Isolation of Human Monoclonal scfv Antibody Specifically Recognizing the D 2 - 5 - Ht 1a Heteromer.

Antibody phage display has become a useful technique for discovering and optimizing target - specific monoclonal antibodies suitable for many applications, including therapeutic ligands, which may act as direct pharmacological compounds or may be used as targeting ligands for controlled drug delivery. Recently, the D 2 - 5 - HT 1A heteromer, which is formed by the dopamine D 2 and serotonin 5 - HT 1A receptors has attracted attention as a potential target of antipsychotic drugs. Therefore, the aim of the study was to identify scFv monoclonal antibodies that are able to specifically recognize epitopes formed within the heteromer structure. Because both receptors are membrane proteins, it is important to conduct bio - panning experiments in the most natural conditions, in which the presented antigens (D 2 - 5 - HT 1A heteromers) are in their native form and possibly in their best - preserved spatial structure. It has been shown here that phage display methodology can be successfully used in the preparation of monoclonal antibodies against dimers of membrane proteins. To separate phages specifically binding the D 2 - 5 - HT 1A heteromer, the selection process using CHO+ cells with overexpression of both receptors was conducted. Phages that were bound to receptor monomers or other CHO - K1 cell surface proteins were eliminated as a result of negative selection by using CHO - cells expressing separate receptor monomers.


Introduction
Nanoparticulate systems with functionalized surfaces for targeted drug delivery play central roles in modern therapies [1,2,3]. In our previous studies, we focused on the encapsulation of clozapine (one of the most important antipsychotic drugs) into biocompatible polymeric nanocapsules formed by polyelectrolyte multilayer shells [4,5]. Promising nanocarriers able to cross the blood brain barrier (BBB) were obtained [6]; however, to achieve higher selectivity, additional functionalization of the nanocarrier is needed.
This necessity is well exemplified by clozapine, which is effectively used in the clinic; however, clozapine displays many serious side effects that most likely could be eliminated if we knew the precise mechanism responsible for its efficacy. Recently, we have shown that heteromers formed by the dopamine D 2 and serotonin 5-HT 1A receptors (D 2 R and 5-HT 1A R, respectively) might be important site of action of clozapine [7]. Using the conventional route of administration, this drug (as well as other antipsychotics), addressed only for a small part of the cell population in the brain, is non-specifically delivered into all brain areas, causing undesirable side effects.
Higher selectivity may be achieved using compounds directly targeting D 2 -5-HT 1A heteromers because these heteromers can be formed only on neurons co-expressing receptors engaged in the complex formation.
Human monoclonal scFv (single-chain variable fragment) antibodies may be used as targeting ligands to decorate the outer surface of nanovehicles. ScFvs are small antibody fragments that compose the variable regions of the heavy (VH) and light (VL) chains of immunoglobulins with a flexible peptide linker designed to connect the two chains in such a way that the antigen binding site is retained in a single co-linear molecule [8]. ScFvs can be obtained from phage display libraries [9,10]. ScFvs have the potential to be very useful for the targeted delivery of drugs to specific cells or tissues. In comparison to the much larger Fab, F(ab) 2 , and IgG forms of monoclonal antibodies, from which they are derived, scFvs have lower retention times in non-target tissues, faster blood clearance, better tissue penetration and reduced immunogenicity, making them attractive for therapeutic applications [11].
Therefore, in the present study, we focused on the production of an scFv antibody that specifically binds the D 2 -5-HT 1A heteromer. The antibody should be able to recognize the heteromer formed by both receptors, and it should not recognize the monomeric forms of the receptors. The obtained antibody would be used to modulate the surface properties of nanocapsules, which could then be exploited as a novel nanocarrier of antipsychotics. In this way, we can obtain efficient delivery of the encapsulated drugs preferentially to the defined site in selective tissues. To produce specific scFv antibodies with defined a D 2 -5-HT 1A heteromer selectivity, the antibody phage display libraries

Analysis of Specificity of Obtained scFvs Monoclonal Antibodies
The

Results and Discussion
Phage display is a powerful tool for the identification of peptides, proteins or antibodies with affinity for a specific target [13]. The advantage of the method is the possibility of the production of monoclonal  immunize  an  animal  due  to  their  toxicity, non-immunogenicity or presence in complexes on the surface of cell membranes [14]. Therefore, in the present study, we used a phage display technique to prepare a novel human monoclonal scFv antibody that specifically recognized the D 2 -5-HT 1A heteromer. To obtain such properties, the antibody should recognize the structural, spatial epitope formed within the heteromer structure. Both receptors mentioned above are membrane proteins belonging to the GPCR family.
Therefore, it was impossible to perform a selection process using recombinant antigen since obtaining such an antigen in a soluble form with the correct spatial structure is too difficult to achieve in the case of membrane proteins.
In the present study, to separate phages The process was performed on cells growing on the plate surface. The process provided the best conditions for the formation of the spatial D 2 -5-HT 1A antigen and minimized the risk of isolation of antibodies that recognize undesired antigens [15]. Several parameters affect biopanning efficiency, including antigen concentration, temperature, washing stringency (washing number and composition of wash buffer) as well as blocking and elution buffer composition [16].
Elimination of nonspecific binding without losing rare specific clones remains a serious challenge [16]. Therefore, in the present study, we  The activity of phage display-derived antibodies was determined using ELISA (Fig 2). of the receptors, was isolated. In the future, to obtain the best parameters of antigen-antibody interaction, the modifications of selected antibodies will be conducted using random mutagenesis. Eventually, the obtained antibody would be used as a targeting ligand to functionalize the surface of nanocapsules, which would then be exploited as a novel nanocarrier of antipsychotics.