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Nanobody Development Services

TekBiotech provides customers with high-affinity and high-specificity antibody drug pre-discovery technology services based on the M13 phage antibody display system, providing strong support for customers' subsequent CAR-T/CAR-NK lead sequence design, antibody humanization, drug target antibody development, bispecific antibody development, and efficient blocking neutralizing antibody development and other downstream R&D work. TekBiotech has 10 years of project development experience and experience in drug antibody discovery, and can provide customers with high-quality camel-derived VHH nanoantibody development services for targets including but not limited to proteins, peptides, small molecules, viruses, membrane proteins, etc.

█ Nanoantibody Development Technology Service

TekBiotech can provide customers with camel-derived VHH nanoantibody library construction and screening services. This technology platform uses M13 phage to insert the VHH nanoantibody gene from camel into the N-terminus of the P3 protein of M13 phage through multiple rounds of PCR, so that the nanoantibody gene is displayed on the phage surface along with the expression of the coat protein, and finally the antibodies that bind to the target antigen are screened out through a screening method similar to "fishing". TekBiotech can provide customers with high-quality alpaca/camel immunization services, nanoantibody library construction based on phage display technology and screening services (immune library capacity is 10^9-10^10 (natural library capacity is 10^10-10^12), library diversity>90%, insertion rate 95%, positive rate>95%). Compared with the traditional hybridoma method, phage display technology is a high-throughput method: the fusion efficiency of the former is generally<0.4%, that is, there are approximately 4 fusion hybridoma cells among 1,000 fusion cells; while phage display can easily obtain 10^8-10^9 correct antibodies with different sequences, and the experiment can be paused or started at any step. It is a modular experimental operation, saving time and development costs. The phage display discovery path of camel-derived VHH antibodies (also known as nanobodies) currently widely used in CAR-T/CAR-NK therapeutics is shown in Figure 1:

Nanobody Development Services-tekbiotech1.png

Figure 1 VHH nanoantibody discovery service process based on phage technology platform

█ Nanoantibody Development Service Content and Cycle

Steps

Service Content

QC Testing

Cycle

Step 1: Antigen Preparatio

*Antigen Type:

(1) Recombinant Protein Preparation

(2) Small Molecule (Transformation) + Coupling

(3) Peptide Synthesis + Coupling

(4) Customer Provides Inactivated Virus

(5) Customer Provides Packaged mRNA

(1) Recombinant Protein (Purity>85%)

(2) Small Molecule Purity>90%

(3) Peptide Purity>90%

4-6 Weeks

Step2: Animal Immunization

(1) Animals are immunized 4 times, with one booster shot, for a total of 5 shots;

(2) Collect negative serum before immunization, and collect blood for the 4th shot for ELISA to test serum titer;

(3) If the titer of the 4th shot serum antibody meets the requirements, then boost the immunization again 7 days before blood collection. If it does not meet the requirements, continue routine immunization;

(4) If the titer is qualified, collect blood to isolate monocytes;

(1) Animals: clear background;

(2) Immunization: protein/virus antigen titer detection; peptide/small molecule antigen titer detection;

8-10 Weeks

Step3: Template cDNA

Preparation

(1) PBMC total RNA extraction;

(2) High-fidelity RT-PCR to prepare cDNA;

(1) PBMC cell quality control;

(2) Total RNA quality control;

(3) cDNA quality control;

1 day

Step4: Phage Display

Library Construction

(1) Using cDNA as template, PCR amplifies VHH gene;

(2) Phagemid construction and transformation: VHH gene splicing phagemid vector,

electroporation transformation TG1 host bacteria, and construction of antibody library;

(3) Identification: Randomly select clones, PCR identification of positive rate +

insertion rate;

(4) Auxiliary phage preparation: M13 phage amplification + purification;

(5) VHH display library rescue;

(1) Library positive rate detection;

(2) Library insertion rate detection;

(3) Library capacity detection + sequence detection;

2-3 Weeks

Step 5: Library Screening

(1) Antigen coating (single protein screening, the default is solid phase screening or

magnetic bead sorting);

(2) Default 3-5 rounds of screening: pressure screening, maximally remove

non-specific antibodies;

(3) Pick a single clone to amplify phage + induce expression + ELISA to detect

positive clones;

(4) Pick all positive clones for gene sequencing;

(1) ELISA positive standard setting;

(2) VHH screening standard setting;

2-3 Weeks

Step 6: Drugability

Evaluation

(1) Construct a suitable expression vector with the obtained antibody sequence for

expression + affinity purification + antibody protein quantification;

(2) ELISA verification of antibody-antigen binding;

(3) BLI method verification of antibody affinity;

(4) Cell function verification: flow blocking verification;

(1) Recombinant antibody expression quality control;

(2) EC50 verification;

(3) Rapid affinity determination results;

(4) Blocking verification results;

4-6 Weeks

After conducting preliminary drugability evaluation for customers, TekBiotech can also provide one-stop technical services such as antibody humanization, antibody affinity maturation, CAR-T/CAR-NK lead sequence design and cell killing verification. We can design reasonable solutions and customize them according to customer needs to help customers' scientific research projects and drug antibody development.

█ Advantages of Nano-antibody Development Service


With an immune base, a variety of camel-derived immune options are available, including but not limited to llama, Alpaca and camel, etc., and the animal source background is clear	A variety of antibody library phage display options: VHH antibody library display, scFv antibody library display, etc.	A variety of target antibody discovery services are available: protein, peptide, small molecule, virus, membrane protein, mRNA, etc.	Large library capacity: immune library capacity 10^9-10^10, natural library capacity 10^10 -10^12

Flexible library screening methods: solid phase screening, liquid phase screening, cell screening selection, magnetic bead screening, etc.	Experimental records are traceable: QC quality control standards (immunity titer, PBMC quality control, library quality control and screening verification quality control), Chinese and English experimental reports, original experimental records	One-to-one personalized program customization (including immunization program, library construction program, screening program and subsequent in vitro expression verification program, etc.) to meet the scientific research project needs of various customers	A series of supporting downstream drug antibody development services can be provided, including antibody in vitro expression verification, antibody humanization, antibody affinity maturation, bispecific antibody development, CAR-T lead sequence molecular design, etc.

Nanobody Development Services Frequently Asked Questions

How do we select immunogens for the VHH antibody discovery service?

In the immune response process, we use a diverse range of immunogens, which can be categorized into natural, recombinant, synthetic and small molecule antigens based on their properties. Among them, natural antigens include viral immunogens, whose purification steps are cumbersome and more difficult and costly than those of other immunogens. Viral immunogens are further subdivided into whole virus inactivated vaccines, subunit vaccines, viral vector vaccines and mRNA vaccines. Whole virus inactivated vaccines are effective in triggering an immune response, but the virus is completely inactivated. Adenoviruses, which trigger an immune response through their ability to replicate and express themselves, have shown to be highly effective and long-lasting, but are also complex to prepare and require strict biosafety controls. mRNA vaccines are introduced into cells to induce expression, which then triggers a response. As for small molecule or peptide antigens, they can be synthesized in vitro, but the design process can be complex. Small molecule antigens, such as peptides and nucleotides, can only be used in conjunction with a carrier because they lack immunogenicity themselves.
What is VHH antibody?

The VHH antibody is an antibody that researchers have found to be missing VL, which has a simple structure consisting of only two VHs. Nano-antibodies are composed of VHH, that one is easy to recognize and deliver. Later, antibodies with this structure were found in other animals such as alpacas and sharks. Although there is no VL structural domain, it is still able to bind to antigen and is a single structural domain antibody with high stability. Tektronix can provide our customers with a series of services including library construction and screening, VHH nanobody construction, etc. Through nanobody synthesis and phage display development platforms, we are able to efficiently construct and screen phage antibody libraries that include many different types of phage antibodies to provide our customers with proprietary and highly specific antibody solutions.
What are the key components of the VHH Antibody Discovery Service?

First, Tektronix Biologics selects healthy, disease-free alpacas for immunization and stimulates a response from these animals, which are capable of producing single domain antibodies. Single B cells are isolated from PBMC, RNA is extracted and reverse transcribed into cDNA, which is then used as a template and screened for the target VHH sequence by gel electrophoresis. The screened VHH antibody sequences were sequenced and analyzed then VHH antibody sequence design was performed. By introducing certain specific mutations for screening and other methods, Tektronix obtained higher stability VHH antibody mutants. The screened VHH sequences are then inserted into appropriate expression vectors (e.g. plasmids, viruses, etc.) for VHH antibody expression. Finally, through phage display technology, the VHH antibody is allowed to be expressed on the surface of the phage, and then the nano-antibodies capable of reacting are utilized for screening. Teckbio sequences and verifies them to ensure the correctness of the sequence and functional activity.
Advantages of VHH Antibody Discovery Service

VHH technology takes advantage of the numerous advances in genetic engineering that allow genes to be routinely spliced together and rearranged to provide a tool with superior binding power that is easily purified and also contains marker tags. Using high-throughput techniques, VHH antibodies with specific functions can be selected from a large number of candidates in a short period of time.VHH can be produced in unlimited quantities\economically, is more stable when exposed to heat and solvents, and can be genetically manipulated for a myriad of uses including scaffolding, labeling, and alteration of specific amino acids.VHH is suitable for use in, among other things, electrochemical biosensors and devices. We hypothesize that higher densities of bound structural domains will provide outstanding advantages in terms of increased signal and therefore higher sensitivity.VHH antibodies are utilized frequently in diagnostics and therapeutics, treatment of central nervous system disorders, etc.VHH antibodies are particularly useful for monitoring mycotoxins in food and feed, as they can be easily genetically engineered and have excellent stability.
Advantages of VHH structure? And its application?

Its unique molecular structure gives it good tissue penetration, shorter half-life and higher renal clearance across the blood-brain barrier, and severe thermal stability.VHH has excellent temperature stability. Further examination of the crystal structure of VHH showed that although VHH lacks the hydrophobic clefts normally formed by VL and VH chains, the CDR of VHH is adaptive and may bind in a variety of ways. Both stacking and charge-charge interactions occur in molecules that are both aromatic and hydrophilic. Some researchers have used triethylamine to elute VHH phage fusions bound to immobilized caffeine/BSA antigens, immunized them, and eluted caffeine-selective VHH from libraries VHH has been shown to be heat-stable, with more than 90% of activity restored by incubation at temperatures up to 90°C. The VHH has been shown to be thermally stable, with more than 90% of activity restored by incubation at temperatures as high as 90°C. The accuracy of this analysis was demonstrated by testing for caffeine in the range of commercially available beverages where caffeine was not intentionally spiked. These thermally stable caffeine-selective VHHs and their use in disposable lateral chromatography devices have been patented for monitoring caffeine in beverages at home and in restaurants.

How do we select immunogens for the VHH antibody discovery service?

In the immune response process, we use a diverse range of immunogens, which can be categorized into natural, recombinant, synthetic and small molecule antigens based on their properties. Among them, natural antigens include viral immunogens, whose purification steps are cumbersome and more difficult and costly than those of other immunogens. Viral immunogens are further subdivided into whole virus inactivated vaccines, subunit vaccines, viral vector vaccines and mRNA vaccines. Whole virus inactivated vaccines are effective in triggering an immune response, but the virus is completely inactivated. Adenoviruses, which trigger an immune response through their ability to replicate and express themselves, have shown to be highly effective and long-lasting, but are also complex to prepare and require strict biosafety controls. mRNA vaccines are introduced into cells to induce expression, which then triggers a response. As for small molecule or peptide antigens, they can be synthesized in vitro, but the design process can be complex. Small molecule antigens, such as peptides and nucleotides, can only be used in conjunction with a carrier because they lack immunogenicity themselves.
What is VHH antibody?

The VHH antibody is an antibody that researchers have found to be missing VL, which has a simple structure consisting of only two VHs. Nano-antibodies are composed of VHH, that one is easy to recognize and deliver. Later, antibodies with this structure were found in other animals such as alpacas and sharks. Although there is no VL structural domain, it is still able to bind to antigen and is a single structural domain antibody with high stability. Tektronix can provide our customers with a series of services including library construction and screening, VHH nanobody construction, etc. Through nanobody synthesis and phage display development platforms, we are able to efficiently construct and screen phage antibody libraries that include many different types of phage antibodies to provide our customers with proprietary and highly specific antibody solutions.
What are the key components of the VHH Antibody Discovery Service?

First, Tektronix Biologics selects healthy, disease-free alpacas for immunization and stimulates a response from these animals, which are capable of producing single domain antibodies. Single B cells are isolated from PBMC, RNA is extracted and reverse transcribed into cDNA, which is then used as a template and screened for the target VHH sequence by gel electrophoresis. The screened VHH antibody sequences were sequenced and analyzed then VHH antibody sequence design was performed. By introducing certain specific mutations for screening and other methods, Tektronix obtained higher stability VHH antibody mutants. The screened VHH sequences are then inserted into appropriate expression vectors (e.g. plasmids, viruses, etc.) for VHH antibody expression. Finally, through phage display technology, the VHH antibody is allowed to be expressed on the surface of the phage, and then the nano-antibodies capable of reacting are utilized for screening. Teckbio sequences and verifies them to ensure the correctness of the sequence and functional activity.
Advantages of VHH Antibody Discovery Service

VHH technology takes advantage of the numerous advances in genetic engineering that allow genes to be routinely spliced together and rearranged to provide a tool with superior binding power that is easily purified and also contains marker tags. Using high-throughput techniques, VHH antibodies with specific functions can be selected from a large number of candidates in a short period of time.VHH can be produced in unlimited quantities\economically, is more stable when exposed to heat and solvents, and can be genetically manipulated for a myriad of uses including scaffolding, labeling, and alteration of specific amino acids.VHH is suitable for use in, among other things, electrochemical biosensors and devices. We hypothesize that higher densities of bound structural domains will provide outstanding advantages in terms of increased signal and therefore higher sensitivity.VHH antibodies are utilized frequently in diagnostics and therapeutics, treatment of central nervous system disorders, etc.VHH antibodies are particularly useful for monitoring mycotoxins in food and feed, as they can be easily genetically engineered and have excellent stability.
Advantages of VHH structure? And its application?

Its unique molecular structure gives it good tissue penetration, shorter half-life and higher renal clearance across the blood-brain barrier, and severe thermal stability.VHH has excellent temperature stability. Further examination of the crystal structure of VHH showed that although VHH lacks the hydrophobic clefts normally formed by VL and VH chains, the CDR of VHH is adaptive and may bind in a variety of ways. Both stacking and charge-charge interactions occur in molecules that are both aromatic and hydrophilic. Some researchers have used triethylamine to elute VHH phage fusions bound to immobilized caffeine/BSA antigens, immunized them, and eluted caffeine-selective VHH from libraries VHH has been shown to be heat-stable, with more than 90% of activity restored by incubation at temperatures up to 90°C. The VHH has been shown to be thermally stable, with more than 90% of activity restored by incubation at temperatures as high as 90°C. The accuracy of this analysis was demonstrated by testing for caffeine in the range of commercially available beverages where caffeine was not intentionally spiked. These thermally stable caffeine-selective VHHs and their use in disposable lateral chromatography devices have been patented for monitoring caffeine in beverages at home and in restaurants.

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