An engineered lipocalin specific for CTLA-4 reveals a combining site with structural and conformational features similar to antibodies.
Biomolecular reagents that enable the specific molecular recognition of proteins play a crucial role in basic research as well as medicine. Up to now, antibodies (immunoglobulins) have been widely used for this purpose. Their predominant feature is the vast repertoire of antigen-binding sites that arise from a set of 6 hypervariable loops. However, antibodies suffer from practical disadvantages because of their complicated architecture, large size, and multiple functions. The lipocalins, on the other hand, have evolved as a protein family that primarily serves for the binding of small molecules. Here, we show that an engineered lipocalin, derived from human Lcn2, can specifically bind the T cell coreceptor CTLA-4 as a prescribed protein target with subnanomolar affinity. Crystallographic analysis reveals that its reshaped cup-like binding site, which is formed by 4 variable loops, provides perfect structural complementarity with this "antigen." Furthermore, comparison with the crystal structure of the uncomplexed engineered lipocalin indicates a pronounced induced-fit mechanism, a phenomenon so far considered typical for antibodies. By recognizing the same epitope on CTLA-4 that interacts with the counterreceptors B7.1/B7.2 on antigen-presenting cells the engineered Lcn2 exhibits strong, cross-species antagonistic activity, as evidenced by biological effects comparable with a CTLA-4-specific antibody. With its proven stimulatory activity on T cells in vivo, the CTLA-4 blocking lipocalin offers potential for immunotherapy of cancer and infectious disease. Beyond that, lipocalins with engineered antigen-binding sites, so-called Anticalins, provide a class of small ( approximately 180 residues), structurally simple, and robust binding proteins with applications in the life sciences in general.
Schönfeld
D
D
Munich Center for Integrated Protein Science and Lehrstuhl für Biologische Chemie, Technische Universität München, Freising-Weihenstephan, Germany.
Matschiner
G
G
Chatwell
L
L
Trentmann
S
S
Gille
H
H
Hülsmeyer
M
M
Brown
N
N
Kaye
P M
PM
Schlehuber
S
S
Hohlbaum
A M
AM
Skerra
A
A
D000209:Acute-Phase Proteins / Q000235:genetics; D000906:Antibodies / Q000737:chemistry; D015703:Antigens, CD / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D060908:CTLA-4 Antigen; D018360:Crystallography, X-Ray; D000939:Epitopes; D006801:Humans; D007202:Indicators and Reagents / Q000138:chemical synthesis / Q000737:chemistry; D000071068:Lipocalin-2; D054834:Lipocalins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D015202:Protein Engineering; D011518:Proto-Oncogene Proteins / Q000235:genetics
D000209:Acute-Phase Proteins; D000906:Antibodies; D015703:Antigens, CD; D060908:CTLA-4 Antigen; C556706:CTLA4 protein, human; D000939:Epitopes; D007202:Indicators and Reagents; C489506:LCN2 protein, human; D000071068:Lipocalin-2; D054834:Lipocalins; D011518:Proto-Oncogene Proteins