US Patent No. 10,428,332

HYBRID IMMUNOGLOBULINS WITH MOVING PARTS


Patent No. 10,428,332
Issue Date October 01, 2019
Title Hybrid Immunoglobulins With Moving Parts
Inventorship Daniel Capon, Hillsborough, CA (US)

Claim of US Patent No. 10,428,332

1. A process of making a product comprising:a) transfecting a cell with a recombinant nucleic acid which encodes a chimeric polypeptide comprising, from its N terminus to its C terminus,
(i) an N-terminal signal sequence, contiguous with
(ii) such stretch of consecutive amino acids capable of forming a disulfide bond, contiguous with
(iii) a C-terminal intein-containing binding domain,
under conditions such that:
(i) the cell expresses the chimeric polypeptide;
(ii) the N-terminal signal sequence is cleaved from the chimeric polypeptide, so as to produce a second chimeric polypeptide comprising the stretch of consecutive amino acids that is capable of forming a disulfide bond contiguous with the C-terminal intein-containing binding domain; and
(iii) the cell secretes the second chimeric polypeptide,
b) isolating the secreted second chimeric polypeptide produced in step (a);
c) treating the second chimeric polypeptide from step (b) with a cysteine or selenocysteine, so as to cause:
(i) thio-mediated cleavage of the C-terminal intein-containing binding domain from the stretch of consecutive amino acids; and
(ii) ligation of the cysteine or selenocysteine to such stretch of consecutive amino acids within such second chimeric polypeptide, at the C-terminal side thereof;
d) recovering the product of step (c) which is the stretch of consecutive amino acids comprising at its C-terminal side the additional cysteine or selenocysteine, and
e) then either:
(i) combining the product recovered in step (d) with a second product made using the process of steps (a)-(d) under conditions resulting in the formation of an —S—S—, —S—Se—, —Se—Se—, or —Se—S— bond between the respective C-terminal residues of the first product and the second product;
(ii) combining the product recovered in step (d) with a second, different stretch of consecutive amino acids comprising an N-terminal S-terminus or Se-terminus, under conditions resulting in the formation of an —S—S—, —S—Se—, —Se—Se—, or —Se—S— bond between the C terminus of the product and the N-terminal S-terminus or Se-terminus of the second, different stretch of consecutive amino acids; or
(iii) combining the product recovered in step (d) with a solid surface under conditions resulting in attachment of the product to such surface at its C-terminus, wherein the attachment is mediated by an —S—S—, —S—Se—, —Se—Se—, or —Se—S— bond formed between the cysteine or selenocysteine of the product and a cysteine or selenocysteine of the solid surface.