Research and technology background

Evolutionary Molecular Engineering

Epsilon Molecular Engineering Inc. was founded as a biotechnology start-up company emerged from Saitama university which have used the core technology of evolutional molecular engineering. From 1985, Saitama university, Prof. Husimi and his team have been researching the Evolutionary Molecular Engineering. In the same time, Prof. George P. Smith (He received the Norvel Prize of Chemistry in 2018) has started researching Phage display method for peptide and antibody. In vitro virus (mRNA display) method which is one of the cell free genotype/phenotype linking systems similar to phage display method  have developed first in the world by Dr. Nemoto  and Prof. Husimi in 1997. mRNA display method has ten thousand times efficiency than phage display method. However, mRNA display method is rack of stability than other methods. Thus, we developed cDNA display method as genotype-phenotype linking strategy which has much larger diversity and higher quality than phage display method and mRNA display method.

Biopolymers such as nucleic acids (DNA/RNA) and proteins have been gaining the high functions through 4 billion years 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 evolve the molecules under the lab setting with high speed, and enable to gain the evolved molecules which are new functional biopolymers.

In EME, we use one of the evolutionary molecular engineering technics; 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 through high-though put screening platform (it has developed by EME), and these candidates are applied for innovation of new medications.

Paradigm shift in biopharmaceutical developments: appearance of single domain antibodies (VHH)

Paradigm shift in biopharmaceutical developments: appearance of single domain antibodies (VHH)

Since the 1980s, biotechnology-based recombinant proteins, such as insulin and erythropoietin, were 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. 

On the other hand, VHH was discovered in 1993 by Prof. Hamers at Vrije Universiteit Brussel in Belgium(Hamers-Casterman, C., et al. 1993). Although it has many features favoring drug discovery, Major pharmaceutical companies did not consider VHH as attractive new biomolecule 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 2019, FDA approved Caplacizumab as the first VHH antibody drug against von Willebrand Factor for the treatment of acquired thrombotic thrombocytopenic purpura. Recently, many pharmaceutical companies have shifted their attention to VHH from antibody drug as new biopharmaceuticals.

Clinical stages of VHH

31 products are in clinical development (As of 2019/10)

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VHH Drug Target Indication Status
Caplacizumab Von Willebrand factor Thrombotic thrombocytopenic purpura (TTP) Launch
2018:EME
2019:FDA
Ozorlalizumab TNF-a Rheumatoid Arthritis PII
Vobarilizumab IL-6R Rheumatoid Arthritis
SLE
PII
PII
ALX-0171 RSV Lower Respiratory Infection PII
ALX-0761
/MSB0010841
IL-17A, IL-17F,
IL-17A/F
Psoriasis PII
CAR-T CD19/CD20 Refractory B-cell lymphoma PI
M6495 ADAMTS-5 Osteoarthritis PI
203027 Rotavirus Diarrhoea PII
68-GaNOTA Her2 VHH Her2 Brain metastasis of breast cancer PII

Referred from http://tabs.craic.com

What is a VHH antibody?

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 calld VHH with a molecular weight of 12−15 kDa (C).

What is a VHH antibody?

Comparison between IgG antibody (A), heavy-chain antibody (B), VHH (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 only three CDRs (yellow parts in the figures below). CDR3, in particular, is an important region for antigen binding. VHH preferentially recognizes clefts and cavities in the target molecules.

What is a VHH antibody?02

Conformation of VHH

VHH are able to bind to the epitopes where are structurally different from previous antibodies bind.

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. On the other hand, VHHs are favor to bind to hollows and gaps, but also bind to the convex areas and nearly flat surface structures. Moreover, VHH are also able to recognize the low molecular particles.

VHH are able to  bind to  the epitopes where are structurally different from previous antibodies bind.

Reference:
Structure and development of single domain antibodies as modules for therapeutics and diagnostics. Hoey R.J., Eom H., Horn J.R., Experimental Biology and Medicine, 244, 1568-1576 (2019)

Distribution of CDR3 lengths

Distribution of CDR3 lengths

We analyzed approximately four hundred VHH information registered in the data base. The figure above shows the distribution of the number VHH clones registered and the lengths of VHH CDR3. It indicates that the lengths of VHH CDR3 shows (which are able to bind target molecules (antigen) random distribution although conventional antibodies shows a normal distribution. We can see the random peaks on the graph above, and this is one of the characteristics of VHH.
Additionally, according to farther analyzation of CDR-3 of VHH, VHH are likely to form specific structures of CDR3 based on the lengths of CDR and amino acid sequences of specific regions.

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 pair 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 the core technology page.

The phage display method and mRNA display method are commonly used for screening of functional cyclic peptides and peptide aptamers. Especially, the mechanism of screening cyclic peptides with mRNA display method is that synthesize the cyclic peptides spontaneously with artificial amino acids which are prepared with genetic reprograming. On the other hand, in EME, we synthesize cyclic peptides with linkage between two cysteines using thiol group (disulfide linkage) or a chemical linkage agent. Moreover, in EME, we have been working for improvement of formation efficiency of cDNA display library, condition and etc. for synthesizing cyclic peptides.

Macrocyclic peptide-based inhibition and imaging of hepatocyte growth factor, Sakai K., et al., Nat Chem Biol., 6, 598-606 (2019) Reprogramming the Translation Initiation for the Synthesis of Physiologically Stable Cyclic Peptides, Goto Y., et al., ACS Chem. Biol., 3, 120-129 (2008)
Ribosomal Synthesis of Bicyclic Peptides via Two Orthogonal Inter-Side-Chain Reactions, Sako Y., et al., JACS, 130, 7232-7234 (2008)