GenScript 2^nd Annual Gene & Cell
Engineering Virtual Summit

New Innovations in Cell Therapy

Dr. Fang provided an overview of the progress in therapeutic modalities from small molecules to biological or live therapies such as cell therapies. Additionally, he offered a perspective on the complexities of developing cell therapies, which require an expansive toolbox, such as engineered antibodies, signaling modules, and immune response enhancing modules. Furthermore, cell therapies may be implemented through autologous or allogeneic approaches and may also involve engineering immune cell types beyond T cells, such as NK cells. Lastly, manufacturing at scale and under GMP conditions is a critical and challenging aspect of developing live therapies.

Legend Biotech has a robust preclinical and clinical pipeline for cell therapy development targeting hematological malignancies and solid tumor indications. Its autologous CAR T-cell therapy cilta-cel or Carvykti (ciltacabtagene autoleucel) recently received FDA approval for use in previously treated, relapsed, or refractory multiple myeloma patients. Legend’s core technology platforms enable the development of autologous and allogeneic cell therapies based on a broad range of lineages, including αβT, NK, and γδT cells. CAR design is VHH-based, which provides several advantages, such as improved expression and stability, enhanced access to complex epitopes, engineering flexibility enabling multi-epitope targeting, and enhanced immune synapse formation. Cilta-cel's design is based on two VHH domains targeting BCMA antigens in B plasma cells, conferring improved avidity. Lastly, Dr. Fang discussed the difficulties in targeting solid tumors and how Legend Biotech is approaching the challenges to cell therapy imposed by immunosuppressive and physical barriers to cell infiltration, among others.

CAR T cell therapy - The long-sought cure for cancer

Dr. Melenhorst shared clinical work done in collaboration with Dr. Carl June’s team at the University of Pennsylvania involving the dosing of chronic lymphocytic leukemia (CLL) patients with autologous CD19 targeted CAR T cells over a decade ago. CLL primarily affects males and accounts for 25% of all newly diagnosed leukemias. Several therapies for CLL include small molecule cell signaling inhibitors and antibody-based therapies, but these are not curative. In contrast, cell therapies such as CAR T cells targeting CD19 have led to significant tumor regression. Positive clinical outcomes have correlated with CAR T cell expansion in peripheral blood and marrow and concomitant enhanced functionality, confirmed by elevated levels of cytokines such as IL6 and IFN-gamma. In some study participants, which remain in remission, CAR T cells have persisted and are detectable even after four years post-treatment. In addition, a higher proportion of early memory T cells with a CD8+, CD27+, and CD45RO- phenotype correlate well with CAR T cell efficacy and induced remission. These findings have helped develop biomarkers for CAR T cell therapy efficacy in CLL. To this date, Melenhorst’s team continues to follow study participants to understand the genetic and immune-related basis for long-term CAR T cell persistence. Overall, long-term remission in CLL patients correlates with the persistence of a CD4 CAR T population having a restrained activation phenotype.

Development of a T cell engineering platform for fully-individualized neoantigen-specific TCR therapy

Neogene leverages its T cell engineering platform to develop personalized T cell therapies. Their workflow starts with neoantigen identification, for which Dr. Kong’s team relies on analyzing patient-derived tumor samples. Neoantigens are tumor-specific novel antigens derived from cumulative mutations common in carcinogenesis. Patient-derived tumor samples also enable the team to identify the TCR sequences of neoantigen binding T cells in parallel. Once neoantigen-TCR pairs are elucidated, autologous T cells expressing neoantigen-specific TCRs are engineered for personalized immunotherapies.

To engineer T cells, Neogene leverages CRISPR tools and non-viral gene delivery strategies using DNA templates. Specifically, the team electroporates the Cas9/guide RNA ribonucleoprotein complex together with DNA templates for precise TCR knock-in editing, achieving ~24% mean editing efficiency. Nevertheless, editing efficiency may vary from 5-40% in individual experiments. Thus standardization of editing efficiencies was highlighted by Dr. Kong as a challenge to overcome for successfully delivering engineered T cells to the clinic. To standardize and maximize editing efficiency, his team has developed a drug-based selection strategy to enrich edited T cells in vitro. Lastly, securing a reliable source of GMP-grade DNA repair templates is critical for developing cell therapies, representing another significant challenge in manufacturing cell therapies. Therefore, Neogene has entered an agreement with GenScript to develop GMP-grade linear closed DNA repair templates (GenWand™) to enable cell therapy manufacturing activities. Dr. Kong found that a high T cell editing efficiency (~80%) could be achieved by using these dsDNA repair templates, following edited T cell enrichment as per their established drug-based selection strategy.

Polymeric nanoparticles for non-viral gene delivery

Viral vectors such as retroviruses, adenoviruses, and adeno-associated viruses (AAVs) are commonly used at the clinic in gene and cell therapy approaches due to their high transduction efficiency. Nonetheless, non- viral alternatives for gene delivery are desirable to avoid potentially severe outcomes associated with the use of viral vectors. Additionally, viral vector payload capacity and production cost limitations further highlight the need for alternative gene delivery approaches. Among different synthetic vehicles developed to date, lipid nanoparticles (LNPs) have shown significant success in delivering vaccine products, such as the recent mRNA-based vaccines developed against SARS-CoV-2.

LNPs offer several advantages, such as targeting flexibility based on surface chemistry and large payload capacity. Nevertheless, to bypass some of the limitations associated with LNPs, including instability and high production cost, Dr. Zhou’s research has focused on developing strategies for polymer-based gene delivery.

Synthetic polymers that either condense or encapsulate genetic material for delivery may be based on the use of polylysine (PLL) or polyethylenimine (PEI). These polymeric nanoparticles are positively charged and easily traverse the cell membrane but also induce toxicities. Dr. Zhou’s team has been developing strategies to curve associated toxicity and improve uptake of polymeric nanoparticles, thus expanding their potential for in vivo gene delivery. For example, his team has been modifying polymeric nanoparticles, such as Poly(lactic-co-glycolic-acid) PLGA-based nanoparticles, by addition of different moieties that facilitate cellular transport, endosomal escape, and tissue targeting, thus improving gene delivery efficiency. Lastly, Dr. Zhou has also synthesized various poly(amine-co-ester) polymers for gene delivery applications.

Discovery of a novel C*07:02-restricted epitope on MAGE-A1 and pre-clinical development of an enhanced TCR-T cell therapy candidate for the treatment of solid tumors

One of the challenges in solid tumors is the scarcity of therapeutic targets identified to date. Additionally, targeting tumor-associated antigens (TAAs) in solid tumors may lead to toxicities affecting the safety of TCR-T cell therapies. Therefore, as part of TScan Therapeutics, Dr. Wang is developing a proprietary genome-wide screening platform, TargetScan, which facilitates identifying cognate TCR antigens.

In the TargetScan platform, an extensive library covering the entire human proteome, organized as overlapping 90 mer peptides, may be screened for TCR binding in vitro. In addition, beyond non-mutated sequences, the library includes sequences for retroviruses, non-coding ORFs, and shared neoantigens, among others, which provides more opportunities to identify clinically relevant targets.

In one approach, Dr. Wang leverages this extensive library to screen T cells derived from patients responding to check-point inhibitor treatment, thus enabling the discovery of TCR-antigen pairs with therapeutic value. For example, by screening T cell repertoires from head and neck cancer patients responding to combined treatment with Ipilimumab and Nivolumab, Dr. Wang’s team has identified MAGE-A1 and SHH as targets that may hold therapeutic potential. MAGE-A1’s expression in most normal tissues is limited, while it’s highly expressed in melanoma, cervical, and head&neck tumors, providing new opportunities for developing new TCR-T cell therapies. Through preclinical in vitro and in vivo studies, Dr. Wang has shown that TCR-T cells targeting the identified MAGE-A1 epitope become activated, leading to cytotoxic activity and tumor regression.

Physicochemical and Functional Characterization of Different Engineered Cas9 variants and CRISPR-Cas9 Ribonucleoprotein (RNP) Complexes.

In his talk, Dr. Camperi discussed strategies involving liquid chromatography and capillary electrophoresis (CE) based methods leveraged by his group at Genentech to evaluate engineered Cas9 variants and ribonucleoproteins (RNPs). Engineering of Streptococcus pyogenes (Sp) Cas9 aims to resolve some of the challenges associated with undesirable activities limiting clinical applications, such as off-target cleavage. Nevertheless, high-quality characterization methods to meet regulatory requirements are needed. To this end, at Genentech, scientists have developed a strategy that relies on CE-SDS to assess the purity and stability of Cas9 variants. Additionally, in vitro DNA cleavage assays allow scientists to evaluate the functional properties of engineered Cas9 proteins. Lastly, mass spectrometry enables the team to elucidate protein heterogeneity resulting from different post-translational modifications (PTMs), supporting batch-to-batch consistency. Particularly to meet regulatory requirements, verifying the Cas9 protein’s identity is critical, which they achieve through peptide mapping assays.

Characterization of RNP complexes requires evaluating different factors for the protein and guide RNA components. For example, Dr. Camperi shared that Cas9 is analyzed for PTMs and aggregation while guide RNAs are assayed for the impact of aggregates and secondary structures. Nevertheless, an approach to evaluate the effects of each component on RNP complex formation was missing, which the team has addressed through new HPLC methods for physicochemical and functional characterization. Overall, these strategies enable Dr. Camperi and colleagues to identify critical factors that may help optimize the formation of functional RNP complexes to support improved editing activity.

Rapid, gentle, efficient and scalable cell engineering with microfluidic vortex shedding and Hydropore

Indee Labs is the developer of Hydropore, a microfluidics-based device enabling more effective engineered cell therapies. This technology rivals more commonly used approaches such as viral transduction and electroporation, each having critical disadvantages such as high cost and low yield of modified cells. Hydropore is a simple instrument that effectively and gently enables genome engineering.

Currently, Indee Labs is leveraging its Hydropore technology to develop cell therapies for autoimmune diseases and cancer. Hydropore works by running cells together with editing constructs and complexes through a vortex, which permeabilizes the cellular membrane and enables the transit of RNA, DNA, and proteins into cells. The platform has been validated for engineering various cell types, including PBMCs, enriched T cells, NK cells, and commonly used cell lines. Head-to-head comparison against other technologies, such as electroporation, revealed that while Hydropore may lead to lower editing efficiency, cells have a better recovery and greater viability. Thus overall, it enables a better yield of edited cells. Indee Labs collaborates with various academic institutions to validate Hydropore as an effective technology to support developing functional CAR T cells while reducing manufacturing timelines.

TRACeR: Antigen-centric MHC recognition by a novel engineered protein platform

Presentation of antigens by Mayor Histocompatibility Complex (MHC) class I and II molecules is a central step in adaptive immune responses. Conventional T cell antigen recognition relies on the interaction between T cell receptors (TCRs) with antigen peptides loaded onto MHC molecules. Nevertheless, TCRs bind with low affinity and specificity to MHC-loaded antigen peptides. Enabled by protein engineering approaches, Dr. Huang’s lab has developed a new platform for antigen-centric MHC recognition. Rather than engineering TCR mimetic antibodies, his approach aims to modify superantigen proteins, which naturally interact with TCR and MHC molecules and activate CD4+ T cells.

Dr. Huang’s team is developing a TCR-like binder able to recognize antigens in an MHC class-independent manner. Leveraging computational approaches, he has identified superantigen protein designs enabling access to both MHC classes and peptide antigens. Phage display screens of superantigen variants, constructed through the use of GenScript’s precision mutant libraries, enabled Dr. Huang to test their designs and identify superantigen variants that bind specifically to different antigens, such as NY-ESO-1, EBV, and SARS-CoV-2 Spike protein. This new TRACeR platform would enable developing new specific binders of TCR antigens for various therapeutic applications and modalities, such as drug conjugates, Fc fusions, and CAR-T cells.