Kuo Mei-Chang

Potential therapeutic recombinant proteins comprised of peptides containing recombined T cell epitopes

The complete primary structure of Fel d I2 has been determined and shown to be comprised of two separate polypeptide chains (designated chain 1 and 2). Overlapping peptides covering the entire sequence of both chains of Fel d I have been used to map the major areas of human T cell reactivity. The present study describes three non-contiguous T cell reactive regions of < 30 aa in length that were assembled in all six possible configurations using PCR and recombinant DNA methods. These six recombinant proteins comprised of defined non-contiguous T cell epitope regions artificially combined into single polypeptide chains have been expressed in E. coli, highly purified, and examined for their ability to bind to human cat-allergic IgE and for human T cell reactivity. Several of these recombined T cell epitope-containing polypeptides exhibit markedly reduced IgE binding as compared to the native Fel d I. Importantly, the human T cell reactivity to individual T cell epitope-containing regions is maintained even though each was placed in an unnatural position as compared to the native molecule. In addition, T cell responses to potential junctional epitopes were not detected. It was also demonstrated in mice that s.c. injection of T cell epitope-containing polypeptides inhibits the T cell response to the individual peptides upon subsequent challenge in vitro. Thus, these recombined T cell epitope-containing polypeptides, which harbor multiple T cell reactive regions but have significantly reduced reactivity with allergic human IgE, constitute a novel potential approach for desensitization to important allergens.

Purification and immunochemical characterization of recombinant and native ragweed allergen Amb a II

The complete sequence of a cDNA encoding Amb a II and its relationship to the Amb a I family of allergens has recently been described [Rogers et al. (1991) J. Immun. 147, 2547-2552; Griffith et al. (1991a), Int. Archs Allergy appl. Immun. 96, 296-304]. In this study, we present results generated with rabbit antipeptide antisera that recognize Amb a II or Amb a I, but not both. The specificity of two anti-Amb a II antipeptide sera, anti-RAE-50.K and anti-RAE-51.K, was verified on Western blots of recombinant Amb a II and Amb aI.1. These two sera, directed against separate regions of the Amb a II molecule, detected three individual 38-kDa Amb a II isoforms on 2D Western blots of aqueous ragweed pollen extract. These Amb a II isoforms have pI in the 5.5-5.85 range and can be easily distinguished from Amb a I isoforms with pI in the 4.5-5.2 range detected by an anti-Amb a I specific peptide antiserum. The Amb a II isoforms have also been individually purified from pollen, positively identified as Amb a II by amino acid sequencing, and visualized as separate bands on IEF gels. An analysis of Amb a II cDNA sequences generated by PCR led to the prediction of three Amb a II isoforms with pI of 5.74, 5.86 and 5.97 that are very similar to the pI deduced from 2D Western blot analysis. Recombinant Amb aI.1 and Amb a II have been expressed in E. coli, purified in their denatured form, and examined by ELISA for their capacity to bind pooled allergic human IgE. Purified native Amb a and Amb a II from pollen were shown to have very similar IgE-binding properties. In contrast, Amb a II had a markedly reduced IgE-binding capacity as compared to Amb a I.1. These data suggest that recombinant Amb a I.1 and Amb a II, isolated in a denatured form, differ significantly in their IgE-binding properties whereas the native molecules isolated from pollen do not.

Rabbit-mouse hybridomas secreting intact rabbit immunoglobulin

Rabbit-mouse hybridomas offer the potential for production of monoclonal rabbit antibodies by immortal cell lines. In previous studies, it was possible to produce and stabilize rabbit-mouse hybrid cells secreting either a rabbit heavy or light chain. These have been useful for structural characterization of the individual rabbit immunoglobulin polypeptides and for isolation of large amounts of immunoglobulin mRNA for molecular studies. For some studies, however, it would be useful to have intact rabbit immunoglobulin molecules comparable to the myeloma proteins available in the human and mouse. The availability of rapid, sensitive and specific assays for rabbit heavy and light chains and allotypes located on specific chains has now permitted the early identification of clones secreting intact rabbit immunoglobulin. Vigorous cloning efforts have resulted in isolation and partial stabilization of three such clones. The first, H105, secretes a product with a kappa light chain bearing the b6 allotype and a mu-chain bearing the a1 allotype. Biochemical and serologic analyses of the product show that it is secreted as a fully assembled IgM pentamer and that the rabbit heavy and light chains are covalently associated. No rabbit J-chain gene was detected in H105 by Southern blot analysis. The second hydridoma, H134, secretes a product with a mol. wt of 150 K, consisting of a b4 light chain and an a1 heavy chain. The third, H171, secretes an alb4 IgG with antibody specificity for group C streptococcal carbohydrate. An additional rabbit-mouse hybridoma, H89, have been produced which secretes a rabbit heavy chain lacking group a allotypic activity. The rabbit heavy chain, which is associated with a mouse light chain, has an N-terminal amino acid sequence identical to a2-positive molecules although thorough serologic analysis revealed no group a allotypic activity.