Artificial proteins with lowered adjuvanticity

FIELD: medicine.

SUBSTANCE: invention concerns immunology area. Versions of the artificial fused protein consisting of an antibody (or its fragment) and cytokine, fused through a link peptide are offered. The antibody or its fragment is chosen from an antibody 225, 425, KS 1/4, 14.18, anti-CDx-antibody where x has the whole value 1-25. Each of versions of the fused protein has lowered quantity T-epitopes, at least, in the component of the fused protein presented by an antibody, and as consequence, possesses the lowered adjuvanticity, in comparison with an initial molecule. Identification of T-lymphocyte epitopes is performed by the automated calculation of sizes for the binding centres of class II MHC molecules with the subsequent experimental test of the obtained versions of protein for presence of the lowered adjuvanticity. The automated way of T-epitopes calculation is based on use of the Bjom's function modified in such manner that contribution of Van-der-vaals repulsion and lipophilic interaction in pairs between all lipophilic atoms of the chosen segments of the fused protein and a binding groove of a MHC P molecule is taken into account. Also a way of protein construction on the basis of the modified function Bjom's function with the subsequent experimental test of the received versions for presence of the lowered adjuvanticity is revealed, and also application of the fused protein for preparation of a pharmaceutical composition for tumour treatment is in addition considered.

EFFECT: invention use allows obtaining the fused proteins with the lowered adjuvanticity and, basically, keeping identical biological activity in comparison with a parent molecule; it can be used in treatment of tumours.

4 cl, 6 dwg, 22 tbl, 19 ex

 

The text descriptions are given in facsimile form.

1. Artificial protein formula:
A-Ln-X
where a means the Fc part of the antibody molecules or whole antibody or its fragments sFv, Fab, Fab', F(ab')2selected from the group including:
monoclonal antibody 225, and derivatives
monoclonal antibody 425 and derivatives
monoclonal antibody KS 1/4 and derivatives
monoclonal antibody 14.18 and derivatives
anti-CDx-antibody, where x is an integer 1-25;
X means a cytokine selected from the group comprising IL-2, IL-12, EPO, G-CSF, GM-CSF, TNFα, endostatin and angiostatin,
L means of the linker peptide,
n=0 or 1,
derived from the parental artificial fused protein having an amino acid sequence that differs from the sequence specified parent synthetic fused protein and exhibiting reduced immunogenicity by reducing the number of T-cell epitopes on the number of epipo parent fused protein by exposure to the immune system of this type, where these T-cell epitopes have a peptide sequence that has the ability to communicate with the communicating groups of molecules sit in class II, and at least And no or a reduced number of T-cell epitopes, and the specified immunogene modified artificial protein get the using method, providing
(i) identifying one or more potential T-cell epitopes within the amino acid sequence of the fused protein, where this identification includes:
selection of protein segments, overlapping by 1-5 amino acid residues, where the length of each segment is 13 amino acid residues;
consistent calculation of the binding constant of each segment and the binding groove of the MHC molecules of class II using the evaluation function Behm, modified by the fact that additionally takes into account the contribution of the energy of the van der Waals repulsion and lipophilic interactions between all pairs of lipophilic atoms selected segments of the protein and all lipophilic atoms binding grooves of MHC molecules class II, optionally, the comparison of calculated data with experimental data on the affinity of peptides to MHC class II;
the choice of the segments with the highest values of the binding energy or optional, the choice of segments with values of the binding energy in excess of the value of experimental data:
(ii) change in silico from 1 to 9 amino acid residues within the identified potential sequences of T-cell Kapitonov at amino acid residues, eliminating the ability of these epitopes to contact MHC class who II, with preservation of protein function;
(iii) receiving options modified sequences of T-cell epitopes by recombinant DNA;
(iv) test the above options on the presence of reduced immunogenicity at a constant therapeutic activity and, if necessary,
(v) repeating steps (i)-(iv).

2. Fused protein according to claim 1, in which X is IL2.

3. Method of constructing fused protein according to claim 1, including:
(i) identifying one or more potential T-cell epitopes within the amino acid sequence of the fused protein, where this identification includes:
selection of protein segments, overlapping by 1-5 amino acid residues, where the length of each segment is 13 amino acid residues;
consistent calculation of the binding constant of each segment and the binding groove of the MHC molecules of class II using the evaluation function Behm, modified by the fact that additionally takes into account the contribution of the energy of the van der Waals repulsion and lipophilic interactions between all pairs of lipophilic atoms selected segments of the protein and all lipophilic atoms binding grooves of MHC molecules class II, optionally, the comparison of calculated data with experimental data on the affinity of peptides to MHC class II;
the choice of segments with you the most is okimi values of the binding energy or optional, the choice of segments with values of the binding energy in excess of the value of experimental data:
(ii) change in silico from 1 to 9 amino acid residues within the identified potential sequences of T-cell epitopes at amino acid residues, eliminating the ability of these epitopes to contact MHC class II, with preservation of protein function;
(iii) receiving options modified sequences of T-cell epitopes by recombinant DNA;
(iv) test the above options on the presence of reduced immunogenicity at a constant therapeutic activity and, if necessary,
(v) repeating steps (i)-(iv).

4. The use of fused protein according to claim 1 for the preparation of pharmaceutical compositions for the treatment of tumors.
Priority items:

19.02.2001 according to claims 1, 2, 4;

05.04.2001 according to claim 3.



 

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