Research and technology background

Evolutionary Molecular Engineering

Our technology and evolutionary molecular engineering

    Biopolymers such as nucleic acids (DNA/RNA) and proteins have been gaining the high functions through 4 billion years of history. Nowadays, researchers are able to synthesize the DNA, RNA, and protein with their purpose sequences. However, designing nucleic acids and proteins for the purpose function, in other words, structurally approaching molecular design is very hard yet. Evolutionary molecular engineering is the technology which evolves the molecules under the lab setting with high speed, and enables the evolved molecules which are new functional biopolymers. In EME, we use one of the evolutionary molecular engineering techniques; cDNA display method as our core technology. cDNA display method allows us to gain specific and high affinity (up to nM level of KD) VHH antibody and cyclic peptide by high-throughput screening platform (it has been developed by EME), and these candidates are applied for innovation of new medications.

The history of developing cDNA display method

    Epsilon Molecular Engineering Inc. was founded as a biotechnology start-up company emerged from Saitama university which has used the core technology of evolutionary molecular engineering. From 1985, Saitama university, Prof. Hushimi and his team have been researching evolutionary molecular engineering. Recently, evolutionary molecular engineering has been highlighted in the world. For instance, Prof. George P. Smith (He received the Norvel Prize of Chemistry in 2018) has started researching the Phage display method for peptides and antibodies. In vitro virus (mRNA display) method which is one of the cell free genotype/phenotype linking systems developed first in the world by Dr. Nemoto and Prof. Hushimi in 1997. mRNA display method has ten thousand times efficiency than phage display method. However, the mRNA display method is less stable than other methods. Thus, we developed cDNA display method as genotype-phenotype linking strategy which has much larger diversity and higher quality. The cDNA display method can be replaceable for mRNA display because of its stability.

Display Technology

What is cDNA display

cDNA display is our protein display method which is genotype/phenotype linking systems. cDNA display has high stability and it handles high diversity.

The Advantages of cDNA display

Restriction of Displayed protein

No restriction for proteins with cytotoxicity

cDNA display enables to display proteins which have cytotoxicity for cells and microbes.

Stability

Very high stability

Displayed protein is linking to cDNA display by linker. Therefore, cDNA display is very stable in alkaline pH, high temperature, and RNase.

Diversity

Very huge diversity

Available to hundle approximately 10¹³⁻¹⁴

Comparison of cDNA display and other protein display method

Display method	Structure	Genotype	Diversity (/mL)
Yeast display		Plasmid DNA	10⁸
Phage display		ssDNA	10⁸
cDNA display		cDNA	10¹³⁻¹⁴
Ribosome display		mRNA	10¹³
mRNA display		mRNA	10¹³

EME’s linker technology

For our cDNA display method, the puromycin linker is the key technology of genotype-phenotype linking strategy. As this puromycin linker, we use our unique puromycin linker which contains 3-cyanovinylcarbazole nucleoside (cnvK), and it is called cnvK linker. Firstly, the cnvK linker is hybridized with specific region of mRNA. After that, the cnvK linker is hybridized to the mRNA and photo-cross-linked by UV irradiation then the mRNA-cnvK linker cross-linked product is formed. These steps largely save the time for the preparation of cDNA display molecules.

The advantages of cnvK linker are as follows;

• cnvK linker is able to be ligated with mRNA by photo-cross linkage instead of conventional enzymatic ligation. Save the reaction time (enzyme: approximately 1hr ⇒ photo-cross linkage: few minutes)
• Normally using enzyme for the concatenate cause contamination of samples. However, using cnvK linker does not require application of enzyme, and reduces the contamination. Therefore, using cnvK linker can improve the reaction rate of cDNA display molecules because of reduction of contamination.
• cnvK is useable for both in-vitro selection experiment and evaluation of binding affinity

The method of genotype-phenotype linking strategy with puromycin linker

    After the preparation of the mRNA-cnvK linker, the mRNA-cnvK linker is added to cell free translation reaction mixture. Ribosome starts translation from 5’ ends side of mRNA, and polypeptides are synthesized. Because ribosome doesn’t translate the sequence of cnvK linker, ribosome stops translation when ribosome reaches the sequence of cnvK linker. At the same time, puromycin (aminoacyl tRNA analog) in the cnvK linker is integrated into ribosome. When the cnvK linker is integrated into ribosome, the peptide transfer reaction is occurred. Because of the peptide transfer reaction, the C-termini of synthesized polypeptide chain and puromycin linker is connected with covalent bond. Therefore, this synthesized compound is mRNA-cnvK linker polypeptide. In EME, we use our unique linker “photo-cross linkage puromycin linker (cnvK linker)” and “cDNA display synthesizer.” These technologies allow the auto preparation of large diversity (10¹³⁻¹⁴) of cDNA library.

VHH / Peptide

What is VHH？

    Camelids such as alpacas (Vicugna pacos) and llamas (Lama glama) produce not only IgG antibodies consisting of heavy chains (H chains) and light chains (L chains) (A) but also antibodies consisting of only heavy chains (heavy-chain antibodies, HCAb) (B). The single variable domain of HCAb is called VHH with a molecular weight of 12−15 kDa (C). VHH has high antigen specificity and affinity like that of a conventional IgG antibody. While an IgG antibody forms antigen-binding sites with the three complementarity determining regions (CDRs) of each of VH and VL, VHH recognizes antigens with three CDRs. CDR3, in particular, is an important region for antigen binding. VHH preferentially recognizes clefts and cavities in the target molecules.

VHH binds to several types of epitopes

    Structure of antigen binding site (paratope) which CDR of VHH form have much larger diversity than conventional antibodies. Conventional antibodies bind to the convex areas of antigen. On the other hand, VHH favors to bind to hollows and gaps of antigen, but also binds to the convex areas and nearly flat surface structures of antigen. Moreover, VHH recognizes the low molecular particles.

Paradigm shift in biopharmaceutical developments: appearance of VHH

    Since the 1980s, biotechnology-based recombinant proteins, such as insulin and erythropoietin, have been developed as new medications. Subsequently, in 1987, the first humanized antibody technology was developed then the antibody-drug discovery got the spotlight as next-generation biomedicines. Research and development of antibodies against many target molecules have been promoted along with the development of genome-based drug discovery since the 1990s, and antibody drugs accounted for 62% of the biopharmaceutical market in 2018. While antibody drugs are expected to be developed as the next-generation antibodies in the future based on innovative technology and concepts, we have faced the limitations of antibody drug industry.

Conversely, VHH was discovered in 1993 by Prof. Hamers at Vrije Universiteit Brussel in Belgium(Hamers-Casterman, C., et al. 1993). Although VHH has many features favoring drug discovery, major pharmaceutical companies did not consider VHH as an attractive new biomolecule in the 1990s because VHH was discovered at the same time when antibody drugs attracted much attention as next-generation biopharmaceuticals. However, a biotech start-up company started aiming to commercialize VHH in Belgium as one of the low-molecular antibody drugs, then research and development of VHH were started. In 2018, FDA approved Caplacizumab as the first VHH antibody drug against von Willebrand Factor for the treatment of acquired thrombotic thrombocytopenic purpura. After that, many of VHH antibody medications continuously have been moving to clinical trial phases, and VHH has been highlighted as one of the next generation biomolecules for new drug discovery.

Cyclic peptides/peptide aptamers

In EME, we have a library of cyclic peptides. The greatest feature of this cyclic peptide library is that we synthesize the cyclic peptides with disulfide linkage or chemical linkage agents between pairs of cysteines. These amino acids of cyclic peptides are randomized. Thus, we can have several lengths and sequences of cyclic peptides. For more information, please see our page “Library”. Also, we synthesize cyclic peptides with linkage between two cysteines using thiol group (disulfide linkage) or a chemical linkage agent. Based on these methods, we have been improving efficiency of forming a cDNA display library for screening cyclic peptides.

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