Process for the synthesis of PMPC and Tmab polymersPMPCs. (A) Synthesis process of thiol-reactive maleimide-modified PMPC. MPC was first polymerized after RAFT polymerization to obtain carboxyl-terminated PMPC. Then, the terminal group was converted to an amino group with ethylenediamine modification, followed by the conversion of the amino group to maleimide group by Sulfo-SMCC to obtain maleimide-modified PMPC. (B) Synthetic process of TmabPMPC production. The interchain disulfide bonds of Tmab were first cleaved by TCEP to create thiol groups, followed by reaction with maleimide-modified PMPC to obtain Tmab.PMPC. Credit: Frontiers of cell biology and development (2023). DOI: 10.3389/fcell.2023.1214118
The blood-brain barrier blocks the entry of antibodies into the brain. This limits the potential use of therapeutic antibodies to treat brain diseases, such as brain tumors.
Elsewhere in the body, more than 100 therapeutic antibodies approved by the U.S. Food and Drug Administration are used by medical teams to treat cancers and autoimmune, infectious and metabolic diseases. Finding ways to transport therapeutic antibodies across the blood-brain barrier, from the peripheral bloodstream to the central nervous system, could create effective treatments that work in the brain.
In a new study published in the journal Frontiers of cell biology and development, researchers at the University of Alabama at Birmingham report that site-directed addition of an FDA-approved biodegradable polymer to the hinge and near-hinge regions of the therapeutic antibody trastuzumab effectively facilitated the cerebral administration of this human monoclonal IgG1 antibody. Trastuzumab is used to treat breast cancer and several other cancers.
Preliminary work on this new platform included in vitro experiments and in mouse models. The researchers say the delivery system still needs to be optimized and tested further, but note that their simple methodology converts therapeutic antibodies into a brain-delivered form that maintains the medical functionality of the antibody.
“Concerns about brain entry haunt the development of therapeutic antibodies targeting brain diseases, preventing medical translations of laboratory-generated antibodies into clinical practices,” said Masakazu Kamata, Ph.D., head of the study and associate professor. at the UAB Department of Microbiology. “In this context, this simple methodology has great potential to serve as a platform not only to reuse current therapeutic antibodies, but also to encourage the design of new antibodies for the treatment of brain diseases.”
The biocompatible polymer used was poly 2-methacryloyloxyethylphosphorylcholine, or PMPC, with chain lengths of 50, 100, or 200 monomers. Researchers had previously discovered that this non-immunogenic polymer, which the FDA approved as a coating material for transplantable devices, could bind to two receptors located on the brain’s microvascular endothelial cells that make up the blood-brain barrier, and that these cells could then move the polymer. across the blood-brain barrier by transcytosis. Transcytosis is a specialized transport by which extracellular cargo is brought inside the cell, transported through the cytoplasm to the other side of the cell, and then released.
The UAB researchers were able to cleave four interchain disulfide bonds in the trastuzumab IgG1 hinge and in regions near the hinge, creating thiol groups. Each thiol group was then conjugated to a chain of the PMPC to create trastuzumab molecules with one of three chain lengths, which they designated as Tmab-PMPC50, Tmab-PMPC100, and Tmab-PMPC200.
Each of these modified antibodies still maintained specific binding of trastuzumab to cells expressing the HER2 antigen, the target of trastuzumab. Tmab-PMPC50 and Tmab-PMPC100 were internalized into HER2-positive cells and promoted antibody-dependent cell death, which is the medical functionality by which trastuzumab kills HER2+ breast cancer cells.
The researchers then showed that PMPC conjugation of trastuzumab enhanced blood-brain barrier penetration through blood-brain barrier epithelial cells via the transcytosis pathway. Translocable Tmab-PMPC100 was best for effective penetration of the blood-brain barrier while retaining trastuzumab epitope recognition, the ability of the antibody to bind to its antigenic target.
In a mouse model, Tmab-PMPC100 and Tmab-PMPC200 were approximately five times better in brain penetration than native trastuzumab. In preliminary in vitro experiments and in mouse models, polymer-modified trastuzumab did not induce neurotoxicity, did not show adverse effects on the liver, and did not disrupt barrier integrity. hemato-encephalic.
“These results collectively indicate that PMPC conjugation allows for efficient delivery of therapeutic antibodies, such as trastuzumab, to the brain without induction of adverse effects, at least in the liver, blood-brain barrier, or brain,” said Kamata.
Others have also studied ways to get cargo-like antibodies across the blood-brain barrier, the researchers noted.
In the work that led to the current study, the UAB researchers showed that they could wrap various macromolecular cargoes in PMPC shells, and these nanocapsules demonstrated prolonged blood circulation, reduced immunogenicity, and improved brain delivery in mice and non-human primates.
This system, however, had drawbacks. The nanocapsules required the addition of targeting ligands to bring them to their disease target and degradable cross-linkers that would enable release of the cargo at that site. Unfortunately, disease-associated microenvironments often lack conditions that can trigger cross-linker degradation.
Other researchers seeking to break the blood-brain barrier have investigated various ligands other than PMPC to stimulate transport, such as ligands derived from microbes and toxins, or endogenous proteins like lipoproteins. These typically have undesirable surface properties, such as being highly immunogenic, highly hydrophobic, or charged. PMPC does not exhibit these undesirable traits.
Co-authors with Kamata in the study “Site-oriented conjugation of poly(2-methacryloyloxyethyl phosphorylcholine) for enhanced antibody delivery to the brain” are Jie Ren, Chloe E. Jepson, Charles J. Kuhlmann, Stella Uloma Azolibe and Madison T. Blucas, UAB Department of Microbiology; Sarah L. Nealy and Eugenia Kharlampieva, UAB Department of Chemistry; Satoru Osuka, UAB Department of Neurosurgery; and Yoshiko Nagaoka-Kamata, UAB Department of Pathology.
More information:
Jie Ren et al, Site-oriented conjugation of poly(2-methacryloyloxyethylphosphorylcholine) for enhanced brain delivery of antibodies, Frontiers of cell biology and development (2023). DOI: 10.3389/fcell.2023.1214118
Provided by the University of Alabama at Birmingham
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