Humanized antibody conjugates and related methods, assays, reagents, and kits
The invention provides synthetically and recombinantly-derived humanized antibody conjugates and related methods, diagnostic assays, reagents, and kits. In one embodiment, the invention provides humanized antibody conjugates comprising a human immunoglobulin fragment which is bound by a cross-linking functional group to a non-human antibody fragment (e.g., a non-human monoclonal antibody fragment) comprising an antigen-binding amino acid sequence.
1 . A humanized antibody conjugate which comprises a human antibody fragment that is bound by a cross-linking functional group to a non-human antibody fragment, wherein the non-human antibody fragment contains an amino acid sequence which binds to an antigen epitope.
2 . The humanized antibody conjugate of claim 1 , wherein the human antibody fragment is a Fc fragment of either human IgG or human IgE and the non-human antibody fragment is a Fab2 fragment.
3 . The humanized antibody conjugate of claim 1 , wherein the human antibody fragment is a Fc fragment of human IgG and the non-human antibody fragment is a Fab2 fragment derived from a murine Mab or rat Mab.
4 . The humanized antibody conjugate of claim 1 , comprising a Fc fragment of either human IgG or human IgE and a non-human Fab2 antibody fragment, wherein the non-human Fab2 fragment is obtained by digestion of a non-human Mab by an aspartic proteinase.
5 . The humanized antibody conjugate of claim 4 , wherein:
(a) the non-human Mab is a murine Mab or rat Mab, and (b) a reduced thiol (disulfide) bond on the non-human Fab2 fragment forms a cross-linking functional group with a thiol (disulfide)-binding moiety that has been derivatized onto the Fc fragment.
6 . The humanized antibody conjugate of claim 5 , wherein:
(a) the non-human Mab is a murine Mab;
(b) the thiol (disulfide) bond on the non-human Fab2 fragment has been reduced by a reducing agent; and
(c) the thiol (disulfide)-binding moiety which has been derivatized onto the Fc fragment is a maleimide group formed by the reaction of the Fc fragment with a maleimide-containing hetero-bifunctional cross-linking agent.
7 . The humanized antibody conjugate of claim 6 , wherein:
(a) the Fab2 fragment is obtained by digestion of a murine Mab in pepsin;
(b) the Fc fragment is obtained by digestion of human IgG in papain or ficin;
(c) the reducing agent is MEA; and
(d) the hetero-bifunctional cross-linking agent is SMCC.
8 . The humanized antibody conjugate of claim 7 , wherein:
(a) the murine Mab is NS5; and (b) the Fab2 fragment is obtained by digesting NS5 in pepsin.
9 . The humanized antibody conjugate of claim 6 , wherein the antigen is a virus or an allergen.
10 . The humanized antibody conjugate of claim 9 , wherein the antigen is a hepatitis C virus or a pollen.
11 . The humanized antibody conjugate of claim 10 , wherein the antigen is NS5.
12 . A method of making a humanized antibody conjugate which binds to an antigen epitope comprising:
reacting a Fc fragment derived from either human IgG or human IgE with a Fab fragment that is derived from a non-human Mab,
wherein the non-human Mab contains an amino acid sequence that binds to the antigen epitope to form a cross-linking functional group that binds the non-human Fab and human Fc fragments.
13 . A method of making a humanized antibody conjugate which binds to an antigen epitope comprising:
(a) digesting a murine or rat Mab in an aspartic proteinase to form a non-human Fab2 fragment which contains an amino acid sequence which binds to the antigen epitope;
(b) digesting a human IgG or IgE in a cysteine (thiol) proteinase to form a human Fc fragment;
(c) reducing a thiol (disulfide) bond on the non-human Fab2 fragment by reacting the non-human Fab2 fragment with a reducing agent;
(d) derivatizing the human Fc fragment with a thiol (disulfide)-binding moiety by reacting the human Fc fragment with a hetero-bifunctional cross-linking agent; and
(e) reacting the reduced non-human Fab2 fragment and derivatized human Fc fragment to form a cross-linking functional group between the human Fc fragment thiol (disulfide)-binding moiety and the non-human Fab2 fragment reduced thiol (disulfide) bond.
14 . The method of claim 13 , wherein:
(a) a murine Mab is digested in pepsin to form the non-human Fab2 fragment;
(b) a human IgG is digested in papain or ficin to form the human Fc fragment;
(c) the non-human Fab2 fragment is reduced with MEA;
(d) the human Fc fragment is derivatized with a maleimide moiety through reaction with a hetero-bifunctional cross-linking agent; and
(e) the cross-linking functional group is formed between the reduced thiol (disulfide) bond on the non-human Fab2 fragment and the human Fc fragment maleimide moiety.
15 . The method of claim 14 , wherein the non-human Fab2 fragment and the human Fc fragment are reacted in a weight ratio of from about 1.5:1 to about 5:1.
16 . The method of claim 15 , wherein the non-human Fab2 fragments and the human Fc fragments are reacted in a weight ratio of from about 2:1 to about 3:1.
17 . The method of claim 15 , wherein the human IgG is digested in papain and the hetero-bifunctional cross-linking agent is SMCC.
18 . The method of claim 17 , wherein the murine Mab binds to an epitope that is the same as or equivalent to the epitope of NS5.
19 . An analytical kit comprising a control reagent comprising a humanized antibody conjugate of claim 1 .
20 . An analytical kit comprising a control reagent comprising a humanized antibody conjugate of claim 6 .
21 . An analytical kit comprising a control reagent comprising a humanized antibody conjugate of claim 9 .
22 . An analytical assay control reagent comprising a humanized antibody conjugate of claim 1 .
23 . An analytical assay control reagent comprising a humanized antibody conjugate of claim 6 .
24 . An analytical assay control reagent comprising a humanized antibody conjugate of claim 9 .
25 . A method for evaluating the performance of an analytical assay comprising using a control reagent comprising a humanized antibody conjugate of claim 1 .
26 . A method for evaluating the performance of an analytical assay comprising using a control reagent comprising a humanized antibody conjugate of claim 6 .
27 . A method for evaluating the performance of an analytical assay comprising using a control reagent comprising a humanized antibody conjugate of claim 9 .
28 . A analytical kit comprising a control reagent, wherein the control reagent is a recombinantly-derived humanized antibody conjugate.
29 . A method for evaluating the performance of an analytical assay by using a recombinantly-derived humanized antibody conjugate.
30 . The method of claim 29 , further comprising the step of observing of a positive signal.