Methods of screening for agents that inhibit interaction between MHC class I antigens and a cell surface receptor

A method of screening for drugs capable of inhibiting interaction between a major histocompatibility Class I antigen and a cell surface receptor, thereby modulating cell surface expression of said receptor, said method comprising the steps of:

• binding to an insoluble support substantially purified MHC Class I antigen having activity in modulation of cell surface expression of said receptor;
• adding to said bound antigen, a drug candidate and a labeled antibody or peptide in amounts varying relative to one another, said antibody or peptide having binding specificity to a peptide from 8 to 40 amino acids derived from an .alpha.1 or .alpha.2 domain of said major histocompatibility class I antigen having, as the C-terminal sequence, a continuous sequence contained within a sequence selected from the group consisting of:
• G-N-aa.sup.71 -Q-aa.sub.1.sup.73 -F-R-aa.sub.1.sup.76 -aa.sub.1.sup.77 -aa.sup.78 -R-T-aa.sup.81 -L-R-Y-X (SEQ ID NO.32), and W-aa.sup.61 -R-aa.sup.63 -T-aa.sup.65 -aa.sup.66 -aa.sup.67 -K-aa.sup.69 -aa.sup.70 -aa.sup.71 -Q-aa.sub.2.sup.73 -F-R-aa.sub.2.sup.76 -aa.sub.2.sup.77 -L-R-T-L-aa.sup.82 -aa.sup.83 -Y-aa.sup.85 (SEQ ID NO.34)
• where:
• aa.sup.61, if present, is D or E;
• aa.sup.63, if present, is E, I, or N;
• aa.sup.65, if present, is Q, or R;
• aa.sup.66, if present, is I, N, or K;
• aa.sup.67, if present, is A, S, C, or M;
• aa.sup.69, if present, is G, A, T, or P;
• aa.sup.70, if present, is N, Q, K, R, S, or T;
• aa.sup.71, if present, is G, A, S, T, D, or E;
• aa.sub.1.sup.73, if present, is S, T, F, Y, H or W;
• aa.sub.2.sup.73, if present, is S, T, or W;
• aa.sub.1.sup.76, if present, is an aliphatic amino acid other than basic of from 4 to 6 carbon atoms or alanine;
• aa.sub.2.sup.76, if present, is V, or E;
• aa.sup.77, if present, is a polar aliphatic amino acid of from 3 to 6 carbon atoms, or alanine;
• aa.sub.2.sup.77, if present, is N, S, or D;
• aa.sup.78 is a non-polar aliphatic amino acid of from 3 to 6 carbon atoms;
• aa.sup.81 is G, L, I, or V;
• and
• aa.sup.82 is L, or R,
• aa.sup.83 is G, or R;
• aa.sup.85 is Y, or A, and aa85 is the terminal amino acid;
• X is an amino group to provide an amide terminal group, or the amino acids alanine or tyrosine terminating in a carboxy group;
• and
• detecting an amount of labeled antibody or peptide bound in the presence and in the absence of said drug candidate;
• wherein if said detected amount of labeled antibody or peptide is less in the presence of said drug candidate relative to the amount of labeled antibody or peptide detected in the absence of said drug candidate, said drug candidate is capable mediating modulation of said cell surface expression of said receptor.

A method for screening for drugs capable of inhibiting interaction between a major histocompatibility Class I antigen and a cell surface receptor, thereby modulating cell surface expression of said receptor, said method comprising the steps of:

• binding to an insoluble support cells, said receptor being present on the surface of said cells;
• adding to said bound cells, a drug candidate and a labeled antibody or peptide in amounts varying relative to one another, said antibody or peptide having binding specificity to a peptide from 8 to 40 amino acids derived from an .alpha.1 or .alpha.2 domain of said major histocompatibility class I antigen and having, as the C-terminal sequence, a continuous sequence contained within a sequence selected from the group consisting of:
• G-N-aa.sup.71 -Q-aa.sub.1.sup.73 -F-R-aa.sub.1.sup.76 -aa.sub.1.sup.77 -aa.sup.78 -R-T-aa.sup.81 -L-R-Y-X (SEQ ID NO.32), and W-aa.sup.61 -R-aa.sup.63 -T-aa.sup.65 -aa.sup.66 -aa.sup.67 -aa.sup.69 -aa.sup.70 - aa.sup.71 -Q-aa.sub.2.sup.73 -F-R-aa.sub.2.sup.76 -aa.sub.2.sup.77 -L-R-T-L-aa.sup.82 -aa.sup.83 -Y-aa.sup.85 (SEQ ID NO.34)
• where:
• aa.sup.61, if present, is D or E;
• aa.sup.63, if present, is E, I, or N;
• aa.sup.65, if present, is Q, or R;
• aa.sup.66, if present, is I, N, or K;
• aa.sup.67, if present, is A, S, C, or M;
• aa.sup.69, if present, is G, A, T, or P;
• aa.sup.70, if present, is N, Q, K, R, S, or T;
• aa.sup.71, if present, is G, A, S, T, D, or E;
• aa.sub.1.sup.73, if present, is S, T, F, Y, X or W;
• aa.sub.2.sup.73, if present, is S, T, or W;
• aa.sub.1.sup.76, if present, is an aliphatic amino acid other than basic of from 4 to 6 carbon atoms or alanine;
• aa.sub.2.sup.76, if present, is V, or E;
• aa.sup.77, if present, is a polar aliphatic amino acid of from 3 to 6 carbon atoms, or alanine;
• aa.sub.2.sup.77, if present, is N, S, or D;
• aa.sup.78 is a non-polar aliphatic amino acid of from 3 to 6 carbon atoms;
• aa.sup.81 is G, L, I, or V;
• and
• aa.sup.82 is L, or R;
• aa.sup.83 is G, or R;
• aa.sup.85 is Y, or A, and aa85 is the terminal amino acid;
• X is an amino group to provide an amide terminal group, or the amino acids alanine or tyrosine terminating in a carboxy group;
• and
• detecting an amount of labeled antibody or peptide bound in the presence and in the absence of said drug candidate;
• wherein if said detected amount of labeled antibody or peptide is less in the presence of said drug candidate relative to the amount of labeled antibody or peptide detected in the absence of said drug candidate, said drug candidate is capable mediating modulation of said cell surface expression of said receptor.

A method for screening for drugs capable of inhibiting interaction between a major histocompatibility Class I antigen and a cell surface receptor, thereby modulating cell surface expression of said receptor, said method comprising the steps of:

• adding varying amounts of said drug candidate to a solution of first peptide, said first peptide having from 8 to 40 amino acids derived from an .alpha.1 or .alpha.2 domain of said major histocompatibility class I antigen and having, as the C-terminal sequence, a continuous sequence contained within a sequence selected from the group consisting of G-N-aa.sup.71 -Q-aa.sub.1.sup.73 -F-R-aa.sub.1.sup.76 -aa.sub.1.sup.77 -aa.sup.78 -R-T-aa.sup.81 -L-R-Y-X (SEQ ID NO.32), and W-aa.sup.61 -R-aa.sup.63 -T-aa.sup.65 -aa.sup.66 -aa.sup.67 -K-aa.sup.69 -aa.sup.70 -aa.sup.71 -Q-aa.sub.2.sup.73 -F-R-aa.sub.2.sup.76 -aa.sub.2.sup.77 -L-R-T-L-aa.sup.82 -aa.sup.83 -Y-aa.sup.85 (SEQ ID NO.34)
• where:
• aa.sup.61, if present, is D or E;
• aa.sup.63, if present, is E, I, or N;
• aa.sup.65, if present, is Q, or R;
• aa.sup.66, if present, is I, N, or K;
• aa.sup.67, if present, is A, S, C, or M;
• aa.sup.69, if present, is G, A, T, or P;
• aa.sup.70, if present, is N, Q, K, R, S, or T;
• aa.sup.71, if present, is G, A, S, T, D, or E;
• aa.sub.1.sup.73, if present, is S, T, F, Y, R or W;
• aa.sub.2.sup.73, if present, is S, T, or W;
• aa.sub.1.sup.76, if present, is an aliphatic amino acid other than basic of from 4 to 6 carbon atoms or alanine;
• aa.sub.2.sup.76, if present, is V, or E;
• aa.sup.77, if present, is a polar aliphatic amino acid of from 3 to 6 carbon atoms, or alanine;
• aa.sub.2.sup.77, if present, is N, S, or D;
• aa.sup.78 is a non-polar aliphatic amino acid of from 3 to 6 carbon atoms;
• aa.sup.81 is G, L, I, or V;
• and
• aa.sup.82 is L, or R;
• aa.sup.83 is G, or R;
• aa.sup.85 is Y, or A, and aa.sup.85 is the terminal amino acid;
• X is an amino group to provide an amide terminal group, or the amino acids alanine or tyrosine terminating in a carboxy group;
• adding varying amounts of second peptide, said second peptide lacking the ability to bind to said first peptide;
• wherein if aggregation of said first peptide is inhibited in the presence of said drug candidate, and said amount of the drug candidate added to achieve said inhibition is less than said amount of said second peptide which results in non-specific inhibition of aggregation of said first peptide, said drug candidate is capable of mediating modulation of said cell surface expression of said receptor.