A novel synthetic nanobody library

Abstract

The present invention relates to the creation of novel and synthetic nanobody libraries containing nanobodies for use in the diagnosis and treatment of diseases through the modulation of the function of targeted proteins. The creation of a synthetic nanobody library and the ensuring of the specificity of the nanobodies in this library are achieved through modulations in CDR3 regions.

Description

[0001] A NOVEL SYNTHETIC NANOBODY LIBRARY

[0002] Technical Field of the Invention

[0003] The present invention relates to a novel synthetic nanobody library containing nanobodies that can be used in the diagnosis and treatment of diseases by modulating the functions of targeted proteins. The creation of a synthetic nanobody library and ensuring the specificity of the nanobodies in this library is made possible through modifications performed on Complementary Determinant Regions (CDRs) located in the antigen binding region and determining the interaction with the antigen.

[0004] State of the Art

[0005] Nanobodies (VHHs) are the antigen-binding region of heavy-chain antibodies (HCAbs) obtained from camelids and are widely used in research such as protein purification and structural determination due to their small size, high specificity and stability [1], Despite overcoming the limitations of traditional antibody fragments, the immunization of animals for nanobody production and the obtaining of antigen-specific nanobodies remain a costly and laborious process. Therefore, synthetic nanobody libraries constructed using DNA oligonucleotides offer a cost-effective alternative to libraries requiring immunization and hold great potential for identifying nanobodies specific to particular antigens or conformations. The studies conducted examine the design of synthetic nanobody libraries, biopanning methods and the applications of nanobodies obtained from these libraries in research [1],

[0006] The identification of antigen-specific nanobodies relies on the creation of nanobody libraries. Similar to antibody libraries, nanobody libraries are divided into three main groups: immune, naive, and synthetic libraries. Immune libraries are created by immunizing animals such ascamels, llamas, or transgenic mice that produce heavy-chain antibodies (HCAb), and then isolating mRNA from lymphocytes obtained from the blood of these animals and converting it to cDNA. With this method, nanobodies with high binding affinity can be obtained, but it has disadvantages such as the need to create a new library for each antigen, the failure of non-immunogenic antigens to elicit an immune response, and the high cost of animal immunization [1], Naive libraries, on the other hand, are obtained from peripheral blood lymphocytes from unimmunized donors. While this method offers the advantage of not requiring immunization, the absence of in vivo affinity maturation may necessitate additional affinity enhancement steps to obtain nanobodies with high binding affinity. In addition, the accessibility of camelids in biological laboratories may be limited [1],

[0007] Synthetic nanobody libraries are a platform created in a laboratory setting using molecular cloning techniques, enabling the rapid development of binders for specific antigens. Although studies on synthetic nanobody libraries are limited, current research focuses on the construction of these libraries and the selection of antigen- specific nanobodies. For example, Moutel, Bery et al. developed a library they named NaLi-Hl, based on a stable nanobody and by modifying the DNA sequence, they created billions of different variations and screened this library against various target molecules through nanobodies displayed on the surface of bacteriophages [2], Selected nanobodies obtained in vitro using this method have shown similar efficacy to natural antibodies and have even been used to track or inactivate proteins within living cells.

[0008] Synthetic nanobody libraries, such as NaLi-Hl, enable scientists to rapidly develop nanobodies that can bind to novel antigens with high specificity, reducing reliance on animal experiments and saving costs and time in scientific research. The widespread adoption of such synthetic libraries accelerates the development of nanobody-based therapeutics and diagnostic tools, making significant contributions to both basic biology research and medical applications [2],

[0009] The limitations and shortcomings of currently available nanobody libraries include the need for animal immunization of immune nanobody libraries, requiring a new immunization process for each antigen; the low binding affinity of naive nanobody libraries, requiring camel lymphocytes;and the limited diversity and potentially low binding affinity of synthetic nanobody libraries, thus necessitating a nanobody library containing nanobodies with high binding affinity.

[0010] Brief Description and Objects of the Invention

[0011] The invention is a synthetic nanobody library containing nanobodies that can bind to target proteins; said library is characterized by comprising;

[0012] framework regions (FR) with a stable amino acid sequence,

[0013] CDR1 and CDR2 regions, in which amino acid sequences and lengths are kept constant, randomized CDR3 regions which were created and designed with varying lengths

[0014] Said CDR3 region is organized into sub-libraries of varying lengths, and consists of at least three different sub-libraries as follows;

[0015] • The first sublibrary containing CDR3, which carries 9 randomized amino acid regions with amino acid sequence No. 1 and nucleotide sequence No. 2,

[0016] • The second sublibrary, containing CDR3, which carries 15 randomized amino acid regions with amino acid sequence No. 3 and nucleotide sequence No. 4.

[0017] • The third sublibrary containing CDR3, which carries 18 randomized amino acid regions with amino acid sequence No. 5 and nucleotide sequence No. 6

[0018] Amino acid diversity in the CDR3 region is generated by degenerate codon randomization, preferably using the NNK codon strategy.

[0019] These nanobodies, due to their randomized regions, have the capacity to bind to multiple targets and, with the help of their optimization for resistance to high temperatures and pH changes, they treat various diseases and are used in the diagnosis process by inhibiting or modulating the function of target proteins.An object of the present invention is to facilitate the development of intermediary molecules for challenging targets by containing nanobody sequences that can bind with high affinity to intracellular and surface proteins. Thus, it is used in the treatment of diseases by inhibiting and modulating the function of target proteins.

[0020] Another object of the present invention is to use nanobodies with high specificity to bind to biomarkers, thereby facilitating early diagnosis of diseases and improving the sensitivity of diagnostic processes.

[0021] Detailed Description of the Invention

[0022] The present invention is a novel synthetic nanobody library containing nanobodies that can be used to modulate the functions of targeted proteins in the diagnosis and treatment of diseases, and said nanobody library comprising,

[0023] • It is called CKS-Nb-Libl and a nanobody consisting of a CDR3 region having 9 randomized amino acids with the amino acid sequence of Sequence No. 1 and the nucleotide sequence of Sequence No. 2, and CDR1 and CDR2 regions where the amino acid sequences and lengths are fixed, and framework regions where the amino acid sequences and lengths are fixed or,

[0024] • It is called CKS-Nb-Lib2 and a nanobody consisting of a CDR3 region having 15 randomized amino acids with the amino acid sequence of Sequence No. 3 and the nucleotide sequence of Sequence No. 4, and CDR1 and CDR2 regions where the amino acid sequences and lengths are fixed, and framework regions where the amino acid sequences and lengths are fixed or,

[0025] • It is called CKS-Nb-Lib3 and a nanobody consisting of a CDR3 region having 18 randomized amino acids with the amino acid sequence of Sequence No. 5 and the nucleotide sequence of Sequence No. 6, and CDR1 and CDR2 regions where the amino acid sequences and lengths are fixed, and framework regions where the amino acid sequences and lengths are fixed,The scaffold of each library consists of cAbBCIHO nanobodies with amino acid Sequence No. 7, and while the amino acid numbers of the CDR1 and CDR2 regions are kept the same in each library, the CDR3 regions that bind to antigens are kept at different amino acid lengths and contain degenerate sequences to ensure randomization. Diversity in the CDR3 region is generated by degenerate codon randomization, thus preferably using the NNK codon strategy. This increases the diversity of amino acids, resulting in a broader binding repertoire.

[0026] The method for obtaining these novel synthetic nanobody libraries comprising the process steps of:

[0027] i. designing the nanobody sequences through bioinformatics analysis,

[0028] ii. combining the frame sequences and the sequences belonging to the CDR1, CDR2, and CDR3 regions with an average total length of 60 nucleotides and an overlapping region length of 20 nucleotides using "Overlapping Extension Polymerase Chain Reaction (PCR)",

[0029] iii. If the combined sequences are checked using Sanger sequencing and an error is detected in the regions other than CDR3, re-establishing the PCR reaction in the region where the error is located (in step ii) and repeating the process step; if no error is detected, proceeding to step iv.,

[0030] iv. transferring the generated sequences to a plasmid containing fluorescent protein, v. making E. coli competent for transformation and transferring plasmids to E. coli, vi. preparing E. coli cultures of plasmids and isolating nanobodies,

[0031] The chemicals required for the Overlapping Elongation Polymerase Chain Reaction, which combines the frame sequences and the sequences belonging to the CDR regions comprising;

[0032] • 2,5 pL 10X Q5 reaction buffer,

[0033] • 2,5 pL, 10X GC enhancer buffer,

[0034] • 0,5 pL,10 mM dNTP,

[0035] • 0,5 p, Primer I,• 0,5 pL, Primer II,

[0036] • 0,2 pL, Q5 Pfu Polymerase enzyme

[0037] • 18,3 pL Nuclease-free water,

[0038] Due to the use of overlapping elongation polymerase chain reactions, overlapping regions of frame regions are used as the primary components.

[0039] The temperature and cycling conditions required for the reaction were maintained as follows: 95°C for 5 minutes and 1 cycle for denaturation; 95°C for 45 seconds, 60°C for 1.5 minutes, and 72°C for 1 minute, totalling 35 cycles, for the bonding step; and 72°C for 5 minutes and 1 cycle followed by 10°C for 5 minutes and 1 cycle for the elongation step. The 25 pL PCR reaction obtained was analyzed by agarose gel electrophoresis. Amplimers were visualized under ultraviolet light, and gel extraction was performed for Fragment 1 and Fragment 2 PCR products, which were eluted using 50 pL of elution buffer. The purified primary PCR products were then measured in concentration and stored at -20°C. After this step, verification is performed using Sanger sequencing, and then the sequences are transferred to a plasmid, which is then transfected into E. coli bacteria. Sequence-controlled nanobody DNA libraries were converted into highly diverse nanobody libraries using the "Ribosome Display" method.

[0040] Ribosome Display Method

[0041] The method was performed using "PureFrex 2.1". Accordingly;

[0042] Solution I (ribosome + translation factors)

[0043] Solution II (energy system)

[0044] Solution III (amino acids)

[0045] Solution IV (enzymes)

[0046] Solution V (tRNA, etc.)

[0047] RNase Inhibitor

[0048] Template DNAs containing nanobody DNA librariesReactions were established with all these components in the volumes specified below and incubated at 37°C for 6 hours.

[0049] Component Volume

[0050] Solution I 8 pl

[0051] Solution II 4 pl

[0052] Solution

[0053]

[0054] III 4 pl

[0055] Solution IV 2 pl

[0056] Solution V 1 pl

[0057] DNA template 1 pl

[0058] Accordingly, the study primarily focused on in vitro transcription followed by in vitro translation reactions. The products obtained as a result of the reaction were checked using SDS PAGE. The production process then moved to a more advanced stage. For this purpose, plasmids containing these DNAs were transfected into competent cells and their expression was induced. After the necessary incubation for expression, protein isolation and purification were performed and checked using the western blot method.

[0059] The novel synthetic nanobody library has the capacity to bind to multiple targets due to its inclusion of nanobodies carrying randomized CDR3 regions, and with the help of its optimization for resistance to high temperatures and pH changes, it treats various diseases by inhibiting or modulating the function of target proteins and is used in the diagnosis process of diseases.

[0060] REFERENCES

[0061] [1] Liu, B., & Yang, D. (2022). Easily Established and Multifunctional Synthetic

[0062] Nanobody Libraries as Research Tools. International journal of molecular sciences, 23(3), 1482. https : / / doi. org / 10.3390 / ijms23031482

[0063] [2] Sandrine Moutel, Nicolas Bery, Virginie Bernard, Laura Keller, Emilie Lemesre, Ario de Marco, Laetitia Ligat, Jean-Christophe Rain, Gilles Favre, Aurelien Olichon,

[0064] Franck Perez (2016) NaLi-Hl: A universal synthetic library of humanized

[0065] 10 nanobodies providing highly functional antibodies and intrabodies eLife

[0066] 5 : el 6228 https: / / doi.org / ! 0.7554 / eLife.16228

Claims

CLAIMSClaim 1:A synthetic nanobody library containing nanobodies that can bind to target proteins , comprising;- framework regions (FR) with a stable amino acid sequence,- CDR1 and CDR2 regions, in which amino acid sequences and lengths are kept constant, -CDR3 region where diversity is createdand characterized in that said CDR3 region in said synthetic nanobody library consists of randomized amino acid sequences of varying lengths.Claim 2:Synthetic nanobody library according to claim 1, characterized in that; said CDR3 region comprises sub-libraries of varying lengths.Claim 3:Synthetic nanobody library according to claim 2, characterized in that; said sub-libraries consists of at least three synthetic nanobody sub-libraries with CDR3 regions of varying lengths;- The first sub-library comprising a CDR3 region of 9 amino acids in length,- The second sub-library comprising a CDR3 region of -15 amino acids in length,- The third sub-library comprising a CDR3 region of -18 amino acids in length.Claim 4:Synthetic nanobody library according to claim 1-3, characterized in that; amino acid diversity in the CDR3 region is generated by using degenerate codon randomization.Claim 5 Synthetic nanobody library according to claim 4, characterized in that; saiddegenerate codon randomization is performed using the NNK codon strategy.Claim 6: Synthetic nanobody library according to claim 1-5, characterized in that; the nanobody sequences are generated using the overlapping extension polymerase chain reaction (PCR) method.Claim 7: The method for obtaining the synthetic nanobody library according to claim 1-6, characterized by comprising the process step of;- designing the nanobody sequences using bioinformatics analysis,- combining the DNA sequences of the frame regions and the CDR1, CDR2, and CDR3 regions using overlapping extension PCR,- cloning the obtained sequences into an expression vector,- transferring vectors into host cells and producing the nanobodies.

Images