Engineered antibody therapies coming of age for aging brains.

Biological therapies represent more than 30% of drugs in clinical trials, with a significant fraction of these being immune-based. Neuroimmunotherapies have lagged behind immunotherapies for cancer and autoimmune and infectious diseases, but are beginning to emerge, benefiting from molecular/protein technologies and data acquired for other disorders.1 Major questions that should be addressed in the development of neuroimmunotherapeutics targeting proteotoxic neurodegeneration include identification of the nature of the pathogenic species, where and when these species can be targeted, and how to direct the therapeutic to the optimal physiological site. The paper by Spencer et al.2 in this issue provides some answers for targeting of misfolded α-synuclein (α-syn), which accumulates in Parkinson's disease (PD) and dementia with Lewy bodies. Proof of concept is presented for a therapeutic effect of a single-chain Fv antibody fragment (scFv; expressing only antigen-binding domains of IgG) selected against a misfolded oligomeric conformation of α-syn. The scFv was fused to the low-density lipoprotein receptor-binding domain of apolipoprotein B (ApoB) to enhance brain and neuronal penetration. For synuclein-accumulating disorders, a considerable number of studies from multiple systems are consistent with the hypothesis that one major toxic form of misfolded α-syn is oligomeric.3 The Sierks group has been pursuing scFv antibody fragments that can bind to specific conformations of misfolding proteins, selected from phage display library binding to targets deposited on mica, and visualized by atomic force microscopy.4 One such scFv, D5, binds to the oligomeric α-syn 56-kDa pentamer and 28-kDa dimer aggregates in vitro, and a somewhat wider range of higher molecular weight aggregates when tested against brain extracts from PD models.5 The potential of using active or passive immunizations for neurodegeneration has been enhanced by findings that there is a significant representation of pathogenic misfolding proteins in the extracellular milieu. These extracellular forms facilitate spread of pathogenesis, and also offer greater access to therapeutics that are based on binding to the pathogenic species. The Masliah group has previously shown that both active vaccination with α-syn protein, and passive delivery of a presumed protective monoclonal antibody that binds to the C terminus of the protein, offer at least partial protection against neuropathological and functional phenotypes in their α-syn–overexpressing (ASO) line D mouse model.6,7 There are data suggesting that the mechanism of protection is clearance of the antigen–antibody complex by microglial cells, via an Fc receptor–mediated process. In this issue, Spencer et al., following preliminary characterization of the secretable fusion scFv in B103 cells, show that intraperitoneally delivered lentiviral constructs provide a sustained source of modified scFv (sD5-apoB) secreted into the systemic circulation of line D ASO mice. Deletion of the Fc domain of the scFv antibody fragment reduces the chances of pathogenic inflammation following administration in vivo; however, it also eliminates a major effector mechanism of clearance. The authors found that fusion of the antibody fragment to a 38–amino acid low-density lipoprotein receptor-binding domain of apoB, previously reported as a brain-penetrating peptide,8,9 enhanced both brain uptake following systemic delivery and α-syn clearance once the bifunctional protein reached the brain. The paper provides convincing data that the secretable fusion scFv results in lower levels of brain α-syn, reduced neuronal toxicity, and improved cognitive function in a Morris water-maze test. Delivery of a scFv D5 construct without the fusion to the apoB showed much lower levels of protection. The final sets of Spencer and colleagues' experiments approached the question of the mechanism of clearance of what is apparently either extracellular or membrane-bound oligomeric α-syn. To test the hypothesis that clearance occurs via a specific autophagy pathway, the authors used a series of colocalization experiments with ESCRT (endosomal sorting complexes required for transport)/autophagy pathway markers, and examined the effects of lentiviral-mediated overexpression or knockdown of CHMP2B (charged multivesicular body protein 2B), which can modify this pathway. In these experiments, complexes of fusion antibody with target α-syn were added to the media of transduced B103 cells, and localization was assessed after 6 hours. The data are consistent with the proposed mechanism of ESCRT or autophagy, although (as the authors acknowledge) they fall short of a full proof. For both α-syn diseases and related proteotoxic neurodegenerations, it is increasingly clear that there is extracellular presence of the misfolded proteins. Numerous potential immunotherapeutic targets have been described, including several for Alzheimer's disease/dementias; both amyloid-β42 and Tau have been reported to spread from the cells where they initially misfold.10 Huntington's disease (HD), in which cells appeared to be the least likely to extrude aggregates, was recently shown to propagate to neighboring neurons in an elegant series of transplant experiments.11 It will be important to localize the toxic species both physically and temporally during disease. La Spada has proposed classifying these spreading neurodegenerative proteins according to the requirement for physical or functional connections (HD), paracrine spread (which would include PD), or prionlike spread, in which the misfolded protein appears to act at distance.12 These classes may require different therapeutic approaches, although it is possible that the paracrine and prionlike spreads overlap, and that all could be targeted near the extruding cells. The concept of using carrier peptides to deliver proteins to the brain also has more general implications for diseases such as lysosomal storage caused by protein deficiencies.13 In fact, there may be overlap between lysosomal storage and PD, with implications for other neurodegenerations of complex etiology.14 Systemic delivery for neurodegenerative diseases has clear theoretical advantages over approaches that require delivery directly into the central nervous system. Depending on the method of delivery, the drug may also be able to effect important corrections in the periphery that can feed back on the brain syndrome. It is becoming increasingly clear that very few, if any, neurodegenerative diseases are truly limited to intrinsic processes within individual neurons within the central nervous system. Even in the cases where the triggering process may be intrinsic, there can be systemic manifestations that are either primarily due to pleiotropic actions of genes (e.g., HD) or secondary results of inflammatory or other downstream processes. The question of whether to deliver protein-based therapeutics as proteins or as genes that will then be transcribed, translated, and possibly posttranslationally modified in vivo is a critical one. Viral vector-mediated gene delivery has the advantage that production of the critical product will reach a steady state, and exposure should then be relatively uniform and continuous for a long period of time. (In some cases, brain levels could change as a result of changes in blood–brain barrier, but this would also affect proteins that are directly delivered systemically.) Steady-state treatments could prevent buildup of toxic disease products. Gene delivery could also reduce the chances that a bolus-based clearance process could induce acute, undesirable side-effects; e.g., blood vessel leakage caused by acute removal of accumulated misfolded amyloid.10 Gene therapy has the disadvantage that there are currently no approved technologies for turning it off should it prove deleterious. In the study by Spencer et al., the target of the human antibody fragment is an abnormal misfolded species, and the transport component has been optimized to match a normal human process. Any anti-idiotypic response would be to the hypervariable region, which is unique to the antibody fragment, limiting damage to only the novel scFv fusion protein. Advantages to delivery of the therapeutic as a protein include a potentially shorter path to clinical trial approval because there might be tighter control of the final dosage, with brain levels to some extent dependent on the status of the blood–brain barrier, as noted above. Also, no further product will be present once the administered dose has been degraded in vivo. Data on the effective or permissible range of dosing for antibody fragments are not yet available. (In the cancer studies, the fragments are generally conjugated to toxins so as to kill the cells to which they bind, making those data irrelevant to the questions here.) However, the quantities of purified protein that would be required for frequent infusions would be substantial and costly, whereas a gene therapy virus should need to be delivered only once. Economic considerations of development and administration of this class of biologics are complex. There will be an increasingly critical need for therapeutics that can counteract effects of dysregulated proteostasis for neurological disorders in aging populations.15 Spencer et al. offer insights into testing for specific pathogenic species and proof of concept for using a systemic delivery protocol of a bispecific fusion engineered antibody fragment to clear this species from the neuronal vicinity. This can form the basis of future neuroimmunotherapy approaches for multiple important diseases.