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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.

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