Genetically Encoded Azide Containing Amino Acid in Mammalian Cells Enables Site-Specific Antibody-Drug Conjugates Using Click Cycloaddition Chemistry.
Antibody-drug conjugates (ADC) have emerged as potent antitumor drugs that provide increased efficacy, specificity, and tolerability over chemotherapy for the treatment of cancer. ADCs generated by targeting cysteines and lysines on the antibody have shown efficacy, but these products are heterogeneous, and instability may limit their dosing. Here, a novel technology is described that enables site-specific conjugation of toxins to antibodies using chemistry to produce homogeneous, potent, and highly stable conjugates. We have developed a cell-based mammalian expression system capable of site-specific integration of a non-natural amino acid containing an azide moiety. The azide group enables click cycloaddition chemistry that generates a stable heterocyclic triazole linkage. Antibodies to Her2/neu were expressed to contain N6-((2-azidoethoxy)carbonyl)-l-lysine at four different positions. Each site allowed over 95% conjugation efficacy with the toxins auristatin F or a pyrrolobenzodiazepine (PBD) dimer to generate ADCs with a drug to antibody ratio of >1.9. The ADCs were potent and specific in in vitro cytotoxicity assays. An anti Her2/neu conjugate demonstrated stability in vivo and a PBD containing ADC showed potent efficacy in a mouse tumor xenograph model. This technology was extended to generate fully functional ADCs with four toxins per antibody. The high stability of the azide-alkyne linkage, combined with the site-specific nature of the expression system, provides a means for the generation of ADCs with optimized pharmacokinetic, biological, and biophysical properties.
Allozyne, Inc. , 1600 Fairview Avenue East, Seattle, Washington 98102, United States.
Michael P
MP
VanBrunt
Allozyne, Inc. , 1600 Fairview Avenue East, Seattle, Washington 98102, United States.
Kurt
K
Shanebeck
Allozyne, Inc. , 1600 Fairview Avenue East, Seattle, Washington 98102, United States.
Zachary
Z
Caldwell
Allozyne, Inc. , 1600 Fairview Avenue East, Seattle, Washington 98102, United States.
Jeffrey
J
Johnson
MedImmune, LLC , One MedImmune Way, Gaithersburg, Maryland 20878, United States.
Pamela
P
Thompson
MedImmune, LLC , One MedImmune Way, Gaithersburg, Maryland 20878, United States.
Thomas
T
Martin
MedImmune, LLC , One MedImmune Way, Gaithersburg, Maryland 20878, United States.
Huifang
H
Dong
Allozyne, Inc. , 1600 Fairview Avenue East, Seattle, Washington 98102, United States.
Gary
G
Li
Allozyne, Inc. , 1600 Fairview Avenue East, Seattle, Washington 98102, United States.
Hengyu
H
Xu
Spirogen MedImmune , The QMB Innovation Centre, 42 New Road, London E1 2AX, United Kingdom.
Francois
F
D'Hooge
Spirogen MedImmune , The QMB Innovation Centre, 42 New Road, London E1 2AX, United Kingdom.
Luke
L
Masterson
Allozyne, Inc. , 1600 Fairview Avenue East, Seattle, Washington 98102, United States.
Pauline
P
Bariola
Spirogen MedImmune , The QMB Innovation Centre, 42 New Road, London E1 2AX, United Kingdom.
Arnaud
A
Tiberghien
Spirogen MedImmune , The QMB Innovation Centre, 42 New Road, London E1 2AX, United Kingdom.
Ebele
E
Ezeadi
Spirogen MedImmune , The QMB Innovation Centre, 42 New Road, London E1 2AX, United Kingdom.
David G
DG
Williams
Spirogen MedImmune , The QMB Innovation Centre, 42 New Road, London E1 2AX, United Kingdom.|UCL Cancer Institute , 72 Huntley Street, London WC1E 6BT, United Kingdom.
John A
JA
Hartley
Spirogen MedImmune , The QMB Innovation Centre, 42 New Road, London E1 2AX, United Kingdom.
Philip W
PW
Howard
Allozyne, Inc. , 1600 Fairview Avenue East, Seattle, Washington 98102, United States.
Kenneth H
KH
Grabstein
MedImmune, LLC , One MedImmune Way, Gaithersburg, Maryland 20878, United States.
Michael A
MA
Bowen
Allozyne, Inc. , 1600 Fairview Avenue East, Seattle, Washington 98102, United States.|MedImmune, LLC , One MedImmune Way, Gaithersburg, Maryland 20878, United States.
Marcello
M
Marelli
D000596:Amino Acids / Q000737:chemistry* / Q000235:genetics; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000627:therapeutic use; D001386:Azides / Q000737:chemistry* / Q000378:metabolism; D057930:Click Chemistry; D061565:Cycloaddition Reaction; D006801:Humans; D018796:Immunoconjugates / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000627:therapeutic use; D008297:Male; D051379:Mice; D009369:Neoplasms / Q000188:drug therapy; D015202:Protein Engineering; D017207:Rats, Sprague-Dawley; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000276:immunology
D000596:Amino Acids; D000911:Antibodies, Monoclonal; D001386:Azides; D018796:Immunoconjugates; D018719:Erb-b2 Receptor Tyrosine Kinases; C508053:ERBB2 protein, human