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TCR-T Lead Sequence Development Service

With more than ten years of business experience and deep experience in antibody development, TekBiotech's expert team has successfully built an efficient antibody drug candidate molecule platform, which not only covers the development of TCR-T lead sequences, design services for CAR-T and CAR-NK lead antibody sequences, but also widely provides discovery services such as scFv antibodies, VHH antibodies and Fab antibodies, all of which show excellent quality. Relying on advanced phage display technology and single B cell sequencing technology, TekBiotech has made great achievements in the field of antibody development and can quickly provide customers with TCR-T customized development and CAR antibody lead sequence development services.

In the development of TCR-T lead sequences, the correct pairing of TCR-α chain and TCR-β chain is crucial. TechBio uses professional tools to optimize it and can select suitable vectors to optimize its expression conditions. Based on the next-generation sequencing technology, TekBiotech can identify and locate target antigens and sort out specific CD8+T cells by flow cytometry. In addition, through single-cell sequencing technology, TekBiotech can separate tumor-specific TCR encoding genes with high throughput. TekBiotech can insert TCR gene fragments into the phage coat protein (such as pIII) sequence, so that the TCR sequence can be displayed on the phage surface. The constructed phage library can reach 10^8-10^9, ensuring that the library diversity, insertion rate and positive rate exceed 90% to meet the quality requirements of different customers for antibody display libraries.

At the same time, TekBiotech provides comprehensive antibody development downstream services, including the preparation and amplification of TCR-T cells in TCR-T cell therapy, cytokine release testing, in vitro cytotoxicity and proliferation testing, cell viability and biodistribution analysis, etc., to ensure the safety and effectiveness of TCR-T cells, as well as one-stop technical services such as cell killing verification. TekBiotech scientists can analyze and design reasonable solutions based on the specific experimental needs of customers, aiming to provide customers with personalized customized solutions to accurately meet their needs.

█ TCR-T Lead Sequence Development Service

In addition to membrane proteins, proteins in cells can also be recognized by TCR-T cells, which shows that it targets a wider range of targets. And TCR-T can be activated at a lower epitope density. This characteristic also improves the detection and clearance capabilities of TCR-T cell therapy. Generally speaking, its affinity is lower than that of CAR, which means that in TCR-T cell therapy, each TCR-T cell may be allowed to detect and eliminate several antigen-presenting tumor cells. TekBiotech's TCR-T customized development service based on phage display technology uses phage display libraries to obtain corresponding sequences, which can enable cells to have corresponding clearance capabilities. The specific process is shown in Figure 1.

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Figure1 TCR-T Custom Development Service Flowchart Based on Phage Display Technology

█ Service Content and Cycle

Steps	Service Content	Period
Step 1: 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 4th shot serum antibody titer meets the requirements, another booster shot is performed 7 days before blood collection. If it does not meet the requirements, continue routine immunization; (4) If the titer is qualified, blood is collected to isolate T cells;	10 Weeks
Step 2: cDNA Preparation	(1) Extraction of total RNA from T cells (RNA extraction kit); (2) Preparation of cDNA by high-fidelity RT-PCR (reverse transcription kit);	1 Working Day
Step 3: Construction of Antibody Library	Based on phage display technology: (1) Using cDNA as a template, two rounds of PCR are used to amplify the genes of the α and β chains of TCR; (2) Phagemid construction and transformation: TCR gene fragments are spliced into phagemid vectors, electroporated to transform TG1 host bacteria, and antibody library is constructed; (3) Identification: 24 clones are randomly selected, and PCR is used to identify the positive rate + insertion rate; (4) Auxiliary phage preparation: M13 phage amplification + purification; (5) Antibody display library rescue;	3-4 Weeks
Step 4: Antibody Library Screening (3 rounds)	Based on phage display technology: (1) Default 3 rounds of screening (solid phase screening): pressure screening to remove non-specific antibodies to the maximum extent; (2) Pick a single clone to amplify phage + IPTG induced expression + ELISA to detect positive clones; (3) Pick all positive clones for gene sequencing;	4-5 Weeks

█ Service Advantages

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Personalized library

construction service

Mature technology

platform

Traceable experimental

records

Personalized solution

customization

A variety of screening

systems are available

One-stop service

Various service types

Provide a variety of

phagemid vectors

including PMECS,

pComb3X and

pCANTAB 5E, and

modify them

according to

customer needs

The library capacity

can reach 10^8-10^9,

and the insertion rate

is stable over 90%

QC quality control

standards (immunity

titer, multiple quality

control), Chinese and

English experimental

reports, original

experimental records

Immunization scheme,

library construction

scheme, screening

scheme and subsequent

in vitro expression

verification scheme, etc.

Solid phase, liquid

phase, cell screening,

etc., TekBio scientists

make personalized

designs according to

specific projects

Immunogen preparation,

animal immunization,

library construction

and screening, TCR-T

cell preparation and

amplification, cytokine

release test, in vitro

cytotoxicity test, and

cell killing test, etc.

Supporting services

Provide different animal

-derived immune library

construction services

and natural antibody

library screening services

according to customer

needs

TCR-T Lead Sequence Development Service Frequently Asked Questions

If we encounter T-cell unresponsiveness in sequence development service, how can we solve it?

T-cell unresponsiveness represents that T-cells are in a state of hyporesponsiveness, which is generated by the over-activation of the TCR along with the co-stimulatory signals generated by CD28. Genetically encoded T cell molecules may also activate and damage other cells in the body. Teck Biologics proposes three main strategies for restoring the T-cell reservoir: replacement, reprogramming and cell regeneration. Replacement technology utilizes physical means to remove functionally impaired cells from the circulation, ensuring that the remaining cells maintain stable immune effects and memory T-cell proliferation. Reprogramming techniques aim to rescue T cells from dysfunction and failure. Cell regeneration methods reverse the process of thymic degeneration by applying substances or factors that promote cell production.
How can we solve the problem of insufficient affinity in sequence development service?

Affinity is very significant for TCRs, and high affinity can enhance the effectiveness of their therapies. In order to enhance the affinity of TCRs, research has focused on introducing amino acid sequence (AAS) variants into the complementary decision region (CDR) of TCRs. The affinity of these TCRs is in the low mM or even nM range, which exceeds the affinity of most naturally occurring TCRs. TCRs with affinities in the pM range can also be generated using other methods.While these techniques are effective in identifying high-affinity TCR variants, higher affinities are associated with increased cross-reactivity. Therefore, structure-guided approaches have the potential to improve ACT and could substantially reduce the risk of off-target toxicity. Tektronix Biologics has a wealth of project experience to help customers improve affinity and assist experiments with specialized technology.
What is the TCR-T Pilot Sequence?

Engineered T-cells, or TCR-T therapy, as a type of cellular immunotherapy, falls under the category of ACT immunotherapy. This technology is capable of targeting antigens that are not indicated for expression. Initially, this technology was limited to transducing the TCR gene sequence into the patient's peripheral T cells. With advances in technology, it is now possible to screen and identify TCR sequences that pair with specific binding sites, integrate them into the patient's own T cells, and enable the T cells to recognize TCRs with a combination of multiple proteins and HLAs. these modified cells are then reintroduced into the patient's peripheral blood to bind to and effectively clear the tumor-associated sites, achieving the original goal. Teck Biologics provides TCR-T lead sequence development services and has many years of experience in TCR modification.
What is the difference between TCR-T and CAR-T? How to choose between these two approaches?

Nowadays, researchers are developing different ACT technologies, including TIL therapy, TCR-T cell therapy and CAR-T cell therapy.CAR-T technology is mainly used when there is a scarcity of available antigens or when there is tumor heterogeneity or tumor immunosuppression.Biologics of TCR-T technology are now applied to treat some diseases suffering from lung cancer, myeloma or melanoma. TCR-T technology products are more reliable and useful in practice. Compared to CAR-T technology, TCR has a wider range of reactivity and can react with a wider variety of antigens. When TCR-T cells and CAR-T cells target a site, TCR-T cells tend to achieve effective binding and adsorption, so that the carried drug is more evenly distributed everywhere, while CAR-T will make the carried drug adsorbed in the outer layer of tumor cells, and the penetration efficiency to the inside of tumor cells is low.
If there is a TCR mismatch problem in TCR-T lead sequence development service, how can we solve it?

TCR mismatch occurs at TCR-T Pilot Sequence Development Service, which can lead to problems with TCR-T technology recognition sites. A TCR can react with about 106 different epitopes for recognition.TCR sequences we determine the sequence by extracting RNA, PCR amplifying the ends of the template and then sequencing the organism's cells for TCRα and TCRβ.TCR mismatches occur when exogenous sequences are introduced into T cells using molecular techniques, a process that may prevent the organism's own sequences from matching. This mismatch may prompt the TCR to misrecognize the body's normal cells, which in turn triggers an attack on its own normal cells. Reducing mismatches enhances the usefulness of the technology by allowing TCR genes capable of binding to tumor cells to be transformed into T cells via mRNA or viruses, enabling them to localize to tumor cells. Teck Biologics reduces gene mismatches by introducing interchain disulfide bonds, replacing human TCR constant structure domains with mouse TCR sequences, re-manipulating coding TCR constant structure domains, and knocking out endogenous TCRs using genetic engineering techniques.

If we encounter T-cell unresponsiveness in sequence development service, how can we solve it?

T-cell unresponsiveness represents that T-cells are in a state of hyporesponsiveness, which is generated by the over-activation of the TCR along with the co-stimulatory signals generated by CD28. Genetically encoded T cell molecules may also activate and damage other cells in the body. Teck Biologics proposes three main strategies for restoring the T-cell reservoir: replacement, reprogramming and cell regeneration. Replacement technology utilizes physical means to remove functionally impaired cells from the circulation, ensuring that the remaining cells maintain stable immune effects and memory T-cell proliferation. Reprogramming techniques aim to rescue T cells from dysfunction and failure. Cell regeneration methods reverse the process of thymic degeneration by applying substances or factors that promote cell production.
How can we solve the problem of insufficient affinity in sequence development service?

Affinity is very significant for TCRs, and high affinity can enhance the effectiveness of their therapies. In order to enhance the affinity of TCRs, research has focused on introducing amino acid sequence (AAS) variants into the complementary decision region (CDR) of TCRs. The affinity of these TCRs is in the low mM or even nM range, which exceeds the affinity of most naturally occurring TCRs. TCRs with affinities in the pM range can also be generated using other methods.While these techniques are effective in identifying high-affinity TCR variants, higher affinities are associated with increased cross-reactivity. Therefore, structure-guided approaches have the potential to improve ACT and could substantially reduce the risk of off-target toxicity. Tektronix Biologics has a wealth of project experience to help customers improve affinity and assist experiments with specialized technology.
What is the TCR-T Pilot Sequence?

Engineered T-cells, or TCR-T therapy, as a type of cellular immunotherapy, falls under the category of ACT immunotherapy. This technology is capable of targeting antigens that are not indicated for expression. Initially, this technology was limited to transducing the TCR gene sequence into the patient's peripheral T cells. With advances in technology, it is now possible to screen and identify TCR sequences that pair with specific binding sites, integrate them into the patient's own T cells, and enable the T cells to recognize TCRs with a combination of multiple proteins and HLAs. these modified cells are then reintroduced into the patient's peripheral blood to bind to and effectively clear the tumor-associated sites, achieving the original goal. Teck Biologics provides TCR-T lead sequence development services and has many years of experience in TCR modification.
What is the difference between TCR-T and CAR-T? How to choose between these two approaches?

Nowadays, researchers are developing different ACT technologies, including TIL therapy, TCR-T cell therapy and CAR-T cell therapy.CAR-T technology is mainly used when there is a scarcity of available antigens or when there is tumor heterogeneity or tumor immunosuppression.Biologics of TCR-T technology are now applied to treat some diseases suffering from lung cancer, myeloma or melanoma. TCR-T technology products are more reliable and useful in practice. Compared to CAR-T technology, TCR has a wider range of reactivity and can react with a wider variety of antigens. When TCR-T cells and CAR-T cells target a site, TCR-T cells tend to achieve effective binding and adsorption, so that the carried drug is more evenly distributed everywhere, while CAR-T will make the carried drug adsorbed in the outer layer of tumor cells, and the penetration efficiency to the inside of tumor cells is low.
If there is a TCR mismatch problem in TCR-T lead sequence development service, how can we solve it?

TCR mismatch occurs at TCR-T Pilot Sequence Development Service, which can lead to problems with TCR-T technology recognition sites. A TCR can react with about 106 different epitopes for recognition.TCR sequences we determine the sequence by extracting RNA, PCR amplifying the ends of the template and then sequencing the organism's cells for TCRα and TCRβ.TCR mismatches occur when exogenous sequences are introduced into T cells using molecular techniques, a process that may prevent the organism's own sequences from matching. This mismatch may prompt the TCR to misrecognize the body's normal cells, which in turn triggers an attack on its own normal cells. Reducing mismatches enhances the usefulness of the technology by allowing TCR genes capable of binding to tumor cells to be transformed into T cells via mRNA or viruses, enabling them to localize to tumor cells. Teck Biologics reduces gene mismatches by introducing interchain disulfide bonds, replacing human TCR constant structure domains with mouse TCR sequences, re-manipulating coding TCR constant structure domains, and knocking out endogenous TCRs using genetic engineering techniques.

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TCR-T Lead Sequence Development Service Frequently Asked Questions

If we encounter T-cell unresponsiveness in sequence development service, how can we solve it?

How can we solve the problem of insufficient affinity in sequence development service?

What is the TCR-T Pilot Sequence?