Despite the rising trend in the therapeutic antibody market, there still remains functional limits to therapeutic antibodies. For example, pharmacokinetics, cell-specificity, and cytotoxicity are some of the major problems faced by researchers as they study the application of antibodies. [1] Therefore, our team wanted to work on designing antibodies that can function inside the cell. By attaching cell-penetrating peptides on them, we thought that the problems of cytotoxicity and lack of specificity would be resolved. Our project was primarily inspired by the research conducted by Lim KJ et al., which utilized BR2, a cell-penetrating peptide with cancer specificity, for efficient delivery of single chain variable fragments into cancer cells. [2] Our team believes that our research can be used tfor further research in efficient disease and cancer-targeting therapeutic antibodies that are not limited to cell surface targets such as Alzheimer's Disease and Type II Diabetes Mellitus.

Antibodies are one of the fastest growing therapeutic agents in the market. However, the targets of currently developed antibodies are restricted to cell surface proteins due to their inability to go and maintain their function inside the cell. The goal of our project is to overcome the limits of current therapeutic antibodies and to design an antibody that can work inside the cell. Typical antibodies contain immunoglobulin fold that requires disulfide bond to maintain its structure and function and lose their ability to bind target antigens in a reducing environment where disulfide bond is broken. We have chosen a hyperstable single chain variable fragments(scFv(P5)) that can maintain its structure and function in the reducing environment of cytosol. [3] This antibody was engineered from scFv (F8) that recognize coat protein of the plant virus AMCV by changing residues important for antigen binding. [4] We are attaching cell-penetrating peptide to the N-terminus (CPP-scFv(P5)) to develop an antibody that can go into the cell. [5] We will clone, express, purify and test if CPP-scFv(P5) goes inside a cell by immunostaining and recognizes lysozyme in a reducing environment by size-exclusion chromatography. We will also try to engineer CPP-scFv(P5) to change its antigen specificity for more medically applicable targets by molecular modeling. The development of antibodies that can work inside the cell dramatically broadens the range of target molecules and the diseases that can be treated with therapeutic antibodies. Our experiments will start with proof of principles that hyperstable scFv can function inside the cell and can be further developed to target diverse intracellular target proteins by protein engineering.

[1] Chames P, Van Regenmortel M, Weiss E, Baty D. Therapeutic antibodies: successes, limitations and hopes for the future. Br J Pharmacol. 2009 May;157(2):220-33. doi: 10.1111/j.1476-5381.2009.00190.x. Review. PubMed PMID: 19459844; PubMed Central PMCID: PMC2697811.

[2] Lim KJ, Sung BH, Shin JR, Lee YW, Kim DJ, Yang KS, Kim SC. A cancer specific cell-penetrating peptide, BR2, for the efficient delivery of an scFv into cancer cells. PLoS One. 2013 Jun 11;8(6):e66084. doi: 10.1371/journal.pone.0066084. Print 2013. Erratum in: PLoS One. 2013;8(11). doi:10.1371/annotation/fb854e6a-cc9e-4446-b50a-5318cffb68c5. PubMed PMID: 23776609; PubMed Central PMCID: PMC3679022.

[3] Donini M, Morea V, Desiderio A, Pashkoulov D, Villani ME, Tramontano A, Benvenuto E. Engineering stable cytoplasmic intrabodies with designed specificity. J Mol Biol. 2003 Jul 4;330(2):323-32. PubMed PMID: 12823971.

[4] Yu W, Zhan Y, Xue B, Dong Y, Wang Y, Jiang P, Wang A, Sun Y, Yang Y. Highly efficient cellular uptake of a cell-penetrating peptide (CPP) derived from the capsid protein of porcine circovirus type 2. J Biol Chem. 2018 Sep 28;293(39):15221-15232.

[5] Tavladoraki P, Girotti A, Donini M, Arias FJ, Mancini C, Morea V, Chiaraluce R, Consalvi V, Benvenuto E. A single-chain antibody fragment is functionally expressed in the cytoplasm of both Escherichia coli and transgenic plants. Eur J Biochem. 1999 Jun;262(2):617-24.

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