Mirac proteins
This disclosure relates to a method of generating conditionally active biologic proteins from wild type proteins, in particular therapeutic proteins, which are reversibly or irreversibly inactivated at the wild type normal physiological conditions. For example, evolved proteins are virtually inactive at body temperature, but are active at lower temperatures.
1. A method of preparing a conditionally active therapeutic antibody, the method comprising steps of:
(i) selecting a subject, a site of administration, a site of action and an antigen;
(ii) evolving a DNA which encodes a parent antibody that binds the antigen using one or more evolutionary techniques to create mutant DNAs;
(iii) expressing the mutant DNAs to obtain mutant antibodies including at least one said conditionally active therapeutic antibody;
(iv) subjecting the mutant antibodies to a screening assay under a value of a normal physiological condition that is within a normal range of the physiological condition at a site of administration of the mutant antibody and a screening assay under a value of an aberrant condition that deviates from a value of a physiological condition that is within a normal range at a normal tissue or a normal organ at the site of action;
(v) subjecting mutant antibodies determined to be active under the value of the aberrant condition in step iv to a second screening assay under the value of the normal physiological condition that is within the normal range of the physiological condition at the site of administration of the mutant antibody to determine if the activity under the value of the aberrant condition is reversible; and
(vi) selecting from the mutant antibodies the conditionally active therapeutic antibody which exhibits:
(a) an increased binding activity to the antigen in the assay at the value of the aberrant condition, compared to the binding activity of the same conditionally active antibody in the assay under the value of the normal physiological condition that is within the normal range of the physiological condition at the site of administration of the conditionally active antibody in the subject,
(b) a decreased binding activity to the antigen in the assay under the value of the normal physiological condition that is within the normal range of the physiological condition at the site of administration of the conditionally active antibody in the subject compared to the binding activity of the parent antibody in the same assay, and
(c) which is reversibly activated at the aberrant condition,
wherein the normal physiological condition and the aberrant condition are both a same condition selected from temperature, pH, osmotic pressure, oxidative stress, osmolality, and electrolyte concentration.
2. The method of claim 1 wherein the antigen is an inflammatory response mediator and the conditionally active therapeutic antibody is an antibody fragment.
3. The method of claim 1 , wherein the condition is pH.
4. The method of claim 3 , wherein the value of the normal physiological condition is a normal physiological pH and the value of the aberrant condition is a pH in a tumor microenvironment.
5. The method of claim 1 , further comprising a step of glycosylating the conditionally active therapeutic antibody.
6. The method of claim 1 , further comprising a step of codon optimizing the conditionally active therapeutic antibody for an organism or cell.
7. The method of claim 1 , further comprising a step of converting the conditionally active therapeutic antibody to a pharmaceutically acceptable salt.
8. The method of claim 1 , further comprising a step of formulating the conditionally active therapeutic antibody in a cream, ointment, solution or hydrogel.
9. The method of claim 1 , wherein the selecting step comprises testing for improved expression of the at least one mutant antibody in a host cell or host organism as compared to the expression of the parent antibody in the host cell or the host organism.
10. The method of claim 9 , further comprising a step of introducing at least one non-natural amino acid into the conditionally active therapeutic antibody to produce a mimetic protein by a protein chemical synthesis technique or a recombinant technique.
11. The method of claim 1 , wherein the assays are carried out in assay media comprising an electrolyte selected from calcium, sodium, potassium, magnesium, chloride, bicarbonate and phosphate.
12. The method of claim 1 , wherein the evolving step comprises one or more of (i) substituting one or more nucleotides for a different nucleotide; (ii) deleting one or more nucleotides, and (iii) adding one or more nucleotides.
13. The method of claim 12 , wherein the evolving step employs site-saturation mutagenesis.
14. The method of claim 1 , wherein the method further comprises a step of humanizing the conditionally active therapeutic antibody.
15. The method of claim 1 , wherein the method further comprises a step of converting the conditionally active therapeutic antibody to a mimetic form.
16. The method of claim 1 , wherein the method further comprises a step of covalently attaching the conditionally active therapeutic antibody to a nucleotide or a nucleotide derivative.
17. The method of claim 1 , wherein the method further comprises a step of formulating the conditionally active therapeutic antibody and at least one pharmaceutically acceptable carrier into a pharmaceutical composition.
18. The method of claim 15 , wherein the converting step comprises replacing at least one naturally occurring amino acid residue in the conditionally active therapeutic antibody with a non-naturally occurring amino acid residue.