected with CA215 and CD80 and all induced the expansion of only Vbeta6-, Vbeta7- and Vbeta 18-bearing T cells. Fab-SEA mutant fusion proteins may provide attenuated therapeutic agents that, while still able to specifically target high affinity T cells for MHC class II-independent local tumor killing, will not induce excessive systemic toxicity.

Targeted superantigens for immunotherapy of haematopoietic tumours.

With the exception of childhood common acute lymphoblastic leukaemia (cALL), treatment of other hematopoietic B cell lineage tumours such as non-Hodgkin's lymphoma (B-NHL), adult ALL and multiple myeloma (MM) is unsatisfactory. Similarly, the therapeutic outcome of acute and chronic myeloid leukaemia (AML, CML) is frequently dismal. At the same time, leukaemia/lymphoma cells represent ideal targets for immunotherapy. The present review summarizes our preclinical experience with a novel type of cytotoxic T cell based immunotherapy for B-lineage and myeloid tumours. Staphylococcal enterotoxin-derived superantigens (SAgs) are among the most potent T cell activators known, linking the T cell receptor to HLA-DR on natural target cells. SAgs were genetically engineered to reduce DR binding and were then fused to Fab parts of tumour-directed monoclonal antibodies (mAbs). Using these "targeted" SAgs, highly efficient lysis of B-lineage (B-NHL, B-CLL, ALL, MM) and myeloid (AML, CML) tumour cells by T-cells was achieved in vitro and in an animal model. We are entering an interesting era of innovative cancer therapy based on novel man-made biotherapeutic agents.

Staphylococcal enterotoxin-A-induced in-vitro adhesion of HL-60 cells to endothelial cells involves both selectin and integrin families of cell adhesion molecules.

In-vivo exposure to the bacterial superantigen Staphylococcal enterotoxin-A (SEA) induces an inflammatory response characterized by rapid extravasation of leucocytes and release of excessive amounts of cytokines. We have utilized an in-vitro adhesion assay to understand the molecular mechanisms responsible for SEA-induced extravasation of leucocytes. Stimulation of human umbilical cord endothelial cells (HUVEC) with increasing concentrations of recombinant SEA (rSEA) did not influence the in-vitro adhesion of HL-60 cells to HUVEC, whereas stimulation of HUVEC by interleukin (IL)-1beta supported adhesion of HL-60 cells. Increased adhesion of HL-60 cells to HUVEC was noted upon stimulation of endothelium with culture medium obtained from human peripheral blood mononuclear cells (PBM) stimulated with recombinant SEA for 24 (CM-SEA 24 h), 72 (CM-SEA 72 h) and 120 h (CM-SEA 120 h), but not after stimulation with culture medium obtained from control human peripheral blood mononuclear cells (CM), suggesting that soluble factors present in the supernatants play a major role in SEA-induced cell adhesion. While CM-SEA 24 and 72 h induced both a rapid (4 h) and delayed type of adhesion, CM-SEA 120 h only induced a delayed type of adhesion. Stimulation of PBM by SEA resulted in increased levels of IL-1beta, IL-2 and interferon (IFN)-gamma after 24h. Further stimulation for 72-120h resulted in a significant increase in the levels of IL-1beta, IFN-gamma and tumour necrosis factor (TNF). Stimulation of PBM with SEA also resulted in increased levels of soluble and L-selectin in the cell supernatants. Increased cell-surface expression of E-selectin, ICAM-1, HLA-DR and VCAM-1 was detected on HUVEC stimulated with CM-SEA media. While E-selectin and VCAM were induced on HUVEC within a few hours, induction of ICAM and HLA-DR required a longer induction period. Adhesion of HL-60 cells to HUVEC treated with CM-SEA was inhibited by monoclonal antibodies (MoAbs) against both the selectin and integrin families of cell adhesion molecules, suggesting that multiple pathways contribute to SEA-induced leucocyte extravasation. The results suggested that selectin-dependent adhesion was more prominent during the early phase while integrin-induced adhesion occurred at a later stage.

Perforin and IFN-gamma are involved in the antitumor effects of antibody-targeted superantigens.

The bacterial superantigen staphylococcal enterotoxin A (SEA) is a potent inducer of cytokine production and cytotoxic T cell responses. To target a T cell attack against tumor cells we have genetically engineered a fusion protein of SEA and the Fab part of the tumor-reactive mAb C215. Injection of this Fab-SEA fusion protein to mice carrying lung metastases of the poorly immunogenic B16 melanoma transfected with the C215 Ag resulted in infiltration of cytokine-producing T cells, perforin-containing CTL, and a marked tumor elimination. Fab-SEA therapy induced substantial levels of IFN-gamma and TNF-alpha in serum. In the present study we have characterized the molecular mechanisms of the antitumor effect induced by Fab-SEA treatment in vivo. Neutralization of cytokines by specific Abs demonstrated a major role for IFN-gamma in the suppression of tumor growth. In addition, a minor contribution of TNF-alpha was recorded. Injections of Fab-SEA into normal mice induced strong CTL activity but failed to promote cytotoxic function in perforin knockout mice. Also, a markedly reduced therapy was noted in perforin knockout mice, implicating a role for CTL in Fab-SEA-mediated tumor eradication. The data suggest that Fab-SEA-targeted T cells may suppress tumor growth by both perforin-dependent cytotoxicity and local release of cytokines such as IFN-gamma. The latter mechanism may have an important role in cytostatic effects against Ag-negative bystander tumor cells.

Repeated treatment with antibody-targeted superantigens strongly inhibits tumor growth.

Superantigens (SAg) are microbial proteins with the capacity to activate a large proportion of T cells. We have developed a novel approach for cancer immunotherapy by genetically fusing the SAg staphylococcal enterotoxin A (SEA) to a Fab-fragment of a tumor-specific antibody. Repeated exposure to SEA induces a state of unresponsiveness including cell deletion and functional hyporesponsiveness, i.e., anergy. In this study we have developed improved therapeutic schedules to allow repeated injections of Fab-SEA, limit development of immunological unresponsiveness and promote maximal anti-tumor response. Four daily injections of Fab-SEA to mice carrying B 16-C215 lung metastases resulted in 90-95% reduction in the number of metastases. However, the animals did retain a minimal residual tumor disease. The immune system was in a hyporesponsive state after 4 daily Fab-SEA injections, and further injections did not improve therapy. Two repeated cycles, each comprising 4 daily injections of Fab-SEA, significantly prolonged the survival and resulted in complete cure of a fraction of the animals. A rest period of 10 days between the cycles was required to mount an efficient secondary anti-tumor response. This secondary immune response was characterized by partial recovery of cytokine production i.e., interleukin-2, interferon-gamma and tumor necrosis factor-alpha. Strong CTL activity was detected in animals that had rested for 8 weeks between the 2 cycles. Interestingly, irrespective of the resting period, the CD4+ SEA-reactive T cells expanded in response to all 4 additional Fab-SEA injections both locally and in spleen. In contrast, only marginal expansion of CD8+ T cells was seen if restimulation was given within 1 month. Our data show that potent anti-tumor effector functions can be induced after repeated stimulation cycles with a SAg-monoclonal antibody fusion protein resulting in a CD4+ T cell-dependent cytokine release, prolonged survival and induction of complete cures.

Man-made superantigens: Tumor-selective agents for T-cell-based therapy.

Superantigens (SAgs) are a collection of bacterial and viral proteins with potent immunostimulatory properties. SAgs bind to Major Histocompatibility Complex Class II (MHC II) molecules of antigen presenting cells (APCs) and activate a high frequency of T lymphocytes. To target a T-cell attack against tumor cells we genetically linked tumor-specific antibody Fab fragments to the SAg Staphylococcal enterotoxin A (SEA). Fab-SEA fusion protein efficiently targeted to solid tumors and induced a T-cell-mediated eradication of established metastases in animal models. Successful therapy was T-cell-dependent and required tumor specificity of the Fab moiety of the Fab-SEA fusion protein. Due to the high affinity of SAg for MHC II, a limitation of this approach was retention of Fab-SEA proteins in normal tissues expressing MHC II, which caused systemic immune activation and dose limiting toxicity. We recently solved the structure of SEA and applied structure-based drug design to develop a novel generation of 'man-made' SAg with improved pharmacological and pharmacokinetic properties. Mutation of the major MHC II binding site of SEA substantially reduced retention in MHC II(+) tissues and systemic toxicity, while local immune activation at targeted tumor sites was retained. The Fab-SEA mutants display a 10000-fold higher affinity for tumor tissue compared to normal tissue and the therapeutic window was improved >100-fold compared to native Fab-SEA protein. Thus protein engineering can be applied to convert harmful bacterial toxins into tolerable tumor-specific agents.

A mutation of F47 to A in staphylococcus enterotoxin A activates the T-cell receptor Vbeta repertoire in vivo.

The bacterial superantigen staphylococcal enterotoxin A (SEA) binds with high affinity to major histocompatibility complex (MHC) class II molecules and subsequently activates T cells bearing particular T-cell receptor (TCR) Vbeta chains. Structural and mutational studies have defined two distinct MHC class II binding sites located in the N-terminal and C-terminal domains of SEA. The N-terminal F47 amino acid is critically involved in a low-affinity interaction to the MHC class II alpha-chain, while the C-terminal residues H187, H225, and D227 coordinate a Zn2+ ion and bind with moderate affinity to the beta-chain. In order to analyze whether the SEA-MHC class II alpha-chain interaction plays a role in dictating the in vivo repertoire of T-cell subsets, we studied distinct Vbeta populations after stimulation with wild-type SEA [SEA(wt)] and SEA with an F47A mutation [SEA(F47A)]. Injections of SEA(wt) in C57BL/6 mice induced cytokine release in serum, strong cytotoxic T-lymphocyte activity, expansion of T-cell subsets, and modulated expression of the T-cell activation antigens CD25, CD11a, CD44, CD62L, and CD69. SEA-reactive TCR Vbeta3+ and Vbeta11+ T cells were activated, while TCR Vbeta8+ T cells remained unaffected. The SEA(F47A) mutant protein induced a weaker T-cell response and failed to induce substantial interleukin-6 production compared to SEA(wt). Notably, SEA(F47A) failed to activate TCR Vbeta11+ T cells, whereas in vivo expansion and modulation of T-cell activation markers on TCR Vbeta3+ T cells were similar to those for SEA(wt). A similar response to SEA(F47A) was seen among CD4+ and CD8+ T cells. Activation of TCR Vbeta3+ and TCR Vbeta11+ T-cell hybridomas confirmed that SEA(F47A) activates TCR Vbeta3+ but not TCR Vbeta11+ T cells. The data support the view that the SEA-N-terminal MHC class II alpha-chain interaction defines a topology that is required for engagement of certain TCR Vbeta chains in vivo.

Superantigen-induced lysis of melanoma cells.

Superantigens like the Staphylococcus enterotoxin A (SEA) can direct cytotoxic T lymphocytes expressing certain T cell receptor V beta regions to lyse MHC class II-positive target cells. This superantigen-dependent cellular cytotoxicity (SDCC) has been extended to MHC class II-negative tumour cells by targeting T cells via conjugates of a tumour-specific monoclonal antibody (moAb) and a superantigen. In the present study the MHC class II-negative human melanoma cell lines G361 and MaRI were tested for susceptibility to SDCC in vitro. Antibodies recognizing the disialoganglioside GD3 and the CD10 antigen were linked to SEA either by a recombinant protein A-SEA fusion protein or an anti-kappa moAb-SEA chemical conjugate. Specific lysis of melanoma cells was dose- and effector to target (E:T) cell ratio-dependent. Introduction of a point mutation into the SEA gene (producing SEAm9) in order to reduce MHC II affinity of the superantigen, which has already been shown to severely diminish superantigen-dependent binding and lysis of MHC class II-positive cells, did not influence antibody-targeted SDCC. Cytotoxicity was equal with both antibodies (anti-GD3 and anti-CD10) and independent of whether protein A-SEA, protein A-SEAm9 or anti-kappa-SEA were used.

Superantigen-based immunotherapy: a phase I trial of PNU-214565, a monoclonal antibody-staphylococcal enterotoxin A recombinant fusion protein, in advanced pancreatic and colorectal cancer.

PURPOSE: To establish the maximum-tolerated dose (MTD) and define the toxicities of a single-dose infusion of PNU-214565, a recombinant Escherichia coli-derived fusion protein of Staphylococcal enterotoxin A (SEA) and the Fab-fragment of the C242 monoclonal antibody in patients with advanced colorectal and pancreatic carcinomas. To investigate the capability of PNU-214565 to induce a superantigen (SAg) response resulting in cytokine production and tumor regression. PATIENTS AND METHODS: Twenty-one patients (age range, 39 to 76 years; median, 64; 12 men, nine women; 18 colorectal, three pancreatic cancers) were treated with a single 3-hour infusion of PNU-214565, with doses ranging from 0.01 to 1.5 ng/kg. All patients had prior chemotherapy and a good performance status Eastern Cooperative Oncology Group [ECOG] performance status [PS] = 0 [n = 10]; PS = 1 [n = 11]), 10 had prior radiation, and 18 had prior surgery. RESULTS: Fever and hypotension were the most common toxicities. Fever of any grade occurred in 16 of 21 patients (76%): four of 21 (19%) with grade 2 and two of 21 (9.5%) with grade 3. Hypotension of any grade occurred in 13 of 21 (62%): four of 21 with grade 2 and one of 21 (5%) with grade 3. Interleukin-2 (IL-2) and tumor necrosis factor alpha (TNF alpha) induction correlated with toxicity. In the two patients with grade 3 fever, peak IL-2 and TNF alpha levels were 2.9 IU/mL and 165 pg/mL, and 8.3 IU/mL and 245 pg/mL, respectively. Transient, > or = 50% decreases in circulating monocytes were observed in 17 of 21 patients as early as 0.5 hours (median time, 2 hours) from the start of infusion. Decreases (mean 33%) in circulating lymphocytes were observed in seven of 21 patients. All three patients with grade 3 toxicity were treated at the 0.5-ng/kg dose. The significance of baseline anti-SEA, human antimouse antibody (HAMA), CA242-soluble antigen levels, and T-cell receptor variable beta region (TCR V beta) subsets and histocompatibility leukocyte antigen-DR (HLA-DR) genotypes was assessed as possible predictors of toxicity. All toxicities were transient and easily managed. No grade 3 toxicity occurred at the higher dose levels. CONCLUSION: PNU-214565, a SAg-based tumor targeted therapy, is safe when given as a single 3-hour infusion at doses up to 1.5 ng/kg. The MTD for a single dose was not determined. The safety of a repeated dose schedule is currently under investigation, beginning with doses determined to be safe in this trial.

Functional characterization of the interaction between the superantigen staphylococcal enterotoxin A and the TCR.

In this report, we show that despite an overall amino acid residue identity of more than 80% between the staphylococcal enterotoxins (SE) A and E, these proteins markedly differ in their absolute requirement for the MHC class II during T cell activation. The superantigens were produced as C215Fab-SE fusion proteins and analyzed for their ability to activate T cells in a MHC class II-independent manner, using C215 Ag expressing cell lines as pseudo super-APCs. C215Fab-SEA, but not C215Fab-SEE, induced T cell cytotoxicity and proliferation in these MHC class II-independent systems. Introduction of a region from SEA, comprising amino acids 20-27, to SEE transferred the ability to engage T cells in the absence of MHC class II. Analysis of the Vbeta specificity of the chimeric SEA/SEE molecules and a panel of SEA mutants demonstrated that the site for TCR interaction covers the edge surrounding the shallow cavity on top of the SEA molecule.

Tumor therapy with an antibody-targeted superantigen generates a dichotomy between local and systemic immune responses.

Repeated injections of a fusion protein containing the superantigen staphylococcal enterotoxin A (SEA) combined with a Fab fragment of a tumor-specific antibody is a highly efficient immunotherapy for mice expressing lung melanoma micrometastasis. In the present study, the systemic and local immune responses generated by this therapy were analyzed at a cellular level. Two distinct but coupled immune reactions occurred after repeated therapy. Tumor necrosis factor and macrophage inflammatory protein-1 alpha and -1 beta were immediately synthesized, in the absence of T lymphocytes, at the local tumor site in the lung. This was followed by the induction of VCAM-1 adhesion molecule expression on pulmonary vascular endothelial cells. Concurrently, the early response in the spleen was characterized by the induction of selective T cells producing interleukin (IL)-2. The primed and expanded SEA-reactive V beta 3- and V beta 11-expressing T lymphocytes accumulated to the tumor area only after Fab-SEA therapy and were not present in the lung when SEA, Fab fragment, or recombinant IL-2 was injected. The tumor-infiltrating T cells produced large amounts of interferon-gamma, but no IL-2 or Th2 type of lymphokines were detected at the tumor site in the Fab-SEA-targeted antitumor immune response. These results emphasize the necessity to investigate several sites of antigen presentation to elucidate the effects of immunotherapy.

Prevention of superantigen-induced tolerance in vivo by interleukin-2 treatment.

Injection of the superantigen staphylococcal enterotoxin A (SEA) activates both CD4+ and CD8+ T cells expressing certain families of T cell receptor (TCR) variable-region beta (V beta) chain. T cells respond with profound cytokine production and induction of cytotoxicity. Repeated injections, however, cause deletion and anergy of both CD4+ and CD8+ T cells, resulting in reduced frequency of SEA-responsive cells TCR-V beta11+ as well as reduced cytokine levels in serum upon challenge with SEA. Exogenous interleukin-2 (IL-2) in vivo rescued SEA-responsive CD4+ and CD8+ cells from SEA-induced deletion and/or increase expansion of SEA-primed cells as well as preventing downregulation of endogenous IL-2 production in vivo. Combined treatment with SEA and IL-2 also superinduced production of important cytokines for the cytotoxic function of T cells, tumour necrosis factor alpha, interferon gamma and IL-6, on a cellular level. These studies show that continuous stimulation with IL-2 in vivo could be useful for superantigen-based immunotherapy by induction of excessive T cell activation and by prevention of the development of T cell deletion and anergy.

Molecular characterization and role in T cell activation of staphylococcal enterotoxin A binding to the HLA-DR alpha-chain.

Superantigens bind to MHC class II-positive cells and stimulate T lymphocytes expressing specific V beta regions of the TCR. Two distinct regions of staphylococcal enterotoxin A superantigen (SEA) have been shown to affect the binding to MHC class II molecules. Results presented here demonstrate for the first time that the SEA-DR interaction can be affected by mutations on the class II alpha-chain. Furthermore, we have precisely mapped the interaction of the SEA N-terminal domain with the alpha1 domain of HLA-DR. Scatchard analysis using DAP cells transfected with mutant class II molecules showed a role for residue DR alpha K39 in the binding of SEA. Also, complementation experiments using mutant SEA molecules revealed an interaction between SEA residue F47 and position alphaQ18 on an outer loop of HLA-DR. These interactions between SEAF47 and the DR alpha-chain are critical, as they allow the recognition by an otherwise nonreactive V beta1+ T cell hybridoma and induction of tyrosine phosphorylation through the TCR.

Immunomodulation of autoimmunity by linomide: inhibition of antigen presentation through down regulation of macrophage activity in the model of experimental autoimmune encephalomyelitis.

Linomide (quinoline-3-carboxamide, LS-2616), a synthetic immunomodulator, protects animals against a variety of experimental autoimmune diseases. In experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis (MS), linomide blocks both the clinical and histological signs of the disease, without inducing generalized immunosuppression. In the first clinical trial in patients with MS, linomide was shown to inhibit the progression of the disease. In the present study we investigated several aspects of the mechanisms of action of this immunomodulator. We found that linomide can inhibit acute EAE even when given as pretreatment, prior to induction of disease (days - 10 to 0). This inhibitory effect was reversed by adoptive transfer of naive spleen cells. A short course (7 days) of linomide treatment also inhibited EAE, especially when administered immediately after disease induction. Spleen cells from linomide-treated mice failed to present myelin antigens to T-cell lines in vitro. The defective antigen presentation was normalized by anti-oxidants such as 2-mercaptoethanol. The proportion of Mac1+ cells in the spleens of linomide-treated mice was significantly reduced and macrophage growth was inhibited in long term cultures of spleen cells derived from linomide-treated animals. Our findings suggest that the effect of linomide on EAE may be attributed, at least in part, to inactivation of antigen presenting cells, possibly following a short period of over-stimulation and increased oxidant production. This mechanism may play a universal role in the regulation of autoimmune reactivity and merits further investigation.

Costimulation by B7-1 and LFA-3 targets distinct nuclear factors that bind to the interleukin-2 promoter: B7-1 negatively regulates LFA-3-induced NF-AT DNA binding.

We have characterized the regulation of nuclear factors involved in transcriptional control of the interleukin-2 (IL-2) promoter-enhancer activity in Jurkat T cells stimulated with superantigen presented on HLA-DR transfectants combined with the ligands LFA-3 (CD58) and B7-1 (CD80). Gel shift analyses showed that NF-AT was strongly induced in LFA-3-costimulated Jurkat T cells, suggesting that NF-AT is a key target nuclear factor for the CD2-LFA-3 pathway. Studies using HLA-DR-B7-1-LFA-3 triple transfectants showed that the LFA-3-induced NF-AT DNA binding activity was negatively regulated by B7-1 costimulation. In contrast, induction of a CD28 response complex containing only c-Rel proteins was seen after B7-1 costimulation. Both LFA-3 costimulation and B7-1 costimulation induced the AP-1 and NF-kappaB nuclear factors. Distinct compositions of the NF-AT complexes were seen in B7-1- and LFA-3-costimulated cells. LFA-3 induced primarily Jun-D, Fra-1, and Fra-2, while B7-1 induced June-D-Fos complexes. In contrast, AP-1 and NF-kappaB complexes induced in B7-1- and LFA-3-costimulated T cells showed similar contents. Transient transfection of Jurkat T cells with a construct encoding the IL-2 enhancer-promoter region (position -500 to +60) linked to a luciferase reporter gene revealed that B7-1 costimulation was required to induce strong transcriptional activity. Combined B7-1-LFA-3 costimulation resulted in a synergistic increase in IL-2 transcriptional activity. Multimers of the AP-1, NF-AT, NF-kappaB, and CD28 response elements showed distinct kinetics and activity after LFA-3 and B7-1 costimulation and revealed that B7-1 and LFA-3 converge to superinduce transcriptional activity of the AP-1, NF-AT, and CD28 response elements. Transcriptional studies with an IL-2 enhancer-promoter carrying a mutation in the CD28 response element site revealed that the activity was reduced by 80% after B7-1 and B7-1-LFA-3 costimulation whereas the transcriptional activity induced by LFA-3 was unaffected. Our data strongly suggest a selectivity in induction of nuclear factors by the CD2-LFA-3 and CD28-B7-1 pathways. This selectivity may contribute to regulation of the levels of IL-2 induced by LFA-3 and B7-1 costimulation and favor autocrine and paracrine T-cell responses, respectively.

A superantigen-antibody fusion protein for T-cell immunotherapy of human B-lineage malignancies.

The bacterial superantigen staphylococcal enterotoxin A (SEA) is an efficient activator of cytotoxic T cells when presented on major histocompatibility complex (MHC) class II molecules of target cells. Our previous studies showed that such SEA-directed T cells efficiently lysed chronic B-lymphocytic leukemia (B-CLL) cells. Next, we made a mutated SEA-protein A (SEAm-PA) fusion protein with more than 1,000-fold reduced binding affinity for MHC class II compared with native SEA. The fusion protein was successfully used to direct T cells to B-CLL cells coated with different B lineage-directed monoclonal antibodies (MoAbs). In this communication, we constructed a recombinant anti-CD19-Fab-SEAm fusion protein. The MHC class II binding capacity of the SEA part was drastically reduced by a D227A point mutation, whereas the T-cell activation properties were retained. The Fab part of the fusion protein displayed a binding affinity for CD19+ cells in the nanomolar range. The anti-CD19-Fab-SEAm molecule mediated effective, specific, rapid, and perforin-like T-cell lysis of B-CLL cells at low effector to target cell ratios. Normal CD19+ B cells were sensitive to lysis, whereas CD34+ progenitor cells and monocytes/macrophages were resistant. A panel of CD19+ B-cell lines representing different B-cell developmental stages were efficiently lysed, and the sensitivity correlated with surface ICAM-1 expression. The anti-CD19-Fab-SEAm fusion protein mediated highly effective killing of tumor biopsy cells representing several types of B-cell non-Hodgkin's lymphoma (B-NHL). Humanized severe combined immune deficiency (SCID) mice carrying Daudi lymphoma cells were used as an in vivo therapy model for evaluation of the anti-CD19-Fab-SEAm fusion protein. Greater than 90% reduction in tumor weight was recorded in anti-CD19-Fab-SEAm-treated animals compared with control animals receiving an irrelevant Fab-SEAm fusion protein. The present results indicate that MoAb-targeted superantigens (SAgs) may represent a promising approach for T-cell-based therapy of CD19+ B-cell malignancies.

Genetically engineered superantigens as tolerable antitumor agents.

Superantigens (SAg) are a family of bacterial and viral proteins with strong immunostimulatory properties. SAg bound to major histocompatibility complex (MHC) class II molecules activate a high frequency of T cells and represent the most potent known activators of T cells to date. To explore the use of SAg for T cell-based tumor therapy we have created a tumor-reactive SAg by engineering a fusion protein composed of a tumor-reactive mAb (C215Fab) and the bacterial SAg staphylococcal enterotoxin A (SEA). A point mutation D227A was introduced at the major MHC class II binding site in SEA to reduce systemic toxicity. Treatment of tumor bearing mice with the Fab-SEA D227A fusion protein resulted in profound antitumor effects with a markedly reduced toxicity as compared with the wild-type Fab-SEA fusion protein. The reduced toxicity was probably due to a weak distribution of the SEA D227A fusion protein in tissues with a high MHC class II expression and low systemic cytokine levels as exhibited in mice and rabbits. The data presented demonstrate the efficacy of immunoconjugates containing a mutated SAg in directing a T cell attack against tumor cells with minimal systemic immune activation.