Cross-linking of major histocompatibility complex class II molecules by staphylococcal enterotoxin A superantigen is a requirement for inflammatory cytokine gene expression.

Staphylococcal enterotoxin A (SEA) has two distinct binding sites for major histocompatibility complex (MHC) class II molecules. The aspartic acid located at position 227 (D227) in the COOH terminus of SEA is one of the three residues involved in its interaction with the DR beta chain, whereas the phenylalanine 47 (F47) of the NH2 terminus is critical for its binding to the DR alpha chain. Upon interaction with MHC class II molecules, SEA triggers several cellular events leading to cytokine gene expression. In the present study, we have demonstrated that, contrary to wild-type SEA, stimulation of the THP1 monocytic cell line with SEA mutated at position 47 (SEAF47A) or at position 227 (SEAD227A) failed to induce interleukin 1 beta and tumor necrosis factor-alpha messenger RNA expression. Pretreatment of the cells with a 10-fold excess of either SEAF47A or SEAD227A prevented the increase in cytokine messenger RNA induced by wild-type SEA. However, cross-linking of SEAF47A or SEAD227A bound to MHC class II molecules with F(ab')2 anti-SEA mAb leads to cytokine gene expression, whereas cross-linking with F(ab) fragments had no effect. Taken together, these results indicate that cross-linking of two MHC class II molecules by one single SEA molecule is a requirement for cytokine gene expression.

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.

Antibody-targeted superantigens induce lysis of major histocompatibility complex class II-negative T-cell leukemia lines.

CTLs bearing certain T-cell receptor V beta-regions are directed by the bacterial superantigen Staphylococcus enterotoxin A (SEA) to lyse MHC class II-positive cells. In order to extend superantigen-dependent cytotoxicity to MHC class II-negative carcinoma cells, covalent conjugates of superantigen and mAbs against surface markers of these cells have been used. We now describe a novel strategy which allows rapid selection of mAb suitable for superantigen targeting against MHC class II-negative tumor cells. A recombinant fusion protein of protein A and SEA binding to the mAbs CD7 or CD38 was able to mediate T cell-dependent lysis of MHC class II-negative Molt-4 and CCRF-CEM acute lymphatic leukemia cell lines. Lysis was dose dependent and correlated with E:T cell ratio. In contrast, SEA alone did not induce any significant lysis. In order to decrease the MHC class II affinity of the protein A-SEA complex, a point mutation was introduced into SEA (protein A-SEA mu9). The mutated fusion protein had similar potency as protein A-SEA against Molt-4 cells but was 100-fold less active against MHC class II-positive cells. Considering the efficiency and specificity of the mutated SEA protein interacting with mAb in targeting T lymphocytes against MHC class II-negative leukemia cells while only marginally affecting normal MHC class II-positive cells, we suggest the development of SEA-mAb fusion proteins as a potential adjuvant therapy of leukemias.

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.

T cell activation pathways: B7, LFA-3, and ICAM-1 shape unique T cell profiles.

Two signals are required for induction of cell proliferation and cytokine production in resting T cells. Occupancy of the T cell receptor by antigen/MHC complexes delivers the first signal to the T cell, while the second signal is provided by interaction with costimulatory ligands on APC. CD2, LFA-1, and CD28 are the major costimulatory and adhesive molecules on T cells and bind to the LFA-3, ICAM-1 and B7 ligands, respectively, on APC. LFA-3 plays a central role for naive and memory T helper cells during the early phase of an immune response. The LFA-3/CD2 pathway initiates strong antigen-independent cell adhesion, substantial expansion of naive T helper cells, and induction of large amounts of IFN-gamma in memory cells. The release of IFN-gamma may upregulate expression of ICAM-1 and B7 on APC and allows multiple adhesion pathways to amplify the immune response. The LFA-1/ICAM-1 pathway stimulates adhesion and cell proliferation more efficiently in memory T helper cells than in naive cells. Further, the results suggest that naive T helper cells express functionally inactive LFA-1 molecules on the cell surface, which may have a physiological role in keeping these cells in a resting state. B7 costimulation superinduces IL-2 production in both naive and memory T helper cells and generates long-lasting cell proliferation. This permits transition from an autocrine to a paracrine immune response. Coexpression of B7/LFA-3 provides an optimal APC function and enables a vigorous T cell response to minute amounts of antigen. AP-1 and NF-kappa B transcription factors are involved in the induction of several cytokine gene promoters and play a central role in the regulation of IL-2 gene transcription. LFA-3 costimulation only moderately enhances AP-1 DNA-binding activity and does not influence the NF-kappa B activity induced by TCR engagement, whereas B7 costimulation induces large amounts of NF-kappa B and AP-1 activity in T helper cells. The costimulatory ligands represent a family of adhesion molecules with considerable redundancy. Interfamily redundancy of LFA-3, B7, and ICAM ligands offers an opportunity to regulate distinct T cell response profiles in various microenvironments at separate time points of an immune response.

Antibodies are capable of directing superantigen-mediated T cell killing of chronic B lymphocytic leukemia cells.

The bacterial superantigen staphylococcal enterotoxin A (SEA) is a highly potent activator of cytotoxic T cells when presented on MHC class II molecules of target cells. Our earlier studies showed that such SEA-directed T cells efficiently killed chronic B lymphocytic leukemia (B-CLL) cells. With the ultimate goal to replace the natural specificity of SEA for MHC class II molecules with the specificity of a monoclonal antibody (mAb), we initially made a mutated protein A-SEA (PA-SEAm) fusion protein with > 100-fold reduced binding affinity for MHC class II compared to native SEA. The fusion protein was successfully used to direct T cells to B-CLL cells coated with different B lineage specific (CD19, CD20) or associated (CD37, CD40) mAbs. The PA-SEAm protein was 10-100-fold more potent against mAb coated compared to uncoated HLA class II+ B-CLL cells. No correlation was seen between the amount of mAb bound to the cell surface and sensitivity to lysis. Preactivation of B-CLL cells by phorbol ester increased their sensitivity, and lysis was dependent on ICAM-1 molecules. However, no preactivation of the target cells was needed when a cocktail of two or four mAbs was used. Circulating leukemia and spleen cells were equally well killed. We conclude that the natural target specificity of SEA, MHC class II, can be reduced by mutagenesis and novel binding specificity can be introduced by linkage to tumor reactive mAbs. Our findings encourage the construction of recombinant SEA mutant fusion proteins for specific T cell therapy of hematopoietic tumors such as B-CLL.

Production and characterization of rabbit antibodies against histamine.

Antisera were raised in rabbits against histamine conjugated to human serum albumin (HSA) by the carbodiimide (ECDI) method. The specificity of the antisera was studied in a radioimmunoassay using 125I-protein A for detection of IgG binding. The HIS-HSA antisera reacted with histamine-HSA conjugates prepared by either the carbodiimide or diisocyanate coupling procedure, as well as with carbodiimide-prepared histamine-ferritin and histamine-ovalbumin conjugates. On the contrary, the antisera were unreactive with unconjugated HSA, ECDI-reacted HSA, or HSA conjugated to ethanolamine or pentylamine. Free unconjugated histamine significantly inhibited antibody binding to histamine-HSA and 50% inhibition of antibody binding (IC50) was recorded at 3 mM histamine concn. On a histamine molar concn basis a much lower inhibitory potency of free histamine was recorded, as compared to histamine-protein conjugates (IC50 = 3 X 10(-6) mM). This probably reflected amplification of antibody binding to the multivalent ligand, but possibly also that the protein carrier adds some common features to the antigenic determinant. Histidine, ornithine, glutamine, asparagine, sterylamine and several other amino acids lacked inhibitory effects. Histamine H1 and H2 receptor antagonists inhibited histamine binding to the histamine antibodies. The antagonists varied in their affinity for the histamine antibodies and 50% inhibition of antibody binding was recorded in the range of 1-50 mM concn of the antagonists. Comparing one H1 and one H2 antagonist (diphenhydramine and cimetidine, respectively) two of the sera were preferentially inhibited by cimetidine whereas the third serum seemed to be more prone to inhibition by diphenhydramine.

Antagonistic effects of the staphylococcal enterotoxin a mutant, SEA(F47A/D227A), on psoriasis in the SCID-hu xenogeneic transplantation model.

Psoriasis is a T-cell-mediated immune dermatosis probably triggered by bacterial superantigens. This pathomechanism has been experimentally reproduced in a SCID-hu xenogeneic transplantation model. We analyzed the effects of different bacterial superantigens on the induction of psoriasis in this model. Staphylococcal enterotoxin B and exfoliative toxin triggered the onset of psoriasis when administered repetitively intracutaneously over a period of 2 wk, whereas staphylococcal enterotoxin A representing a distinct subfamily of staphylococcal enterotoxins only mimicked certain aspects of psoriasis. The biologic effects of staphylococcal enterotoxin A were more pronounced when a mutated form, SEA(H187A), of this superantigen with reduced affinity to major histocompatibility complex class II was coinjected. Another mutated variant, SEA(F47A/D227A), exhibiting no measurable major histocompatibility complex class II affinity blocked the effects triggered by wild-type staphylococcal enterotoxin A when injected in a 10-fold higher dose. Inhibition was specific as induction of psoriasiform epidermal changes by staphylococcal enterotoxin B could not be blocked. As staphylococcal enterotoxin A, in contrast to the other superantigens tested, is capable of inducing epidermal thickening but not the typical appearance of psoriasis, we conclude that bacterial superantigens may differ with regard to their effects on human nonlesional psoriatic skin. Staphylococcal-enterotoxin-A-mediated effects were blocked by a genetically engineered superantigen highlighting the potential therapeutic use of mutated superantigens.

Vaccination with B16 melanoma cells expressing a secreted form of interleukin-1beta induces tumor growth inhibition and an enhanced immunity against the wild-type B16 tumor.

We have demonstrated previously that gene transfer of the mature human interleukin-1beta (IL-1beta) gene, fused to a signal sequence (ss), into mouse B16 melanoma cells results in an inhibition of their growth in vivo compared with control B16 cells. We here extend these results to show that intraperitoneal vaccinations with irradiated IL-1beta-secreting cells result in protection against subsequent subcutaneous challenge with wild-type (wt) B16 tumor cells in syngeneic C57BL/6 mice. This protection appears to be long-lasting, because rechallenge of cured mice 4 months after the first challenge also demonstrated resistance. In addition, we demonstrate that mice with established wt tumors subjected to therapeutic vaccinations with irradiated B16/ssIL-1beta cells starting 3 days after challenge isografting have a significantly inhibited tumor growth and 25-40% survival at the challenge doses given. In vitro coculture of spleen cells from B16/ssIL-1beta vaccinated animals and wt B16 cells induced an enhanced proliferative response, which correlated with elevated production of IL-2 and interferon-gamma. A significantly enhanced cytolytic activity against B16 wt target tumor cells was observed when spleen cells from B16/ssIL-1beta vaccinated mice were used as effector cells compared with spleen cells from control vaccinated mice. In vitro depletion experiments using anti-asialo GM1 revealed a prominent role for natural killer cells as effector cells. The data suggest that local IL-1beta secretion during the vaccination phase can provoke or augment protective immune responses to B16 melanoma cells, which are otherwise not recorded in mice bearing B16 tumors.

Characterization of individual tumor necrosis factor alpha-and beta-producing cells after polyclonal T cell activation.

Mononuclear cells from human blood were stimulated to tumor necrosis factor alpha (TNF alpha) or beta (TNF beta) production by the T cell mitogens anti-CD3 antibody (OKT3) or staphylococcal enterotoxin A (SEA). The cells were then fixed and subsequently permeabilized in suspension by the detergent saponin in order to enable TNF alpha- or TNF beta-specific antibodies to enter the cells and interact with cytoplasmic TNF in producer cells. A characteristic morphology of the staining pattern of the two cytokines was noted, with a local accumulation in the cytoplasm in a perinuclear position reflecting the presence of TNF alpha or -beta in the Golgi system. TNF alpha-producing cells appeared 2-3 h after activation of the cultures and increased up to 6 h. The majority of these early TNF alpha-producing cells were monocytes as judged by two-color staining and morphology, but a small fraction of CD4- and CD8-positive T cells was found up to 72 h. TNF beta production started later and peaked 18 or 48 h after OKT3 or SEA stimulation, respectively. The number of TNF beta-producing cells was much larger than that of TNF alpha-producing cells, and approximately 90% of them were CD4-positive T cells. The remaining TNF beta production occurred in CD8-positive T cells and in B cells. Almost every second CD4-positive T cell made TNF beta at the peak of the SEA-induced synthesis. The cytotoxic activity found in the supernatants correlated well with the number of TNF-producing cells found in the cultures. Cells from fresh blood or unstimulated cultures showed no or very few TNF-producing cells.

Activation of alloreactive natural killer cells is resistant to cyclosporine.

BACKGROUND: We have previously shown that cytotoxic T lymphocytes (CTL) with alloreactivity were induced when Wistar Furth (WF; RT1u) rats were immunized with allogeneic Brown Norway (BN; RT1n) cells. In contrast, when BN rats were immunized with WF cells, the allospecific response was confined to alloreactive natural killer (NK) cells, and no CTL activity was observed. In this study, the effect of cyclosporine (CsA) on the activation of alloreactive NK cells in vivo was analyzed. METHODS: Distinct peritoneal effector cells from rats immunized with allogenic cells with or without concomitant CsA and/or interleukin (IL) 2 treatment were tested for specific cytolytic activity. Furthermore, the presumptive role of NK cells in rejection immunity was addressed in a cardiac graft model. RESULTS: The results showed that doses of CsA that completely inhibited the activation of alloreactive CTL, only marginally affected the activation of alloreactive NK cells. We also showed that CsA treatment failed to prolong graft survival in BN recipients of WF hearts. Treatment of BN rats with CsA/IL-2 during immunization with allogeneic WF cells resulted in concomitant induction of alloreactive NK cells and alloreactive CTL. CONCLUSIONS: We have demonstrated that CsA failed to suppress the activation of alloreactive NK cells. Consequently, the cardiac graft survival in the donor-recipient combination known to activate alloreactive NK cells was not significantly prolonged by CsA treatment, emphasizing the involvement of NK cells as effectors in organ rejection. Furthermore, the parallel emergence of alloreactive NK cells and CTL only in the presence of CsA/IL-2 indicated that CsA interfered with alloreactive NK cell-associated suppression of CTL activated by allogeneic tissue.

Proliferation of human CD4+45R+ and CD4+45R- T helper cells is promoted by both IL-2 and IL-4 while interferon-gamma production is restricted to IL-2 activated CD4+45R- T cells.

Recombinant IL-2 (rIL-2) and IL-4 (rIL-4) promote proliferation of human CD4+ T cells activated in the presence of PHA, TPA or OKT-3 monoclonal antibody (MAb), whereas the production of interferon-gamma (IFN) can be induced only by rIL-2. rIL-4 induced strong proliferative responses both in accessory cell independent assays and in the presence of autologous monocytes, but has failed to induce IFN production in any of these systems. The ability of rIL-2 to induce IFN production was strongly enhanced by the addition of monocytes, although a similar proliferative response was recorded in the absence or presence of monocytes. The MAb anti-Tac inhibited the proliferative response and the production of IFN by CD4+ T cells activated in the presence of rIL-2, whereas the proliferative response to rIL-4 was unaffected. CD4+45R+ and CD4+45R- T helper cell subsets proliferated in response to both IL-2 and IL-4. A kinetic analysis demonstrated that the production of IFN throughout a five day activation period was restricted to stimulation of CD4+45R- T cells with rIL-2. This report clearly demonstrates a dissociation of IFN production and T cell proliferation in man. While proliferation can be induced by both IL-2 and IL-4 in both the helper T cell subsets studied, IFN production was induced only in the CD4+45R- subsets and only in response to IL-2.

Locally superantigen-activated peritoneal cytolytic T lymphocytes belong to the CD8+ CD45RC- subset and lyse MHC class II+ tumor cells.

Bacterial encoded superantigens (SA) are capable of activating and targeting cytolytic human and mouse T lymphocytes (CTL) to lyse major histocompatibility complex class II positive (MHC class II+) target cells. In this study both in vitro and in vivo activated rat CTL were directed against MHC II+ tumor targets by bacterial encoded SA. Polyclonal in vitro activation of rat peripheral blood T lymphocytes generated CTL capable of killing MHC class II+ human BSM cells coated by staphylococcal enterotoxin (SE) -A, -E, -D, and TSST-1 but not by SEB or SEC1-3. Allo selective peritoneal CTL generated by intraperitoneal stimulation with allogeneic spleen cells were directed against BSM cells by SEA, -D, and -E but not by SEB, SEC1-3 or TSST-1. Based on the above observations, and in order to locally activate CTL, SEA was chosen for in vivo priming of rats by intraperitoneal inoculation of the toxin. SEA injection generated highly cytolytic CTL, and maximum cytolytic responses were seen at 50-250 micrograms SEA per animal with a peak in response 48-72 hours after injection of the toxin. The cytolytic activity of peritoneal SEA reactive effector cells was confined to the TCR alpha beta+ CD4- CD8+ CD45RC- cell population. MHC class II- colon carcinoma cells were insensitive to lysis by SEA reactive CTL but colon carcinoma cells induced to express MHC class II by interferon-gamma (IFN-gamma) treatment were efficiently lysed in the presence of SEA. Comparison of rat and human MHC II+ colon carcinomas revealed a peak in sensitivity to lysis at 10-100 ng SEA/ml for both tumor targets. These findings suggest that superantigens can be used in local immunotherapy of peritoneal tumors such as ovarian and colorectal carcinomatosis, with inducible or constitutive expression of MHC class II.

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.

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.

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.