Just as heat-seeking missiles race toward the infrared signatures of their targets, chimeric antigen receptor (CAR) T cells home in on cancer-associated or -specific antigens. Once the antigens are engaged, CAR T cells let fly with cytotoxic flak, granules containing perforin and granzymes, while activating supplementary tumor-killing mechanisms such as stromal sensitization and macrophage polarization. It is to be hoped that by the time the cytotoxic smoke clears, the cancer will have been destroyed.

The development of CAR T cells has revolutionized adoptive cellular therapies against cancer. CARs are genetically engineered to combine antigen- or tumor-specific-binding with T-cell activating domains. T cells, obtained from the patient (autologous cells) or from a healthy donor (allogeneic cells), are typically transduced with an engineered vector, expanded, and infused back into the patient for tumor eradication.

In the 10 years since its inception, the CAR T-cell field has progressed rapidly. Two CAR T-cell products have been approved for clinical use, and many more products are undergoing clinical trials or are in development. Although the field initially focused on B-cell malignancies, it is now advancing on solid tumors.

Despite its initial success, the CAR T-cell field must find ways to generate products that are potent, affordable, and available. To achieve enduring success, the CAR T-cell field is undertaking a range of initiatives. These include the engineering of bridging proteins for multiantigen targeting; the creation of nonviral allogeneic off-the-shelf products; the organization of vein-to-vein networks; and the development of precisely tuned therapies, that is, precisely timed and dosed therapies.

Installing a biological rheostat for precise dosing

“Cellular therapy is a living drug,” declares Steve Shamah, PhD, senior vice president, Obsidian Therapeutics. “As with any drug, damage can occur if the therapy is not carefully regulated. Our company focuses on creating controllable cell therapies by engineering CAR T cells or tumor-infiltrating lymphocytes to produce regulatable cytokines and proteins that can enhance functional activity, especially against solid tumors.”

For example, the company is developing a platform that armors CAR T cells with immunomodulatory factors such as interleukin-15 (IL-15) or CD40 ligand. Shamah explains, “These factors can enhance functional activity by driving T-cell expansion, conferring resistance to immunosuppression, improving antigen presentation, and inducing antigen spread. However, both factors can also produce systemic toxicity. Our technology modulates the level and timing of their activity in a fully controlled, dose-dependent manner using an FDA-approved small-molecule drug.”

cytoDRiVE™ platform
Obsidian Therapeutics has developed the cytoDRiVE™ platform, which allows a drug-responsive domain (DRD) to be fused to a therapeutic protein. The DRD confers instability to the therapeutic protein, which is quickly degraded unless a ligand, a small-molecule drug, is present that can bind to the DRD. The small-molecule drug, which stabilizes the therapeutic protein, may be supplied to the degree desired, when desired, to pharmacologically regulate cell and gene therapies.

The Obsidian platform, cytoDRiVE™, adds a drug-responsive domain (DRD) onto a therapeutic protein of interest. DRD tags are misfolded or inherently unstable in the cell. However, they can be reversibly stabilized by the binding of approved small-molecule drugs. When the drug is absent, the DRD-tagged protein is turned “off.” When the drug is present, the DRD-tagged protein is turned “on.” When DRD tags are in place, the concentration of the small-molecule drug serves as a biological rheostat for controlling the dosing of the therapeutic protein.

Preclinical in vivo mouse studies assessed anti-CD19 CAR T cells that were engineered to express an IL-15-DRD that responded to the FDA-approved drug acetazolamide. In these studies, tumor regression was demonstrated.

“Controlling the precise timing and expression level of these immunomodulatory factors in CAR T cells could significantly enhance safety and therapeutic efficacy,” concludes Shamah.

Obsidian is currently focusing on the oncology space, but the company is also exploring other areas such as autoimmunity and even the regulation of transcription factors to enable controllable in vivo
CRISPR-Cas9 gene editing.

Rescuing CAR T cells in relapsing patients

Despite the remarkable success of CAR T-cell therapies, relapses can occur within six months for up to 50% of patients treated with anti-CD19 CAR T-cell therapy.
Failures can occur due to loss of CD19 expression or to continued tumor proliferation. Aleta Biotherapeutics has developed a novel technology to reactivate CAR T cells in relapsed patients.

CD19-targeting CAR T cells
Aleta Biotherapeutics keeps CD19-targeting CAR T cells in the fight against cancer by overcoming the limitations of these cells, which include suboptimal expansion and the loss of CD19 on the targeted B-cell cancers. Essentially, Aleta creates bridges between the anti-CD19 receptors on the CAR T cells and the tumor-associated antigens on the cancer cells. In this image, the targeted antigen is CD20. Notice that the bridge, which engages CD20 (and which is supplied as an injectable biologic), coats the cancer cell with CD19, thereby optimizing anti-CD19 CAR T-cell cytotoxicity.

“Our approach utilizes antigen-bridging proteins to coat tumors with CD19,” says Paul Rennert, PhD, Aleta’s president and CSO. “[The tumors are then] recognized by the patient’s anti-CD19 CAR T cells, essentially creating a cytotoxic synapse that results in tumor cell death.”

To thwart anti-CD19 CAR T-cell therapy relapses, the company developed a bridging protein using the extracellular domain of CD19 and an anti-CD20 antibody domain. CD20 is an antigen present on the majority of B-cell malignancies. Rennert explains that “these injected bridging proteins will coat the patient’s tumor cells with CD19, creating a target to activate or reactivate a patient’s anti-CD19 CAR T cells.”

To show proof-of-principle, the company performed in vivo studies using a half-life-extended form of the bridging protein injected into mice carrying CD20-positive tumor cells and anti-CD19 CAR T cells. Rennert emphasizes, “Our studies demonstrated this strategy can be used to reactivate CD19 CAR T cells to prevent and reverse relapses.”

Other programs in development include a bridging protein for injection to improve outcomes in multiple myeloma patients treated with CAR T cells, and bridging protein programs for HER2-positive breast cancer patients with central nervous system metastases. The company is preparing investigational new drug applications for its technology and plans to start Phase I trials in 2021.

Using spatial transcriptomics to assess tumor infiltration

Assessing whether engineered CAR T-cell and T-cell receptor (TCR) therapies have successfully attacked and penetrated solid tumors (and not normal cells) can be like finding the proverbial needle in the haystack. “Traditional methods using immunohistochemistry are useful for immune profiling, but they cannot differentiate engineered versus endogenous cells,” points out Christopher Bunker, PhD, senior director of business development, Advanced Cell Diagnostics, a Bio-Techne brand. “We developed a means to easily detect and track engineered therapeutic cells and delineate their pharmacokinetics within the tumor microenvironment of intact tumor biopsies, as well as their on-target/off-tumor activity.”

Enter RNAscope®, an RNA in situ hybridization technology that can enable single-cell spatial transcriptomics. RNAscope, Bunker asserts, is “the only off-the-shelf method that can specifically detect engineered CAR T cells and TCR T cells in solid tumor patient biopsies.”

RNAscope, a single-cell spatial transcriptomics platform developed by Advanced Cell Diagnostics, can detect T cells that express chimeric antigen receptors (CARs) and T cells that express engineered T-cell receptors (TCRs) in solid tumor patient biopsies. The technology utilizes pools of paired oligos (ZZ pairs) that hybridize to the target mRNA and lead to signal amplification. Individual transcripts appear as visible and quantifiable dots.

Most cell therapies employ lentivirus transduction. Because CAR or TCR transgenes have unique sequences in the viral untranslated regions, these can be used as tags for identification of engineered cell therapies with RNAscope probes. The technology utilizes pools of paired oligos that can be thought of as a “ZZ” pair, where the paired 3′ ends hybridize to ~50 bases of target mRNA, and where the paired 5′ ends hybridize to a signal amplification module, which is built through sequential hybridization steps. The signal amplification of paired oligos results in an assay able to detect individual transcripts that appear as visible and quantifiable dots.

“It’s a little like planting and lighting Christmas trees,” quips Bunker. “The ZZ pairs plant trees along the mRNA with branches that are decorated either with fluorophores or chromogens.” Although the primary technology currently features four colors, the company has developed a HiPlex (12-plex) assay and foresees even higher-plex assays with different detection methods.

“We envision assays based on our core technologies that enable spatial analysis of perhaps a hundred transcripts in combination with tens of proteins,” Bunker projects. “In the context of cell therapy development, these will enable a more comprehensive understanding of tumor biology and immune cell profiles to determine the most effective treatment strategy for a patient, as well as for monitoring efficacy of solid tumor cellular therapies.”

Choosing T-cell media

Companies developing CAR T-cell products are also eyeing a future involving GMP production. Thus, a critical early question is how to choose the best T-cell medium for manufacturing processes. To test the suitability of a CAR T-cell growing medium, companies must assess factors such as cell viability, cell expansion, cytokine profiles, and cell purity. A medium suitable for a CAR T-cell manufacturing process also needs to support rapid activation and CAR transduction. Additionally, the selected medium needs to be compatible with a variety of donors.

There are many available choices for T-cell culture media ranging from do-it-yourself recipes to commercially available one-size-fits-all complete formulations. CellGenix has developed a novel T-cell medium that avoids the use of human serum. Sebastian Warth, PhD, a senior scientist at CellGenix, explains, “To achieve consistent results, human serum requires extensive testing prior to its use for production of cellular products due to lot-to-lot inconsistencies. Since human serum is a limited resource and might not be available in large quantities, it is unfavorable for commercial-scale manufacturing. Furthermore, the human origin of serum poses a certain risk of containing adventitious agents and is, therefore, a risk to the safety of the T-cell therapy product.”

The company’s TCM GMP-Prototype medium provides a serum-free and xeno-free product for early-onset T-cell expansion. According to Warth, key advantages include “promotion of sustained viability, support for expansion of CD4+ and CD8+ T cells, promotion of a central memory and early differentiated memory T-cell phenotype, and maintenance of a high proportion of cytokine-producing cells including polyfunctional cells. Further, it was optimized for and verified with CAR T cells.”

Generating off-the-shelf allogeneic T cells

While autologous CAR T-cell therapies have proven highly successful, they also require a long and expensive manufacturing process. The dream of being able to utilize off-the-shelf allogeneic T cells is on the horizon.

Devon J. Shedlock, PhD, senior vice president, research and development,
Poseida Therapeutics, reports, “With our technology, we are able to genetically modify cells to create a fully allogenic, or off-the-shelf, product that does not require additional immunosuppression treatment like earlier generation approaches. We also have developed technology to allow us to make hundreds of doses from a single manufacturing run from healthy donors, thereby dropping the cost substantially.”

According to Shedlock, the technology consists of three key aspects: 1) the piggyback® DNA Modification System, 2) the Cas-CLOVER™ site-specific gene editing system, and 3) the Booster Molecule.

The PiggyBac DNA Modification System is a nonviral technology for stably integrating genes into DNA. One key feature is that piggyBac preferentially inserts into less mature T cells, enabling the production of therapies that have a high proportion of stem cell memory T cells, or Tscm cells.

“Viral technologies are virtually excluded from Tscm cells,” Shedlock states. “However, Tscm cells are the ideal cell type for cell-based therapies because they have the ability to engraft and potentially last a lifetime, can produce wave after wave of more differentiated cells to attack the tumor, and are much more tolerable with low levels of adverse events compared to other CAR T-cell products.”

The company’s Cas-CLOVER gene editing technology is a hybrid gene editing technology used to edit the T cells to make allogeneic products. “Cas-CLOVER works well in resting T cells, which is important in preserving Tscm cells in a fully allogeneic CAR T-cell product,” Shedlock elaborates. “It also is a very precise and clean system. This is a particularly important safety issue for allogeneic products that may be given to many patients.”

The Booster Molecule is added during manufacture and is temporarily expressed on the cell surface to allow cell stimulation. Normally when allogeneic CAR T-cell products are created, the T-cell receptor must be eliminated to avoid the graft-versus-host reaction, which is a major safety issue. Importantly, this booster stimulation occurs while preserving the Tscm phenotype.

Poseida Therapeutics expects to launch a clinical trial for its multiple myeloma allogeneic product late this year or early next year. The company will also begin clinical trials later in 2021 on its pan-solid tumor allogeneic program.

Vein-to-vein networks

Creation of partnerships can help drive development of CAR T-cell therapeutics from concept through clinical trials. “Advanced therapies require advanced supply chain and data management,” advises Minh Hong, PhD,  head of autologous cell therapy, Lonza Pharma & Biotech. “Prior biopharmaceutical models of mass production and distribution—and the systems that support them—are not effective for personalized therapeutics. As manufacturing demand increases for autologous cell therapies, there is an overarching need to both industrialize and simplify the entire supply chain ecosystem.”

Hong says the overall project needs to be considered from a more comprehensive perspective: “Due to the criticality of the starting material, everything from cell sourcing, patient coordination and scheduling for collection/infusion, transportation logistics, and manufacturing logistics needs to be coordinated, ensuring the highest standards, regulatory compliance, and safety throughout the process.”

To meet these needs, Lonza is building a network of partners to develop a fully integrated vein-to-vein solution, that is, a system that includes all touch points involved in patient scheduling and sample collection, through material shipping logistics, manufacturing, and eventually the infusion of the cell therapy back into the patient. The partner network, Hong indicates, will help participants define smart workflows and execute an integration strategy. Hong sums up the network’s therapeutic implications as follows: “We believe these partnerships will decrease time to clinical program setup.”

Lonza has more than a 20-year history of providing clinical and commercial manufacturing. Hong asserts, “Our company brings to the table our process development and manufacturing experience along with proprietary solutions including a manufacturing execution system solution, MODA-ESTM, for electronic batch records and manufacturing traceability. In addition, we have announced partnerships with Vineti for a supply chain orchestration system and Cryoport to aid in shipping and logistics.”

Lonza is also looking beyond CAR T-cell therapies. “We would not limit our solutions and partnerships to autologous cell therapies,” Hong declares. “We can envision solutions for our in vivo viral vector manufacturing clients as well as our traditional allogeneic cell therapy clients.”

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