CAR-T Lead Molecule Discovery Service

Tek Biotech is composed of a group of experts in antibody drug discovery, with an average project experience of more than 10 years. Tek Biotech has established a comprehensive antibody drug candidate discovery platform based on phage display technology, and is able to provide a series of therapeutic biomolecule discovery-related services, including but not limited to VHH antibody, scFv antibody and Fab antibody discovery, CAR-T sequence design of CAR-T and CAR-NK antibodies. After about 10 years of development, Tek Biotech has introduced single B-cell sequencing technology on the basis of its antibody phage display technology platform （Phagepro Tech^®）, which enables it to provide customers with high-quality CAR antibody lead sequence discovery services more rapidly.

CAR-T therapy refers to Chimeric Antigen Receptor T-Cell Immunotherapy (CAR-NK is similar, just replacing T cells with NK cells). It is a new type of precision targeted therapy for treating tumors, which has achieved good results in clinical tumor treatment through optimization and improvement in recent years, and it is a very promising new type of tumor immunotherapy that can be precise, fast, efficient, and has the potential to cure cancer.

T-cells, also called T-lymphocytes, are a type of human leukocytes, derived from bone marrow hematopoietic stem cells, which mature in the thymus gland and then migrate to the human blood, lymph and peripheral tissues and organs to perform immune functions. Through genetic engineering technology, technicians have transferred the VHH antibody gene, scFv antibody gene into T cells by suitable methods, such as lentiviral packaging, and expressed them through the membrane localization of VHH or scFv, i.e., a special set of localization navigation device CAR (tumor chimeric antigen receptor), or CAR-T cells, tailored to the T or NK cells against specific tumors, using their CAR-T cells use their “localization navigation device” CAR to specifically identify tumor cells in the body and release a large number of multiple effectors through immune action, thus efficiently killing tumor cells and achieving the purpose of treating malignant tumors.

Compared with traditional scFv antibodies (from common IgG antibodies), VHH antibodies (also known as heavy chain antibodies) from camelids have a molecular weight of about 15kDa, which is more stable and is now widely used in CAR-T lead antibody development. Through its own alpaca base, Tek Biotech is able to provide customers with healthy alpacas of pure background and clear origin for nanobody (VHH antibody) discovery services.

█ Alpaca VHH antibody

Alpaca antibodies have only heavy chains without light chains and are therefore called heavy chain antibodies (hcAb), as shown in Figure 1, D. Alpaca antibodies lack light chains and consist of four HCs and two VHs, where the VH region is also known as a nanobody or a single domain antibody (sdAb). The independent alpaca VH region has complete antigen binding capacity, while unlike artificially modified single chain antibody fragments (single chain Fv, scFv), it tends to aggregate. Compared to conventional antibodies, alpaca antibodies have stable properties, such as remaining active at 60°C and resisting extreme pH environments.

At present, Tek Biotech has successfully established a natural phage antibody display library from 300 alpacas, with an effective library capacity of 10^10, which can shorten the preparation cycle of alpaca monoclonal antibody through the screening of this library. At the same time, using different screening strategies, such as antigen decreasing method, combined with our recombinant protein expression platform, we are able to provide customers with recombinant antibody expression fermentation services up to the level of -5g/L, which largely improves the batch-to-batch consistency and stability of the antibody.

Figure1  A, conventional IgG; B, Fab fragment of conventional antibody; C, single-chain scFv fragment of conventional antibody; 

D, heavy-chain antibody or camelid antibody; E, VH region of camelid antibody or single-domain antibody (sdAb)

█ Preparation Process

Figure 2: Camel derived VHH antibody discovery service based on phage technology platform

█ Service Content

█ CAR-T Cell Therapy Development Process

Figure 3 Tek Biotech CAR-T/NK program service flow

Additional value-added services we can provide for CAR-T/NK:

(1) CAR expression testing

(2) Specificity of target proteins to the antigenic recognition region of the lead antibody

(3) Anti-idiotype testing against the antigen recognition region of the lead antibody

(4) Antigen Recognition Region (ARR) testing of Fab-form antibodies against different target proteins

(5) Cytokine release test

         a) Cytokine type: Y-chain co-receptor family, IL-12 family, IL-1 superfamily, etc.

         b) Methods: enzyme-linked immunospot test (ELISpot), intracellular cytokine staining test (ICS), flow cytometry, etc.

(6) In vitro cytotoxicity test

       Tek Biotech is able to provide in vitro cytotoxicity assays to validate the assessment of cytotoxicity and possible adverse effects of CAR-T cells. For example, some acute targeted/tumor toxicity (cytokine release syndrome, tumor lysis syndrome) or targeted/non-tumor toxicity that may be induced by CAR-T cell therapies, as well as failure of CAR-T cells to immortalize due to the fact that the CAR-T cells undergo antigen-independent proliferation that may increase cell-mediated toxicity.

(7) CAR cell proliferation assays

     Tek Biotech has developed second and third generation CAR products that enhance the persistence of CAR-T cell or NK cell proliferation, and Tek Biotech is able to evaluate this activation and proliferation process through different assays such as MTT, MTS, Cell Counting Kit-8 (CCK-8), 5,6-carboxyfluorescein diethylsuccinimidyl ester acetate (CFSE) binding flow cytometry analysis and other techniques.

(8) Viability and Biodistribution Analysis

       Tek Biotech is able to provide viability and biodistribution analyses to validate and assess the normal proliferative capacity of CAR-T cells under physiological conditions for preclinical CAR-T cell therapies. For example, viability and biodistribution analyses provide important support for evaluating the long-term therapeutic efficacy of CAR-T cells in response to barriers to CAR-T cell therapies with diminished efficacy due to the limited replicative lifespan of CAR-T cells.

█ Service Advantages

-- Adopting mature alpaca immunization procedure combined with phage display technology to build camel-derived immune antibody library, and screening to obtain monoclonal antibodies with high affinity and specificity.

-- Abundant experience and mature technology to ensure stable and reliable product quality.

-- Strict quality control system: standard hardware and facilities platform, meet the key management rules of cGMP documents, and provide handwritten experiment transcripts.

-- Can overcome the problems of target point and high side effects that are difficult to be solved by traditional methods, and is an ideal source of antibody drug preparation.

-- Provide large-scale production service of monoclonal antibodies to meet the demand of industrialized production

-- Complete upstream and downstream services: providing integrated services from antigen preparation, phage library construction, alpaca immunization, antibody screening to antibody humanization, etc.

-- Services can be customized: prepared according to customer needs, tailor-made experimental solutions for each customer to meet the special needs of each customer