Characterization of two distinct MHC class II binding sites in the superantigen staphylococcal enterotoxin A.

Abrahmsén, Lars; Dohlsten, Mikael; Segrén, Sverker; Björk, Per; Jonsson, E; Kalland, Terje

doi:10.1002/j.1460-2075.1995.tb07300.x

Cited by 154 publications

(144 citation statements)

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“…The structure and function properties of SEA have been well characterized (Abrahmsén et al, 1995;Hudson et al, 1995;Schad et al, 1995). To reduce the systemic toxicity, a major drawback in the use of wild-type SEA (Alphaug et al, 1998), a substitution of Asp 227 has been made.…”

Section: Discussionmentioning

confidence: 99%

“…The fusion proteins were produced at Pharmacia & Upjohn (Stockholm, Sweden) essentially as described (Dohlsten et al, 1994;Abrahmsén et al, 1995;Forsberg et al, 1997). The Fvencoding portions of 5T4 were cloned from the 5T4 hybridoma and fused to sequences coding for the constant regions of the murine IgG1/k antibody C242 lacking the interchain disulphide bond.…”

Section: Cloning Expression and Purification Of Fab-sea Fusion Proteinsmentioning

confidence: 99%

“…A region coding for SEA D227A was connected to the Cterminus of the heavy chain ( Figure 1A) via a Gly-Gly-Pro linker. The products were expressed and secreted in the E. coli K-12 strain UL 635 (xyl-7, ara-14, T4 R , ∆ompT) (Abrahmsén et al, 1995) using a plasmid with a lacUV5-promoter. After fermentation, clarified growth medium was applied to a Protein G Sepharose column (Pharmacia Biotech, Uppsala, Sweden) and bound protein eluted with 0.1 M acetic acid, 0.05% Tween 80.…”

Section: Cloning Expression and Purification Of Fab-sea Fusion Proteinsmentioning

confidence: 99%

“…The yield in the E. coli growth medium of 5T4FabV13-SEA D227A was in the order of 0.5 g l -1 . To decrease the MHC class II binding, and subsequently in vivo toxicity, the replacement Asp227Ala was introduced in SEA, yielding SEA D227A (Abrahmsén et al, 1995). The full-length product was recovered in a 2-step purification procedure.…”

Section: E Coli Production Of the Fusion Proteinmentioning

confidence: 99%

“…staphylococcal enterotoxin A, SEA, have been for the treatment of human colorectal cancer (Dohlsten et al, 1995b). However, to decrease the reactivity of the superantigen with MHC class IIbearing cells, the Asp227Ala replacement were introduced to destroy the site having the highest affinity for MHC class II in SEA (Abrahmsén et al, 1995).…”

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Therapy of human non-small-cell lung carcinoma using antibody targeting of a modified superantigen

et al. 2001

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Summary Superantigens activate T-cells by linking the T-cell receptor to MHC class II on antigen-presenting cells, and novel reactivity can be introduced by fusing the superantigen to a targeting molecule. Thus, an antibody-targeted superantigen, which activates T cells to destroy tumour cells, might be used as cancer therapy. A suitable target is the 5T4 oncofetal antigen, which is expressed on many carcinomas. We constructed a fusion protein from a Fab of a monoclonal antibody recognizing the 5T4 antigen, and an engineered superantigen. The recombinant product 5T4FabV13-SEA D227A bound the 5T4 antigen expressed on the human non-small-cell lung cancer cell line Calu-1 with a K d of 1.2 nM while the substitution of Asp227 to Ala in the superantigen moiety reduced binding activity to MHC class II. 5T4FabV13-SEA D227A tumour reactivity was demonstrated in 7/7 NSCLC samples by immunohistochemistry, while normal tissue reactivity was low to moderate. 5T4FabV13-SEA D227A induced significant T-cell-dependent in vitro killing of sensitive 5T4 bearing Calu-1 cells, with maximum lysis at 10 -10 M, while the capacity to lyse MHC class II expressing cells was approximately 1000 times less effective. Immunotherapy of 5T4FabV13-SEA D227A against human NSCLC was investigated in SCID mice reconstituted with human peripheral blood mononuclear cells. Mice carrying intreperitoneally growing Calu-1 cells showed significant reduction in tumour mass and number after intravenous therapy with 5T4FabV13-SEA D227A . Thus, 5T4FabV13-SEA D227A has highly attractive properties for therapy of human NSCLC.

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Section: Discussionmentioning

confidence: 99%

Section: Cloning Expression and Purification Of Fab-sea Fusion Proteinsmentioning

confidence: 99%

Section: Cloning Expression and Purification Of Fab-sea Fusion Proteinsmentioning

confidence: 99%

Section: E Coli Production Of the Fusion Proteinmentioning

confidence: 99%

mentioning

confidence: 99%

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Therapy of human non-small-cell lung carcinoma using antibody targeting of a modified superantigen

et al. 2001

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Repeated treatment with antibody-targeted superantigens strongly inhibits tumor growth

et al. 1998

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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 B16‐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‐γ and tumor necrosis factor‐α. 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. Int. J. Cancer 76:274‐283, 1998.© 1998 Wiley‐Liss, Inc.

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Long-term survival and complete cures of B16 melanoma-carrying animals after therapy with tumor-targeted IL-2 and SEA

et al. 1999

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Characterization of two distinct MHC class II binding sites in the superantigen staphylococcal enterotoxin A.

Cited by 154 publications

References 36 publications

Therapy of human non-small-cell lung carcinoma using antibody targeting of a modified superantigen

Therapy of human non-small-cell lung carcinoma using antibody targeting of a modified superantigen

Repeated treatment with antibody-targeted superantigens strongly inhibits tumor growth

Long-term survival and complete cures of B16 melanoma-carrying animals after therapy with tumor-targeted IL-2 and SEA

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