Peptide-MHC complexes (pMHCs) are central to essential immune decisions, including recognition of infection, autoimmune and tumor eradication. After thymic selection, T cell receptors (TCRs) are the natural receptors that can achieve pMHC recognition, but TCR or other antibody-based TCR mimetic engineering for an arbitrary pMHC remains a challenge. Here we create a novel platform to recognize pMHC. The novel scaffold will simplify the process of creating specific pMHC binders, as the designs will have only one variable loop and no longer require the involvement of six loops like TCRs. We combine computational protein design with experimental high throughput screen techniques to achieve highly specific pMHC recognition of tumor, viral and autoimmune antigens to single-residue mutation sensitivity. This novel platform can potentially be a game changer for new theranogstics development.

• Novel platform for pMHC recognition
• Computational Protein Design combined with high-throughput screening for target specificity

Hydropore is an intracellular delivery platform based on microfluidic vortex shedding. Microfluidic vortex shedding relies on micron-scale posts and high fluid velocities to create hydrodynamic forces within a microfluidic device – these forces are capable of porating the cell membrane allowing for intracellular delivery. The Hydropore platform can be used to rapidly, gently and efficiently deliver nucleic acids and gene editing complexes to a variety of cell types such T cells and regulatory T cells. Additionally, intracellular delivery with Hydropore frequently requires significantly less construct per cell than electroporation and other nascent intracellular delivery platforms.

Genome editing T cells with Cas9 RNP and AAV typically results in >50% knockout and >20% knockin efficiencies. Editing efficiencies can be significantly improved by using homology-directed repair enhancers. Alt-R, for example, increased knockin efficiency from 24.1% to 51.5%. Hydropore-edited T cells also had a >4-fold higher initial cell viabilities when compared to Nucleofection. These very high initial cell viabilities (ie. 1-3 hrs post-processing) enhance the proliferation of Hydropore-edited T cells – a 1.6~1.9-fold increase in knockout and knock-in genome edited T cells was observed by day 7. Cumulatively, this means that Hydropore-edited cells also have improved function – a statistically significant increase in persistence or reduced activation induced cell death was observed within 6 hours for CD19 CAR-T cells. Hydropore has also shown to improve the modification of less frequent or rare cell populations. HLA-A2 CAR mRNA can be delivered to primary human regulatory T cells with >99% efficiency and 95% viability while using 3.8-fold less CAR mRNA per cell. Importantly, the same Hydropore instrument and device can be used to process between 1 and 100 million cells in less than 30 s providing a straightforward path from discovery-stage research to early stage translation.

Hydropore development is ongoing and future work is focused on scaling up and out the Hydropore platform for cell therapy while further verifying the improved function of Hydropore-edited cells in vivo. Ongoing Hydropore platform development is done in collaboration with a variety of pharmaceutical companies, biotechnology startups and academic institutes like Stanford, UC San Francisco and the Medical University of South Carolina.

• Hydropore can be used for the rapid, gentle, efficient and scalable cell engineering often with lower reagent requirements than electroporation and other nascent delivery platforms
• Hydropore improves the in vitro persistence of genome edited, CD-19 CAR-T cells

Advances in gene-editing technology enable efficient, targeted ex vivo engineering of different cell types, which offer a potential therapeutic platform for most challenging disease areas. CRISPR (clustered regularly interspaced short palindromic repeats)-associated proteins (Cas) are powerful gene-editing tools used in therapeutic applications.

The quality of gene-editing reagents (i.e., Cas9 nuclease, single guide (sg)RNA) is associated with the final cellular product quality as they can impact the gene-editing accuracy and efficiency. Efforts to minimize off-target cleavage by CRISPR-Cas9 have motivated the development of engineered Cas9 variants. The wild-type (WT) Streptococcus pyogenes (SpCas9) has been engineered into a high-fidelity Cas9 (SpyFi Cas9) that shows promising results in providing high on-target activity (targeting efficiency) while reducing off-target editing (unwanted mutations). For the first time, we developed ultra-high-performance liquid chromatography (UHPLC) and capillary electrophoresis (CE)-based methods for a full characterization of different engineered Cas9 variants, including determination of purity, size variants, isoelectric points (pI), post-translational modifications (PTMs), and functional activities. The purity and size variant characterization were first determined by CE-sodium dodecyl sulfate (SDS). An in vitro DNA cleavage assay using an automated electrophoresis tool was employed to investigate the functional activity of ribonucleoprotein (RNP) complexes derived from Cas9 variants. The pIs of the engineered Cas9 proteins were determined by imaged capillary isoelectric focusing (icIEF), while intact mass measurements were performed by reversed-phase (RP)-UHPLC coupled with high-resolution mass spectrometry (HRMS). A peptide mapping assay based on LC-UV-MS/MS using endoproteinase Lys-C under non-reducing conditions was developed to confirm amino acid sequences, allowing differentiation of SpyFi Cas9 from WT SpCas9.

To assess the impact of the quality of Cas9 protein and sgRNA in the formation of a Cas9 RNP complex, stability, and functional activities, we then developed a size exclusion chromatography method that utilizes multiple detectors and an in vitro DNA cleavage assay using anion-exchange chromatography. Using these methods, we characterized the formation and stability of Cas9 RNP complexes associated with Cas9 and sgRNA characteristics as well as their functional activities. Multi-angle light scattering characterization showed different types and levels of aggregates in different source sgRNA materials, which contribute to form different Cas9 RNP complexes. The aggregations irreversibly dissociated at high temperatures. When the Cas9 RNP complexes derived from non-heated and heated sgRNAs were characterized, the data showed that specific RNP peaks were impacted. The Cas9 RNP complexes derived from the heated sgRNA retained their biological function and cleaved the double-strand target DNA at a higher rate. This work provides insights into sgRNA properties and handling procedures to better control the Cas9 RNP complex formation.

• LC- and CE-based methods can help establish specifications for these modified Cas9 variants that would meet the requirements of regulatory agencies.
• Understanding the factors that impact the Cas9 RNP complex formation could provide valuable clues to better control gene editing for different applications.

Immune checkpoint therapy is an effective strategy to enhance the anti-tumor activity of T-cells and has led to significant and durable responses in a subset of cancer patients. Identification of the T-cell receptors and relevant cancer antigens that drive these positive anti-tumor responses are of great interest as they could then be developed into TCR-T cell therapy candidates, thus extending these anti-tumor benefits to patients that share the same tumor antigens.

Using a proprietary genome-wide screen termed ‘TargetScan’, we interrogated patient-derived TCRs that underwent clonal expansion in response to immune checkpoint therapy. In one screen in which ten expanded TCRs obtained from a Head and Neck cancer patient who exhibited substantial tumor reduction were evaluated, a cancer testis antigen, MAGEA1, was identified as a target. Deconvolution studies revealed that two distinct TCR clonotypes recognized the same novel C*07:02-restricted epitope, suggesting that a polyclonal response to MAGEA1 may have contributed to the tumor reduction of this patient. Engineered T-cells expressing each TCR exhibited significant activation, cytokine production and cytotoxicity against a panel of HLA-C*07:02-positive cancer cell lines expressing variable levels of MAGEA1.

To ensure these TCRs do not exhibit problematic off-target recognition, each TCR was individually screened against a genome-wide library comprising sequences from every protein in the entire human proteome, identifying TCR-41 as a suitable TCR for clinical development. Additional safety assessments revealed no allo-reactivity relative to a panel of 110 HLA-I molecules, and no discernable recognition of a diverse collection of C*07:02-positive primary normal cells. To further enhance the activity of T cells engineered with this TCR, a transposon-based vector was developed that delivers the TCR gene, along with the genes for CD8 and a dominant-negative form of TGFbR2, into both CD4+ and CD8+ T cells. This therapeutic candidate, term TSC-204-C07, has been advanced to IND-enabling studies to prepare for clinical testing. Overall, these results substantiate the approach of using TargetScan to identify relevant tumor antigens and TCRs from the expanded T cell repertoires of patients exhibiting exceptional responses to immune checkpoint blockade.

• By interrogating cancer patients who are responding to immune checkpoint blockade therapy, we successfully identified TCR and relevant antigens that drive positive clinical response. One such TCR is a high-affinity TCR against an HLA-C07:02 restricted MAGE-A1 epitope. This TCR demonstrates robust cancer cell killing, minimal off-target and allo-reactivity effects, and is under IND-enabling studies for future clinical testing.

Gene therapy holds promise for treating many diseases. However, clinical translation of gene therapy has been limited by the lack of approach for efficient and safe delivery of genetic materials to disease locations. In current clinic, viral vectors are often used because of their high delivery efficiency. Unfortunately, long-term safety of viral vectors remains as a concern. To overcome this problem, development of non-viral vectors using synthetic nanoparticles (NPs) is promising. Compared to viral vectors, synthetic NPs have limited immunogenicity and considerable flexibility for surface engineering. The recent success in use of lipid NPs (LNPs) for delivery of COVID-19 mRNA vaccines highlights the potential of non-viral vectors. Nevertheless, LNPs are limited by thermal instability, inability to prolong gene expression, and high complexity with 4-5 components. For several years, our group has focused on development of simple formulation polymeric NPs for gene delivery. In a recent effort, we synthesized a family of poly(amine-co-ester) (PAE) NPs through polymerization of diethyl sebacate (DES) and N-methyldiethanolamine (MDEA) with lactones. This synthetic approach is unique in that it allows tuning four important parameters in one single molecule: positive charge, molecular weight, hydrophobicity, and solidity. We demonstrated that the gene delivery capacity of polymers is not solely determined by charge, but by a balance of positive charge, molecular weight, and hydrophobicity. As a result, we can design and synthesize polymers for gene delivery with high efficiency but limited toxicity. More recently, we further optimized the chemistry and synthesized a new generation of PAEs containing stimuli responsive components, which can form solid NPs. We demonstrate that, in addition to their high efficiency, the NPs have several major important features, including: 1) remarkable stability, allowing them to be stored as lyophilized powder without compromising efficiency; 2) capacity to produce sustained gene expression over a longer time than LNPs; and 3) simplicity, consisting of one polymer, in contrast to 4-5 components for LNPs. Those features make the new generation PAE NPs ideal for clinical translation.

• Discussion of synthesis and application of a new generation of polymeric nanoparticles that are optimal for gene delivery and clinical translation.

Adoptive transfer of T cell receptor (TCR)-edited T cells to target cancer neoantigens is a promising new immunotherapy approach. We have developed a platform that can identify neoantigen-reactive TCRs in a high-throughput and patient-specific manner. To incorporate these TCRs into a clinical T cell product, we have simultaneously developed an engineering process that uses CRISPR gene editing to introduce neoantigen-reactive TCRs into the endogenous TCRα locus of a patient’s own T cells, with TCR knock-in efficiencies of >40% being achieved. However, editing efficiencies were highly variable across donors, which may limit therapeutic efficacy following clinical implementation.

To address this, we have developed a novel drug-based selection method by incorporating a mutant dihydrofolate reductase (mDHFR) gene into the TCR DNA repair template and subsequently selecting cells with methotrexate, a cytostatic drug that inhibits wildtype but not mDHFR. With only three days of methotrexate treatment, efficient enrichment of TCR-edited T cells to an average purity of 80% was achieved across donors. Notably, TCR expression in the methotrexate-selected mDHFR–expressing T cells was comparable with unselected control cells, and further analysis revealed equivalent proliferation, cytokine secretion and tumor cell killing capacities.

In parallel, to identify and track successfully edited T cells, we aimed to characterize the minimal epitope of the anti-murine TCR Cβ antibody H57 and engineer it into human TCRs. We identified 10 murine amino acids as potential antibody contact sites, which after incorporation into human TCR Cβ enabled H57 antibody binding. Subsequent swapping of the individual residues back to their human counterpart revealed the minimal epitope, which required only 6 murine residues (Mur6). Engineering of the Mur6 epitope into human TCR Cβ facilitated detection of TCR-edited T cells without impacting TCR expression or function.

We have applied TCR gene editing incorporating the Mur6 and DHFRMUT tools for clinical scale manufacturing of multi-specific T cell products that will be utilized in an upcoming clinical study of fully individualized neoantigen-specific TCR therapy.

Taken together, we have developed a novel T cell engineering process that enables efficient selection and tracking of TCR-edited T cells that will be utilized for T cell product manufacturing in an upcoming clinical study of fully individualized neoantigen specific TCR therapy.

• Incorporation of a Mur6 epitope in the β-chain constant domain of the introduced TCR enables quantification of TCR-edited T cells in T cell products and tracking of these cells following infusion.
• Incorporation of a DHFRMUT into the TCR-encoding DNA repair template enables efficient enrichment of TCR-edited T cells with MTX and generates T cell products with enhanced cytokine production and tumor control capacities.
• Application of TCR gene editing to incorporate the Mur6 and DHFRMUT tools for clinical scale manufacturing of multi-specific T cell products that will be utilized in an upcoming clinical study of fully individualized neoantigen-specific TCR therapy.

Enticing the immune system to regain control of a patient’s malignancies has remained a seemingly unsurmountable challenge until discoveries on T cell costimulation and -inhibition provided the critical insight to overcome passive and active immune resistance by cancers. Equipping a patient’s T cell with the ability to target tumor cell lineage-specific surface proteins using an engineered antibody grafted onto T cell signaling domains was truly innovative, as was the provision of costimulation in cis in some of the final designs of chimeric antigen receptors (CAR). Second generation CARs thus designed in the mid 2000s and clinically evaluated by the end of that decade have been transformative and such designs have since delivered on a cure in acute and chronic B cell leukemias as well as in lymphomas. However, much remains to be done. Areas still with a high, unmet need of a cure are myeloma, T cell malignancies, and the many solid tumors. In my talk I will highlight the progress made in CLL and look forwards future developments in this domain.

• Chimeric antigen receptors (CAR) went in rapid succession from research tool to means of fighting cancer
• Only with second generation CARs did we start seeing durable remissions and even cures in B cell cancers
• The road ahead will tell us whether this same technology will tell us whether this technology will be similarly transformative in other areas of unmet need

CAR-T cell therapy is a form of combined gene and cell therapy that has revolutionized cancer treatment. Remarkable efficacy and manageable safety profile have been observed by several FDA-approved cell therapy products against blood cancers, including cilta-cel, an autologous CAR-T cell therapy that targets BCMA. Cilta-cel is a structurally differentiated CAR-T cell therapy containing a 4-1BB (CD137) co-stimulatory domain and 2 BCMA-targeting single-domain antibodies designed to confer avidity. The unprecedented clinical response suggests that cilta-cel has the potential to become the best-in-class BCMA-targeted CAR-T cell therapy for the treatment of MM. With the success of cilta-cel, we have built fully-integrated in-house R&D capacities encompassing drug discovery, pre-clinical studies, translational research, manufacture, and clinical development to efficiently generate and advance a broad portfolio of Phase 1 and earlier stage product candidates targeting various solid and hematologic tumor types that address significant unmet needs among patients. Key components of our research and development capacities include in-house antibody discovery, CAR design and screening, innovative disease-tailored armors, multiple allogeneic platforms, robust CMC manufacture and global clinical development. In this presentation, I will share our integrated R&D capacities and highlight several of our autologous cell therapy assets, LB1908, LB2101, and LB2102 to illustrate our recent progress in targeting solid tumors.

• Review current progress in CAR-T cell therapy approaches against hematological malignancies, including efficacy and adverse events.
• Discuss Cilta-cel as a novel B-cell maturation antigen (BCMA)-targeting autologous CAR T-cell therapy for patients with relapsed/refractory multiple myeloma and unique structural design.
• Indicate the current limitations of CAR-T cell therapies in solid tumors and strategies that circumvent their challenging landscape.