Use of soluble CTLA4 molecule in preparation of medicine composition for treating rheumatic diseases
A rheumatic disease, soluble technology, applied in the field of treatment of rheumatic diseases such as rheumatoid arthritis, can solve the problems of toxic side effects, non-continuous long-term use, and limited therapeutic efficacy of rheumatoid arthritis
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Embodiment 1
[0185] Methods for generating nucleotides encoding CTLA4 molecules of the invention are provided below.
[0186] First, a plasmid encoding CTLA4Ig was constructed, and then it was demonstrated to express CTLAIg as described in US Patent Nos. 5,434,131, 5,885,579 and 5,851,795. Single point mutant molecules (such as L104EIg) were then generated from the CTLAIg coding sequence, expressed and examined for binding kinetics to various B7 molecules. Using the L104EIg nucleotide sequence (included in the sequence shown in Figure 18) as a template to generate two-site CTLA4 mutant sequences (included in the sequences in Figures 19-22), they were expressed as proteins, and their binding kinetics were examined study. Two-site CTLA4 mutant sequences include: L104EA29YIg, L104EA29LIg, L104EA29TIg, and L104EA29WIg. Triple site mutants were also generated.
[0187] Construct CTLA4Ig
[0188] The gene construct encoding CTLA4Ig comprises the extracellular domain of CTLA4Ig and the IgCγ ...
Embodiment 2
[0199] The following examples provide screening methods for identifying the above-described single- and double-site mutant CTLA4 polypeptides expressed from the constructs described above, which have higher binding to CD80 and CD86 antigens compared to non-mutated CTLA4Ig molecules affinity.
[0200] Current in vivo and in vitro studies have shown that CTLA4Ig itself is not sufficient to completely block the stimulation of antigen-specifically activated T cells. In vitro studies using monoclonal antibodies specific for CTLA4Ig and CD80 or CD86 measured the inhibition of T cell proliferation and showed that anti-CD80 monoclonal antibodies did not enhance the inhibition of CTLA4Ig. However, anti-CD86 mAbs did enhance CTLA4Ig inhibition, suggesting that CTLA4Ig is not very effective at blocking CD86 interaction. These data support earlier findings by Linsley et al. ((Immunity, (1994), 1:793-801 )) that approximately 100-fold lower CTLA4Ig concentrations were required for inhibit...
Embodiment 3
[0247] The following provides a description of a Phase II clinical study in human patients using the soluble CTLA4 mutant molecule L104EA29YIg (also known as LEA29Y or LEA) or CTLA4Ig to reduce at least one symptom associated with rheumatoid arthritis, including reducing: joint swelling, Joint tenderness, inflammation, morning stiffness and pain. The CTLA4Ig molecule used here starts with a methionine at position +1 (or an alanine at position -1 ) and ends with a lysine at position +357, as shown in FIG. 24 . DNA encoding the CTLA4Ig molecule in one embodiment was deposited as ATCC 68629. The L104EA29YIg molecule used here starts with a methionine at the +1 position (or an alanine at the -1 position) and ends with a lysine at the +357 position, as shown in FIG. 24 . DNA encoding one embodiment of the L104EA29YIg molecule has been deposited as ATCC PTA 2104.
[0248]In addition, instructions for the use of L104EA29YIg or CTLA4Ig to alleviate at least one biosurrogate marker a...
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