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.
Munich Center for Integrated Protein Science and Lehrstuhl für Biologische Chemie, Technische Universität München, Freising-Weihenstephan, Germany.
D
D
Schönfeld
G
G
Matschiner
L
L
Chatwell
S
S
Trentmann
H
H
Gille
M
M
Hülsmeyer
N
N
Brown
P M
PM
Kaye
S
S
Schlehuber
A M
AM
Hohlbaum
A
A
Skerra
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