The treatment landscape for lymphoma – especially aggressive B-cell lymphomas — has been transformed by adoptive cellular immunotherapy with chimeric antigen receptor (CAR) T cells. Clinical trials with a focus on anti-CD19 CAR-T cell therapy have shown efficacy and long-term remissions in poor-risk cases of diffuse large B-cell lymphoma when no other effective treatment options are available. As adoptive cell therapies raise a lot of interest as therapeutic options against cancer — particularly lymphoma — many of these approaches involve DNA variant library designs and the use of the revolutionary Gibson Assembly® cloning method invented by our Chief Technology Officer, Dan Gibson1,2,3,4.
As a key component in adoptive cellular immunotherapy, nanobodies have been receiving a lot of attention. A recent article from the University of Ghent in Belgium2 discusses an optimized protocol to enhance the process of generating CAR constructs using nanobodies. Despite the clinical success of CAR-modified T cells targeting CD19 for leukemia and lymphoma, toxicities often complicate and limit the benefits and overall effectiveness of CAR T-cell immunotherapy. There is therefore a need for improved designs. To that end, the Gibson Assembly® method was employed to construct novel “nanoCARs,” targeting two clinically relevant antigens, CD20 and CD33. In their paper, the Ghent group reports, “Our technique, using PCR and Gibson Assembly®, allowed us to clone several nanobodies in less than four days…” and enabled the rapid and effective generation of compact CARs2. The design advancements using nanobodies are particularly advantageous compared to the more traditional use of single-chain variable fragments (scFvs) since nanobodies are monomeric structures that will likely not aggregate on the T cell surface, nor induce premature T cell activation and exhaustion. Importantly, they also will not lose affinity, a known side effect in the design of scFvs.
Efforts toward effective CAR design, especially for lymphoma and other tumor types, are a priority. Unfortunately, part of the challenge is that CAR development often involves extensive empirical characterization of antigen-binding domain/CAR constructs for clinical suitability, and designs must limit toxicity. A recent paper from Bloemberg et. al.4 presents a cost-efficient and rapid method for evaluating CARs in human Jurkat T cells using the Gibson Assembly® method. As the scientists note in the publication, “Due to the efficiency of Gibson Assembly® or the single-tube restriction/ligation strategy described here, this protocol was adapted to enable even higher throughput CAR screening.” The group’s high-throughput protocol supported the assessment of CAR-mediated signaling specificity and magnitude in the context of an antigen receptor that is particularly well suited to screening scFvs for their on and off activation properties.
Recent exciting developments have focused on fine-tuning the antigen-binding affinity of a CAR scFv. The process typically involves selecting low-affinity antibody or scFv variants from random or rational libraries and triaging the optimal candidates to a CAR format for further testing (Di Roberto et al.)5. These single-site saturation mutagenesis scanning methodologies will be essential in the development of safe and effective CAR-T cells.
The clinical development of CAR-T cells has undergone exponential growth over the last few years, but much remains to be learned and improved upon for broader effectiveness in a wide range of tumor types. Design strategies to optimize CAR specificity and reduce toxicity will be critical as scientists increase the number of tumor antigens to target. Rapid, high-throughput techniques afforded in part by automating the Gibson Assembly® process — especially in efficient variant library screening using the revolutionary BioXp™ 3250 system — will undoubtedly be effective for accelerating these efforts.
- De Munter, Stijn et al. “Rapid and Effective Generation of Nanobody Based CARs using PCR and Gibson Assembly.” International journal of molecular sciences 21,3 883. 30 Jan. 2020, doi:10.3390/ijms21030883. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7037261/
- Zong, Shan et al. “Very rapid cloning, expression and identifying specificity of T-cell receptors for T-cell engineering.” PloS one 15,2 e0228112. 10 Feb. 2020, doi:10.1371/journal.pone.0228112. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7010234/
- Bloemberg D, Nguyen T, MacLean S, Zafer A, Gadoury C, Gurnani K, Chattopadhyay A, Ash J, Lippens J, Harcus D, Pagé M, Fortin A, Pon RA, Gilbert R, Marcil A, Weeratna RD, McComb S. A High-Throughput Method for Characterizing Novel Chimeric Antigen Receptors in Jurkat Cells. Mol Ther Methods Clin Dev. 2020 Jan 31;16:238-254. doi: 10.1016/j.omtm.2020.01.012. PMID: 32083149; PMCID: PMC7021643. https://pubmed.ncbi.nlm.nih.gov/32083149/
- Di Roberto RB, Castellanos-Rueda R, Frey S, Egli D, Vazquez-Lombardi R, Kapetanovic E, Kucharczyk J, Reddy ST. A Functional Screening Strategy for Engineering Chimeric Antigen Receptors with Reduced On-Target, Off-Tumor Activation. Mol Ther. 2020 Aug 8:S1525-0016(20)30413-5. doi: 10.1016/j.ymthe.2020.08.003. Epub ahead of print. PMID: 32827460. https://pubmed.ncbi.nlm.nih.gov/32827460/