RU2721854C1 - Single-domain antibody specifically binding human erbb3 receptor, without canonical disulphide bond - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to a single-domain antibody specifically bound to extracellular part of human ErbB3. Disclosed single-domain antibody contains hypervariable loops CDR1, CDR2, CDR3 with amino acid sequences SEQ ID NO: 1–3 and do not have canonical disulfide bond.

EFFECT: invention provides extending the range of single-domain antibodies specific binding to human extracellular part ErbB3.

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Description

The invention relates to the field of biochemistry, in particular to a single-domain antibody that binds to the human ErbB3 receptor. The invention has the ability to specifically bind to the extracellular domain of the ErbB3 receptor, which may allow blocking the activity of this receptor, and therefore, the growth of tumor cells with an abnormal amount of ErbB3 receptor on the surface. Also disclosed is a DNA sequence encoding an antibody; an expression vector containing the given DNA sequence, producer strain and method for producing antibodies.

The invention relates to the field of biotechnology, in particular to antibodies that bind to the ErbB3 receptor, also referred to as HER3, which belongs to the group of epidermal growth factor receptors (Human Epidermal Factor receptor, HER). The ErbB receptor group includes four beige: ErbB1 (EGFR, HER1), ErbB2 (HER2), ErbB3 (HER3), ErbB4 (HER4). All of them are receptor tyrosine kinases and are often abnormally expressed on the surface of tumor cells. The first two members of the family (EGFR and HER2) are well studied, anti-EGFR and anti-HER2 therapy are widely used in the treatment of tumor diseases. In the clinic, both antibodies and low molecular weight compounds are used to inhibit EGFR and HER2. The most famous among them are: cetuximab (cetuximab) - an anti-EGFR monoclonal antibody used for various oncological diseases, including brain tumors; trastuzumab (trastuzumab) - anti-HER2 drug used in breast cancer; lapatinib is a selective inhibitor of tyrosine kinases ErbB1 and ErbB2.

For a long time, it was believed that ErbB3, a member of the ErbB family, which was first described in 1990, does not exhibit tyrosine kinase activity and, therefore, cannot participate in the signaling pathways of tumor cells. However, studies have begun to appear in the last decade in which it has been shown that a significant proportion of tumors that develop resistance to drugs that block ErbB1 and ErbB2 receptors have increased expression of ErbB3. ErbB3 is involved in the development of tumors of different locations, including the breast and ovary [1, 2]. In this regard, the need arises for the development of therapeutic antibodies that block the dimerization of the ErbB3 receptor and thereby inhibit the tyrosine kinase activity of the HER2-HER3 heterodimer in tumor cells.

Single domain antibodies, also known as VHH antibodies or nanobodies, were first isolated from the blood serum of members of the Camelidae family (camels, llamas, vicunas, guanacos). They represent the variable domain of the heavy chain. (In the literature, single-domain antibodies are also called "nanobody", mini-antibodies, nano-antibodies, VHH antibodies). Despite the fact that non-canonical VH antibodies of a camel, consisting of only two shortened heavy chains, have great homology with human IgG3 heavy chains, antibodies without a light chain were not found in humans. VHH domains have significant differences in the organization of hypervariable regions (CDRs) compared to human antibodies. The first and third variable loops (CDR1 and CDR3) are longer in comparison with classical antibodies. As a rule, a disulfide bond is formed between the variable loops of single domain camelid antibodies (between CDR1 and CDR3, less often between CDR2 and CDR3). The repertoire of paratopes (antigen-binding combinations) of single-domain antibodies and classic is different. Single domain antibodies have a number of significant advantages: greater thermal and chemical stability; they can be cloned and expressed in bacteria, which significantly reduces the cost of their production. In addition to therapeutic applications, examples of which will be listed below, the literature shows methods of using nanobodies in molecular biology. For example, in article [3] of 2018, a single-domain antibody against fluorescent protein GFP is used to visualize short-lived transcription factors in a living cell. In [4, 5], methods of using nanobodies in structural biology to stabilize proteins are shown. The article [6] demonstrated the successful use of single-domain antibodies for immunoprecipitation and determination of transcription factor binding sites.

To date, there are no antibodies against ErbB3 approved for clinical use. However, more than ten such antibodies are now at different stages of clinical trials. All of them are classic full-sized antibodies, including bispecific, and therefore do not violate the patent purity of the claimed invention. We list a few of them. Seribantumab (WO 2008100624, WO 2012116317) is developed by Merrimack Pharmaceuticals Inc. and Sanofi-aventis, undergoing the second stage of clinical trials, as one of the components of complex therapy. Patritumab (WO 2007077028) is developed by Amgen, Daiichi Sankyo Inc. and U3 Pharma GmbH, in 2018 the third phase of clinical trials was completed, in which the effectiveness of treatment of non-small cell lung cancer in combination with erlotinib (Erlotinib) was tested. Istiratumab (Istiratumab) (WO 2011047180) is a bispecific anti-ErbB1 / ErbB3 antibody developed by Dyax Corp. and Merrimack Pharmaceuticals Inc., completed the second phase of clinical trials.

In turn, single-domain antibodies against ErbB3 are presented in open sources in a significantly smaller amount than classical ones. Security document WO 2011144749 "BIOLOGICAL MATERIALS RELATED TO HER3" describes sequences of polypeptides that are thought to be capable of binding to the human ErbB3 receptor. Security document RU2653443C2 presents a bispecific ErbB2 / ErbB3 antibody, the ErbB3 binding portion of which is constructed based on the VHH domain.

The technical problem solved by this invention is to expand the range of antibodies that bind to the ErbB3 receptor by creating a single domain antibody against the extracellular part of the human ErbB3 receptor, capable of inhibiting heterodimerization with other members of the ErbB family,

The proposed antibody is characterized by the following features:

A) Single domain antibody, K _D = 3100 ± 200 nM, k _a = 7.2 ± 0.2 * 10 ^-8 M ^-1 s ^-1 , and k _d = 2.2 ± 0.2 * 10 ³ s ^-1 , molecular weight 14.4 kDa

B) Hypervariable polypeptide regions (CDRs) have a homology of more than 75% with sequences of SEQ ID No: 1-3

B) The antibody contains an amino acid sequence that has a homology of more than 90% with the sequence of SEQ ID No 4.

In addition, a distinctive feature of the proposed antibodies is the absence of a canonical disulfide bond in the hypervariable regions.

In one implementation, an antibody in accordance with the invention is characterized in that it is monoclonal.

In addition, the invention includes a nucleotide sequence encoding a single domain antibody that binds to the extracellular portion of human ErbB3, characterized by the sequences of SEQ ID No: 1-4.

In addition, the invention includes an expression vector containing a nucleotide sequence encoding a single domain antibody in accordance with the invention.

In addition, the proposed single-domain antibody can be tested as part of antitumor therapy.

In addition, the proposed antibody may be an integral part of a molecule or molecular complex capable of binding to the extracellular part of human ErbB3.

In addition, the invention provides a bacterial producer strain Shuffle, transformed with an expression vector containing nucleotide sequences encoding the following sections of a single polypeptide: single domain antibody characterized by the sequences SEQ ID No: 1-4; TEV protease binding site, ubiquitin-like SUMO protein, N-terminal polyhistidine peptide.

In FIG. 1 presents the results of protein purity analysis by gel permeation chromatography using a Superdex 75 column

In FIG. 2 shows gel electrophoresis of single domain antibody samples after purification.

In FIG. 4 presents: (a) the mass spectrum of the purified single domain antibody obtained using electrospray ionization; (b-c) experimental isotopic distribution after deconvolution (blue curves) and theoretical isotopic distributions (black curves) for the reduced disulfide bond (b) and for the closed disulfide bond (c).

In FIG. 3 shows experimental curves characterizing the binding of a single domain antibody as provided herein to the extracellular domain of ErbB3. The curves were obtained on a Biacore T200 surface plasmon resonance setup (GE Healthcare).

The invention includes a single domain antibody against the extracellular portion of the human ErbB3 receptor capable of inhibiting heterodimerization with other members of the ErbB family.

The term "single-domain antibodies" are equivalent to the terms: "VHH antibodies" or "nanobodies." The term “single domain antibody” is used to refer to a protein molecule consisting of a single polypeptide chain containing frame and hypervariable regions (CDRs), and also capable of binding to an antigen (target molecule).

The term "ErbB3" refers to the third representative of the epidermal growth factor receptor family, the term "HER3" is equivalent to it. Human ErbB3 in the international Uniprot database has the P21860 code. ErbB3 is a membrane protein and has an extracellular portion that can bind ligands, a transmembrane portion, and an intracellular portion that provides phosphorylation and subsequent signal propagation. The single domain antibody provided herein is specific for the extracellular portion of human ErbB3.

The proposed antibody is characterized by the following features:

A) Single domain antibody, K _D = 3100 ± 200 nM, k _a = 7.2 ± 0.2 * 10 ^-8 M ^-1 s ^-1 , and k _d = 2.2 ± 0.2 * 10 ³ s ^-1 , molecular weight 14.4 kDa

B) Hypervariable polypeptide regions (CDRs) have a homology of more than 75% with sequences of SEQ ID No: 1-3

B) The antibody contains an amino acid sequence that has a homology of more than 90% with the sequence of SEQ ID No 4.

Compared with classical immunoglobulins, the characteristic molecular mass of which is 160 kDa, the proposed antibody is significantly smaller and is formed by only one polypeptide chain. Despite this, the affinity of the antibody is quite high, and in terms of thermal and chemical stability it is not inferior to the classical ones.

In some embodiments of the invention, the amino acid sequence of the antibody can be changed while maintaining homology up to a level of 90% with the sequence of SEQ ID No 4. Such changes can be made to the DNA molecule encoding the antibody by genetic engineering. By changes is meant the replacement of some amino acid residues by others, or the insertion of additional amino acid residues, or the removal of certain amino acid residues. By changing the amino acid sequence in the above frameworks, for example, an increase in chemical stability without loss of ability to bind to the original antigen can be achieved. Moreover, the homology of two or more sequences is determined by standard bioinformatics comparison methods.

In addition, a distinctive feature of the proposed antibodies is the absence of a canonical disulfide bond in the hypervariable regions.

In one implementation, an antibody in accordance with the invention is characterized in that it is monoclonal.

In addition, the invention includes a nucleotide sequence encoding a single domain antibody that binds to the extracellular portion of human ErbB3, characterized by the sequences of SEQ ID No: 1-4.

In addition, the invention includes an expression vector containing a nucleotide sequence encoding a single domain antibody in accordance with the invention.

In addition, the proposed single-domain antibody can be tested as part of antitumor therapy.

In addition, the proposed antibody may be an integral part of a molecule or molecular complex capable of binding to the extracellular part of human ErbB3.

In addition, the invention provides a bacterial producer strain Shuffle, transformed with an expression vector containing nucleotide sequences encoding the following sections of a single polypeptide: single domain antibody characterized by the sequences SEQ ID No: 1-4; TEV protease binding site, ubiquitin-like SUMO protein, N-terminal polyhistidine peptide.

The invention is illustrated by the following examples:

Example 1

Cytoplasmic expression of a single domain anti-ErbB3 antibody

For cytoplasmic expression, an expression bacterial SHuffle strain was used. In 100 μl of chemically competent cells, 30 ng of pSol-SUMO-VHH456 plasmid DNA was added. The method of calcium transformation was used. Then the cells were sown on a cup with the antibiotic kanamycin (50 μg / ml).

Several colonies from the dish were transferred to 30 ml of 2xYT medium (1.6% tryptone, 1% yeast extract, 0.5% NaCl, pH 7.2). The preculture grew for about 15 hours to an optical density of 600 nm OD = 8.0 (with constant stirring 200 rpm, 37 ° C). Then it was transferred to a two-liter flask containing 400 ml of 2xYT medium with kanamycin antibiotic (50 μg / ml), diluted 100 times (to OD≈0.08). Upon reaching an optical density of OD = 0.7-0.8, the culture was induced by adding L-ramnose with a final concentration of 5 mM. After induction, the bacterial culture was incubated at 27 ° C for 15 hours with constant stirring at 200 rpm.

After the cells were pelleted in a centrifuge (5 minutes at 10,000 g, with removal of the supernatant). Cells were resuspended in 30 ml metal-chelate chromatography buffer: 50 mM Na2HP04 pH 8.0, 0.3 M NaCl with the addition of 1 mM PMSF, 1 mM EDTA and 1 mM benzamidine as protease inhibitors. The destruction of cells was carried out on ice using an ultrasonic installation. To remove cell membranes, intact organelles, and other insoluble particles, the resulting cell lysate was centrifuged in round-bottom tubes at 40,000 g for 20 minutes at 4 ° C. The supernatant was filtered through a 0.22 μm membrane. Proteins from the resulting clarified lysate were precipitated by slowly adding ammonium sulfate with constant stirring. The clarified lysate with ammonium sulfate was centrifuged for 10 minutes at 40,000 g. The precipitate was redissolved in 20 ml of metal-chelate chromatography buffer (50 mM Na2HPO4 pH 8.0, 0.3 M NaCl, 1 mM PMSF, 1 mM benzamidine)

Example 2

Metal Chelate Purification

Before purification by metal chelate chromatography, the protein solution was transferred to a working buffer (50 mM Na2HPO4 pH 8.0, 0.3 M NaCl, 1 mM PMSF) with 5 mM imidazole added, namely, 20 ml of protein solution were dialyzed at 4 ° C for 12 hours. 2 liters of working buffer using a Thermo Fisher 10K MWCO SnakeSkin Dialysis Tubing membrane. After dialysis, the protein solution was filtered again through a 0.22 μm membrane. The solution was applied to a 1 ml Roche pre-equilibrated metal-chelate chromatographic column using a peristaltic pump. Then washed the column loaded with protein with 20 ml of working buffer with the addition of 3 mm imidazole. Next, the target protein was eluted with 10 ml of working buffer with the addition of 300 mM imidazole, collecting 1 ml fractions.

The impurity content in the obtained fractions was analyzed using SDS gel electrophoresis (denaturing conditions, 12% polyacrylamide gel, DTT as a reducing agent).

Example 3

Purification by ion exchange chromatography

As the final purification, ion-exchange chromatography methods, namely, cation-exchange methods using a MonoS 5/50 GL column manufactured by GE Healthcare, were used. Opposite chromatography was performed on NGC Discover system equipment manufactured by BioRad.

The target single domain antibody, after cutting off the partner protein (SUMO) with a TEV protease, was transferred to a low ionic strength buffer (20 mM Na-acetate buffer pH 6.0) using a Thermo Fisher 7K MWCO dialysis membrane SnakeSkin Dialysis Tubing.

объема колонки. Полученные фракции анализировали с помощью SDS-гель-электрофореза [Фиг. 2] и гель-проникающей хроматографии. Фракции, содержащие целевой белок, переводили в новый буфер (20 mM HEPES рН 7.5, 50 mM NaCl). Для последующих анализов и хранения полученный белок концентрировали в ячейках 10К MWCO Amicon производства компании Millipore. Финальную концентрацию очищенного бежа определяли по поглощению на 280 нм.After loading the sample onto a pre-balanced column using a 5 ml loading loop, the column was washed with 10 volumes of start buffer, then the target protein was washed in a gradient of sodium chloride (from 0 to 0.6 M in 30 volumes) at a flow of 1 ml / min, collecting fractions equal to

column volume. The resulting fractions were analyzed by SDS gel electrophoresis [Fig. 2] and gel permeation chromatography. Fractions containing the target protein were transferred to a new buffer (20 mM HEPES pH 7.5, 50 mM NaCl). For subsequent analysis and storage, the resulting protein was concentrated in 10K cells of Millipore MWCO Amicon. The final concentration of purified beige was determined by absorbance at 280 nm.

Example 4

Characterization of a single-domain antibody by mass spectrometry

Before the experiment, the purified single domain antibody was diluted to a concentration of 1 mg / ml and transferred to deionized water, after which formic acid with a final concentration of 1% would be added to the solution. Mass spectrometric measurements were performed on a maXis Q-TOF apparatus manufactured by Bruker Daltonik, using electrospray ionization. 100 μl of the protein solution was injected at a flow of 50 μl / min. The obtained mass spectra were averaged using DataAnalysis 4.0 Bruker Daltonik software. Further analysis, in particular, fitting of the isotope distribution, was carried out according to the technique published in [7]. Mass spectrometric analysis confirmed the hypothesis that there is no canonical disulfide bond in the hypervariable regions (Fig. 4), which was previously discovered in experiments using the Ellman reagent.

Example 5

Determination of affinity and kinetics of binding by surface plasmon resonance

The affinity and binding kinetics of the purified single domain antibody and the extracellular part of the ErbB3 receptor were measured by plasmon resonance using a GE Healthcare Biacore T200 device using CM5 sensor chips at 37 ° C. The recombinant extracellular domain of ErbB3, previously isolated from CHO cells, was immobilized in a 10 mM Na-acetate buffer pH 4.5 on the surface of the sensor chip.

The purified antibody was dissolved in HBS buffer (10 mM HEPES pH 7.4, 150 mM NaCl), the concentration was determined by absorbance at 280 nm, after which a dilution series was prepared with the addition of BSA (bovine serum albumin) at a concentration of 50 μg / ml. The buffer and protein solutions were filtered through 0.22 μm membranes. The measurements were carried out at a flow of 10 μl / min.

The obtained binding curves were analyzed using the Biacore Evaluation software (GE Healthcare), namely, the key parameters of the affinity of a single domain antibody to the extracellular part of ErbB3 were determined: K _D = 3100 ± 200 nM, k _a = 7.2 ± 0.0002 * 10 ^-5 M ^-1 s ^-1 , and k _d = 2.2 ± 0.2 * 10 ³ s ^-1 . The experimental data and approximation by the Hill equation are presented in FIG. 3.

Bibliography

[1] McIntyre, E. et al. The complete family of epidermal growth factor receptors and their ligands are coordinately expressed in breast cancer // Breast cancer research and treatment. - 2010. - T. 122. - No. 1. - C. 105-110.

[2] Chiu C. G. et al. HER-3 overexpression is prognostic of reduced breast cancer survival: a study of 4046 patients // Annals of surgery. - 2010. - T. 251. - No. 6. - C. 1107-1116.

[3] Bothma J. P. et al. LlamaTags: A Versatile Tool to Image Transcription Factor Dynamics in Live Embryos // Cell. - 2018.

[4] Pardon E. et al. A general protocol for the generation of Nanobodies for structural biology // Nature protocols. - 2014. - T. 9. - No. 3 .-- C. 674.

[5] D Cromie K., Van Heeke G., Boutton C. Nanobodies and their use in GPCR drug discovery // Current topics in medicinal chemistry. - 2015. - T. 15. - No. 24. - C. 2543-2557.

[6] Nguyen-Due T. et al. Nanobody®-based chromatin immunoprecipitation / micro-array analysis for genome-wide identification of transcription factor DNA binding sites // Nucleic acids research. - 2012. - T. 41. - No. 5. - C. e59-e59.

[7] Rhoads T.W. et al. Using theoretical protein isotopic distributions to parse small-mass-difference post-translational modifications via mass spectrometry // Journal of the American Society for Mass Spectrometry. - 2013. - T. 24. - No. 1. - S. 115-124.

Single domain antibody specifically binding human ErbB3 receptor without canonical disulfide bond

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Sequence listing

<110> Federal State Budgetary Institution of Higher Education and Science "St. Petersburg National Research Academic University named after Zh.I. Alferova of the Russian Academy of Sciences ”, Alferov University

<120> Single domain antibody specifically binding human ErbB3 receptor without canonical disulfide bond

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Arg Arg Gly Gly Thr Asn Ser Gly Ser Tyr Tyr Phe Asp Arg Pro Ala

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Claims (1)

A single domain antibody that specifically binds to the extracellular portion of the human ErbB3 receptor containing the hypervariable loops CDR1, CDR2, CDR3 with amino acid sequences of SEQ ID NO: 1-3 and not having a canonical disulfide bond.

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