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Adoptive cell therapy (ACT) has emerged as a highly effective treatment for cancer, particularly for patients with limited treatment options. This innovative approach harnesses the patient’s own immune system to target and attack cancer cells. One form of ACT, known as Engineered TCR therapy, utilizes engineered T cell receptors to specifically target tumor-specific antigens. The process begins with sequencing tumor biopsy samples to identify tumor mutations and analyzing peripheral blood to uncover the TCR repertoire.
TCR repertoire sequencing can be achieved through two main methods: single-cell sequencing and bulk sequencing, each offering unique advantages. Bulk sequencing allows for a broader sampling of the sequence space, but it comes at the cost of losing information about the alpha-beta TCR pairing. On the other hand, single-cell sequencing captures detailed information about alpha-beta chain pairing and receptor composition, but it has a lower throughput compared to bulk sequencing.
The key benefits of Twist’s technology in this context include high diversity and quality in TCR libraries. Combinatorial assemblies can encompass up to 10,000 gene fragment combinations, ensuring a rich and varied representation. NGS-verified libraries guarantee that over 90% of possible variants are present within 10 times of the mean, ensuring reliability and accuracy. The platform also offers customization, allowing users to define combinatorial variants across alpha and beta chains in TCR Libraries.
Twist’s technology is flexible, accommodating sequences up to 1.5 kb in length, and it is designed at scale with diversity across multiple elements of the sequence. This flexibility, combined with high diversity and quality, positions Twist as a valuable partner in advancing Engineered TCR therapy, providing researchers with the tools needed for comprehensive TCR library customization and optimization.
The process of discovering T Cell Receptors (TCRs) involves screening immune cell repertoires and resynthesizing numerous potential binders. TCR Libraries play a pivotal role in this discovery process, enabling the rapid generation of combinations of TCR beta and alpha chains for high-throughput screening.
Two main approaches are employed: Combinatorial TCR Libraries and Paired Pool TCR Libraries. In Combinatorial TCR Libraries (Figure 1a), the alpha and beta chains are shuffled together, creating a library with greater diversity that extends beyond the identified repertoire. On the other hand, Paired Pool TCR Libraries (Figure 1b) preserve the explicit alpha and beta chain pairing identified through sequencing.
The T Cell Receptor (TCR) Discovery workflow involves partnering with Twist Bioscience to identify and develop novel advanced cell therapies using large-scale, highly diverse TCR libraries. Twist provides highly uniform screening libraries, precise user-defined combinations of gene fragments that allow for efficient and comprehensive screening of desired combinations. This partnership facilitates the discovery of novel TCRs, advancing the development of innovative and effective cell therapies for various applications.
Combinatorial TCR Libraries
Paired Pool TCR Libraries
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In recent years, chimeric antigen receptor (CAR) T-cell therapy has emerged as a promising form of immunotherapy, offering new avenues for cancer treatment. Advances in gene transfer technology and gene editing, coupled with innovative CAR designs, have propelled the development of novel cancer therapies. However, despite these strides, several challenges remain, necessitating further research and development efforts to accelerate progress in the field.
CARs typically comprise an extracellular binding domain, a hinge region, a transmembrane domain, and one or more intracellular domains. Fine-tuning each component of the CAR is crucial for enhancing T cell specificity, antigen recognition, and overall T cell function. Studies have demonstrated that even minor modifications to the CAR can significantly impact therapeutic outcomes. Therefore, it is imperative to have a comprehensive library for thorough testing, considering the complexity of each CAR construct.
Twist has pioneered a groundbreaking technology for constructing CAR libraries, allowing seamless shuffling of variants within each domain through a scarless assembly process. The key benefits of this technology include high diversity and quality, with each combinatorial assembly accommodating up to 10,000 gene fragment combinations. NGS-verified libraries ensure that over 90% of possible variants are present within 10 times of the mean. The flexibility and customization options offered by Twist’s technology allow users to design TCR and CAR libraries with user-defined combinatorial variants across specific elements. Additionally, the platform provides flexibility in throughput, enabling the insertion of sequences up to 1.5 kb in length and designing libraries at scale with diversity across multiple elements of the sequence. Twist’s technology represents a significant advancement in the development and optimization of CAR T-cell therapies.
The process begins with synthesizing genes representing different sequence variants for each domain of the Chimeric Antigen Receptor (CAR). These domains, including the extracellular binding domain, hinge region, transmembrane domain, and intracellular domains, can consist of multiple sequence variations. Twist then combines these synthesized genes through a scarless assembly process. This unique assembly method ensures that the combination of these genes results in a highly diverse library for scaffold optimization.
The scarless assembly process enables the seamless merging of the gene variants, creating a comprehensive library that spans a wide range of potential combinations. This technology allows for the exploration of unique domain combinations, unveiling novel functionalities within the CAR scaffold.
The workflow for Chimeric Antigen Receptor (CAR) discovery and optimization involves studying how each domain of the CAR scaffold independently and synergistically influences its functionality. CAR Libraries, generated through this technology, act as a valuable tool for fine-tuning each module. This process helps uncover codependencies among the domains and provides a deeper understanding of their impact on T-cell specificity, antigen recognition, and overall T-cell function.
Researchers can partner with Twist at any stage of their discovery workflow, leveraging both in vivo and in vitro workflows for binder discovery and optimization. Additionally, the use of synthetic libraries from Twist enhances scaffold optimization and validation, providing a versatile and efficient approach to advancing CAR T-cell therapy research and development.
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Introducing the Spread-Out Low Diversity (SOLD) libraries – the latest tool revolutionizing the mapping of protein sequences and exploring the intricate relationship between proteins and their environment. Designed for researchers seeking efficiency, precision, and cost-effectiveness, SOLD Libraries offer a streamlined approach to investigating combinatorial possibilities.
These libraries stand out with their unique features, providing greater flexibility compared to traditional methods. Suitable for sites with scattered diversity, SOLD Libraries require no template, offering a novel and efficient solution. The precision of SOLD Libraries is unparalleled, ensuring no premature stop codons or unwanted codons, along with precise control over amino acid and codon distribution. This precision surpasses traditional methods such as NNK, TRIM, and epPCR.
Ensuring superior quality, all SOLD Libraries undergo NGS verification of modified regions, rigorous quality control, and verification of all variants. Created using Twist’s patented silicon-based synthesis platform, SOLD Libraries guarantee low error rates, making them cloning-ready and a reliable tool for researchers exploring the variant space. SOLD Libraries provide the ultimate combination of flexibility, precision, and quality for an advanced and efficient protein sequence mapping experience.
Twist’s SOLD Libraries stand out as high-fidelity synthetic constructs, consistently showcasing the capability to closely align amino acid distributions with the requested frequencies. The observed variants in SOLD Libraries reveal a uniform distribution with no dropouts, exemplifying the reliability and precision of this synthetic tool.
A distinctive advantage of Twist’s SOLD Libraries lies in their ability to precisely integrate diversity across a wild-type sequence without constraining it to small variant domains. This unique feature enables researchers to simultaneously explore multiple amino acid positions along a sequence, facilitating a rapid and effective investigation and optimization of a protein’s activity. Twist’s SOLD Libraries, with their scattered diversity along the wild-type sequence, offer a powerful and versatile solution for researchers seeking high-fidelity synthetic constructs in their protein sequence studies.
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Twist’s Precision Variant Library Technology stands out as a groundbreaking approach for focused screening in protein engineering. Powered by Twist’s massively parallel silicon-based DNA synthesis platform, this technology delivers highly uniform and accurate oligos, with an impressive 90% of oligos represented within <2.5x of the mean. Additionally, it boasts an industry-leading low error rate of 1:2,000 nt.
The combination of this cutting-edge synthesis platform with Twist’s well-established molecular biology expertise allows the creation of highly diverse gene mutant libraries. These libraries exhibit excellent variant representation and offer highly specific user-defined compositions, free from unwanted bias or motifs. With precise control over codon usage, amino acid distribution, and length variation, Twist’s library technology enables comprehensive exploration of the variant sequence space. The key benefits include high diversity precision, verified quality through rigorous quality control and NGS verification, and unmatched flexibility in designing sequences, domains, and combinations. This technology outperforms alternatives like TRIM and the degenerate approach, providing researchers with a superior tool for efficient and tailored protein engineering studies.
Twist Bioscience’s silicon-based DNA synthesis platform and library technology redefine the landscape of variant libraries, providing scientists with high-quality data in a more efficient manner. In comparison to competing technologies, Twist’s libraries exhibit less than 1% deviation from the designed amino acid frequency, showcasing exceptional precision. The in-silico DNA synthesis platform seamlessly incorporates binding motifs and length variation across multi-domain libraries, empowering scientists to design and customize variant libraries for a comprehensive analysis of the variant space.
Twist libraries address challenges associated with NNK and TRIM libraries by printing variants base-by-base and screening before synthesis, eliminating stop codons, liability motifs, unwanted mutations, and biases early in the process. This approach enriches the library for requested functional variants, reducing the screening burden. Twist’s industry-leading, ready-to-use, highly-diverse, and precisely designed libraries offer scientists more opportunities to achieve their research goals.
The superiority of Twist’s libraries is further emphasized through a comparison with NNK and TRIM technologies. Twist’s molecular biology expertise and single-base control approach result in high-diversity libraries without confounding motifs. The quality is exemplified in a CVL distribution example, where expected variants are present at user-defined ratios. Additionally, Twist’s Precise Variant Libraries allow users to choose unique CDR sequences, which, when combined with machine learning analysis, can be seamlessly incorporated into fully synthetic libraries for refined exploration of the variant space. Twist’s platform ensures uniform synthesis, minimizing bias and dropouts in downstream workflows. Libraries are QC’d with NGS, enabling the identification and removal of mutations that do not yield improved functions in subsequent iterations of library design.
User-Defined CDR Libraries
Cloning Option For Libraries
Libraries QC’d with NGS
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The utilization of single-site variant libraries is proving invaluable for researchers aiming to delve into a protein’s sequence space and understand the intricate relationship between sequence, protein structure, and function. This innovative approach allows for a comprehensive exploration of variants, providing crucial insights into the molecular dynamics of proteins.
The construction of Twist Site Saturation Variant Libraries takes protein engineering to the next level. Leveraging advanced massively parallel oligonucleotide synthesis through Twist’s proprietary silicon-based DNA synthesis platform, these libraries ensure a precise and controlled crafting of variants. With complete mastery over codon usage, high uniformity at each site, and rigorous quality control verified through next-generation sequencing (NGS), these libraries offer researchers the flexibility to conduct experiments with ease. Whether opting for one position per well in a 96-well plate or pooling all positions in a single tube, this methodology enables the screening of 1 to 20 different amino acids at each position, further enhancing the adaptability and efficiency of protein engineering studies.
Site Saturation Libraries, particularly those constructed by Twist, revolutionize the exploration of sequence space in protein engineering by eliminating codon bias and preventing unwanted substitutions. In contrast to conventional methods like degenerate and NNK approaches, Twist Site Saturation Variant Libraries (SSVLs) offer a superior solution. Traditional methods, such as error-prone PCR and degenerate approaches, often suffer from poor repetitive yield, resulting in less than 50% full-length product in typical libraries. In comparison, Twist libraries yield more usable variants, significantly increasing the effective library size. The figure depicting the observed distribution of amino acids across 65 positions in a protein illustrates the efficiency of Twist SSVLs in maintaining the expected ratios of variants, showcasing their ability to provide highly uniform variant libraries.
Comparing key features, Twist SSVLs outshine error-prone PCR and degenerate approaches on various fronts. They eliminate sequence bias, utilize all 64 codons, prevent undesirable motifs, allow codon optimization, and avoid stop codons. This comprehensive set of advantages positions Twist SSVLs as a powerful tool for efficient sampling of a protein’s sequence space in screening assays, demonstrating their superiority in precision, reliability, and overall effectiveness in protein engineering studies.
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