Immunotherapy is the treatment of cancer using antibodies that target points of failure in the life cycle of cancer cells. Many of these therapies involve altering monoclonal antibodies to target specific proteins associated to different types of cancer. Understanding the binding properties of these proteins is of utmost importance.
Pembrolizumab, an igG4 Monoclonal Antibody
Pembrolizumab (KEYTRUDA®) is one of these immunotherapies. Some IgG4 antibodies exhibit unusual surface properties that impact their therapeutic function.
Pembrolizumab is a compact antibody used in the treatment of various cancers, most notably indicated for use when EGFR inhibitors fail to reach treatment outcomes. Scientists identified the programmed cell death 1 receptor, PD-1, a protective mechanism on the surface of cancer cells. Pembrolizumab binds to this receptor allowing for the immune system cells to successfully recognize and destroy cancer cells.
Mechanism of Action : PD-1 an Immune Checkpoint
PD-1 receptors are located on the surface of lymphocytes. By avoiding detection from the immune system, binding to other ligands besides our immune system’s antibodies, this receptor prolongs the life of cancer cells. The chemical signal associated to PD-1 is referred to as an “Immune Checkpoint” since the lack of it prevents cellular destruction.
Examination of protein data-set 4ZQK reveals the mechanism of ligand-receptor binding. PD-L1 fits into a U-shaped fold of the PD-1 receptor. Interesting to note that the active sites listed in the data-set are not located in the ligand-receptor binding region. Instead, alternating amino-acids from each protein fit together by flipping neighboring residues as they find stable connectivity like teeth of a zipper. Without clear active-site ligand binding, it’s possible this conformation is further stabilized by thermic and hydrophobicity congruity of each protein.
Pembrolizumab, an Asymmetrical Monoclonal Antibody
Monoclonal antibodies are uniquely structured to engage multiple antigens, making it a potent therapeutic compound. Pembrolizumab appears to be structurally a bit different than other monoclonal antibodies, adopting an irregular conformation.
In data-set 5DK3, the Pembrolizumab’s Fc domain is rotated about the central axis towards one of the FAB fragments. The Fc domain has a single CH2 domain roated 120 degrees off center. The thin connecting region between the FAB and Fc domains are contracted into a central core of the protein, opening up the Fc domain for enhanced binding properties.
Unique Surface Properties of Pembrolizumad Determine PD-1 Binding
To determine the unique surface properties driving the conformation shown in dataset 5DK3, I’ve investigated three things:
- An analysis of different surface properties may yield insights into binding potential of PD-1.
- Conduct a comparative analysis of data-set 5dk3 to a one of its FAB fragments bound to PD-1.
- Construct a macro-molecular complex of an entire antibody bound to cancer causing antigen.
1. Analysis of Pembrolizumab Surface Properties
Protein behaviors are defined by more than just active sites or amino acid mutations. Conformation states of the protein show us the mechanics of a protein’s function. Interpretation of “active” and “inactive” states of proteins demonstrate the range of movement possible in the protein.
A protein’s surface properties helps define how the protein interacts with surrounding molecules. Here, I’ve selected 3 primary modalities to gain insights into Pembrolizumab’s asymmetrical structure, hydrophobicity, Coulumbic, and bFactor.
The hydrophobicity appears uniform throughout the entire protein. As a soluble antibody, its affinity for water is higher and as expected more blue (phillic) than red (phobic).
The Coulumbic property visualizes the electrostatic potential of the protein. Its positive-neutral-negative charge are visualized in blue-white-red. The variation in this surface pattern appears to have a slightly more negative charge on the left where there is the greatest angle between FAB fragment and Fc domain.
Finally, bFactor visualizes the protein’s thermic properties, its atomic temperature (red is hot, blue is cool). Here we see clear increased temperature within the extended FAB domain region. This is the same region of the protein identified with more negative charge by coulombic analysis.
2. Pembrolizumab FAB Fragment Bound to PD-1
As of the time of publication, no data set of Pembrolizumab exists with a complete antibody bound to one of it’s antigens. To understand how the entirety of the marco-molecule may behave in the presence of PD-1, a theoretical model of that protein complex can yield insights otherwise hidden within the data.
PDB data-set: 5GGS contains the 3D atomic structure of Pembrolizumab’s FAB fragment bound to the PD-1 receptor. In this data-set we can see the exact amino acids involved with binding antigens like PD-1. Again we see similar binding properties of PD-1 to PD-L1.
Standard identification of active residues did not locate amino acids of Pembrolizumab and therefore required a visual inspection to identify which residues most likely involved. There were no clear Pembrolizumab–PD-1 bonds, but rather the same zipper-like alternating amino acids of the two proteins.
While Pembrolizumab and PD-L1 share similar mechanisms for binding, Pembrolizumab does not bind to the same U-shaped region of PD-1. While close, it engages different residues near those engaged by PD-L1, but not the same.
3. Macro-molecular assembly of Pembrolizumab / PD-1 Complex
To further understand the binding properties of Pembrolizumab, it would be interesting to understand how the complete antibody would correlate to the now understood binding configuration of its FAB fragment to PD-1.
Since Pembrolizumab is a monoclonal antibody, protein data-set 5GGS is an ideal model to use for to align PD-1 to the FAB domains of our complete Pembrolizumab model.
Here we can see 5GGS aligned to the contracted FAB fragment of 5DK3. Since this is a monoclonal antibody, the alignment of 5GGS on one FAB fragments should be symmetrical to the other side.
Determining if both FAB Regions Bind Antigen
As expected, both FAB / PD-1 models from 5GGS aligns nicely to the FAB domains of Pembrolizumab. It would seem very likely that the antibody capable of binding two receptors simultaneously. Should this be the case, the thermic properties of each should yield clues about that potential binding.
Upon further inspection, there’s thermic discongruity on the extended FAB domain of the antibody where it would interface with PD-1. As noted, one side expresses an increased atomic temperature while the other cooler with more thermic affinity to PD-1. This may pose a complication for dual binding of PD-1 while making the more reactive antibody core available for binding Fc receptors to the Fc domain of Pembrolizumab.
Monoclonal antibodies show enormous promise for a patient specific targeting of cancer cells. The science behind immunotherapy is as fascinating as it is beautiful.
When considering the macro-molecular structure of PD-1 bound to Pembrolizumab, the surface properties of receptor to antibody may play an important role in determining the binding orientation of Pembrolizumab to the plasma membrane. Due to the asymmetrical alignment of this antibody, this may in fact present therapeutic advantages. In a prone position, as seen above, the Fc region responsible for binding immune cells, is in a favorable position for binding Fc receptors. In this orientation, it exposes the antibody’s reactive core to the extra-cellular space. Further investigations are warranted to determine if and how the second FAB region of Pembrolizumab interacts with extra-cellular proteins. I would be very curious to find out if in fact Pembrolizumab thermic charge changes after binding the first PD-1 receptor, altering the asymmetric structure.
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