The Promise of In Vivo CAR T Cell Therapy

CAR T cell therapy has revolutionized the treatment of relapsed/refractory (r/r) B cell malignancies by utilizing synthetic receptors to redirect T cell function towards tumor surface antigens. These receptors, composed of a targeting moiety typically derived from antibodies, fused with intracellular CD3ζ and co-stimulatory domains, have shown remarkable efficacy, particularly in hematologic cancers such as leukemia, lymphoma, and myeloma. The versatility of CAR molecules extends beyond oncology, with potential applications in autoimmune and infectious diseases.

Despite the therapeutic promise, challenges exist in implementing ex vivo manufactured CAR T cell products on a large scale. Current manufacturing practices involve labor-intensive processes requiring specialized facilities and skilled personnel. Viral transduction methods are commonly employed to modify patient autologous T cells, leading to variability in product quality and yield. Additionally, the time between T cell harvest and infusion can be lengthy, necessitating bridging therapies for rapidly progressing patients.

The cost of CAR T cell therapy is substantial, with list prices exceeding USD $500,000 per patient, not including additional procedures such as leukapheresis, lymphodepleting chemotherapy, and response assessments. The complexity of manufacturing and the associated financial burden limit scalability and pose barriers to accessibility and equity.

Allogeneic CAR T cells, derived from healthy donor T cells, offer a potential universal strategy to overcome these challenges. However, mitigating graft-versus-host reactions and host-versus-graft reactions requires complex genetic engineering maneuvers. Approaches such as virus-specific T cells, gene editing to remove the T cell receptor, and alternative non-TCR expressing lymphocytes have shown promise. To avoid host-versus-graft reactions, strategies like broad HLA-matched donor banks and disruption of genes involved in MHC expression have been explored.

Despite progress, unresolved issues remain, and clinical results have sometimes been limited. Furthermore, allogeneic CAR T cell therapy still requires lymphodepletion, which can lead to prolonged cytopenias and infectious complications.

Innovative solutions are needed to address the challenges of CAR T cell therapy manufacturing and implementation. Two promising technological advances include nanoparticles encapsulating CAR encoding nucleic acid and viral vectors encoding the CAR, which can be infused directly into patients, potentially providing a universal off-the-shelf product. These advancements have the potential to streamline the manufacturing process, reduce costs, and improve accessibility to CAR T cell therapy for a broader patient population.

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Bypassing Ex Vivo Manufacturing with In Vivo CAR T Cell Engineering

In vivo delivery of synthetic immune receptors has the potential to revolutionize cancer treatment and other diseases, facilitated by advancements in viral vector engineering and nonviral nucleic acid delivery platforms like lipid nanoparticles (LNPs) or polymer-based nanoparticles. This approach offers logistical, biological, and safety advantages over ex vivo manufacturing but also presents unique challenges.

Logistical Advantages: Creating a single vector or nanoparticle product for treating multiple patients simplifies manufacturing, reduces costs, and ensures rapid access for patients with aggressive diseases. This streamlined process could democratize access to therapy.

Biological and Safety Advantages: In vivo CAR T cell therapy eliminates the need for lymphodepleting chemotherapy, potentially supporting epitope spreading and broad antitumor immune responses. Off-the-shelf platforms simplify multi-antigen targeting and enable real-time dose adjustments, minimizing toxicities like cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS). Serial dosing provides rest periods, maximizing T cell function and reducing exhaustion. Additionally, in vivo therapy may access various T cell subsets, potentially improving therapeutic outcomes.

Delivery and Expression Challenges: Efficient and specific delivery of genetic cargo to desired cell types is crucial to avoid dilution and off-target effects. Transduction or transfection of tumor blasts or inhibitory immune cells poses risks. However, strategies using T cell-targeted nanoparticles and nucleic acids have shown promise.

Advantages and Challenges of Nanocarriers and Nucleic Acids: Non-viral approaches, particularly mRNA-LNP systems, leverage existing vaccine manufacturing capacity and have proven safety profiles. They offer defined properties and can be produced in a cell-free manner. However, challenges remain in optimizing delivery efficiency and sustaining CAR expression. Strategies like lipid composition optimization, formulation changes, and coencapsulation of DNA plasmids may enhance therapeutic outcomes. Repeat dosing of LNPs has shown promising results in various indications, although concerns like complement activation-related pseudoallergy (CARPA) require management.

In summary, in vivo CAR T cell engineering presents an innovative approach to simplify manufacturing, reduce costs, and improve therapeutic outcomes. While challenges exist in delivery efficiency and sustaining CAR expression, ongoing advancements in nanocarriers and nucleic acids offer promising solutions. Overall, in vivo CAR T cell therapy holds great potential to broaden access to effective treatments for cancer and other diseases.

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T Cell-Targeted Viral Vectors for In Vivo CAR T Cell Therapy

In vivo delivery of CAR genetic cargo using viral vectors, particularly adeno-associated virus (AAV) and lentivirus/retrovirus, offers promising avenues for CAR T cell therapy. Lentiviruses are the primary platform, often pseudotyped with the vesicular stomatitis virus glycoprotein (VSV G) for ex vivo transduction. However, for in vivo applications, alternative pseudotyping strategies have been developed to enhance T cell tropism and transduction efficiency.

Viral Vector Design: Lentiviruses, commonly used for in vivo CAR T cell therapy, have been pseudotyped with various envelopes like those from measles virus, Nipah virus, Sindbis virus, and cocal fusion glycoprotein. These envelopes, along with ligands such as scFv or DARPin, facilitate T cell binding and transduction. AAVs, on the other hand, can display targeting ligands directly on their capsid.

Advantages and Challenges: Both lentiviruses and AAVs have well-established manufacturing protocols and can transduce dividing and non-dividing cells. Lentiviral vectors offer stable integration and long-term expression, while AAVs provide sustained episomal expression. Lentiviral vector engineering allows for multicistronic cassettes, enabling simultaneous provision of genes enhancing T cell function.

However, lentiviral gene transfer carries risks of insertional mutagenesis, necessitating stringent cell type targeting to avoid oncogenic transformation. While improvements like self-inactivated (SIN) vectors have minimized this risk, caution is warranted. Indications for in vivo CAR T therapy extend beyond cancer to autoimmune diseases, fibrosis, and infectious diseases like HIV and hepatitis B. In these contexts, in vivo therapy is advantageous due to its broad applicability and potential to avoid lymphodepletion.

Future Directions: Empirical determination of indications most conducive to in vivo therapy is needed, considering the required durability of CAR expression and depth of target cell depletion. Transient CAR expression may suffice for diseases like fibrosis, while permanent transduction may be necessary for cancer eradication.

In summary, T cell-targeted viral vectors hold promise for in vivo CAR T cell therapy across a range of diseases. While challenges like oncogenic risks exist, ongoing advancements in vector design and careful application can maximize therapeutic benefits while minimizing risks.

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Concluding Remarks and Future Perspectives on In Vivo CAR T Cell Therapy

CAR technology and synthetic biology hold immense potential to revolutionize medicine. While ex vivo cell engineering has shown remarkable clinical outcomes, challenges in manufacturing patient-specific cell products limit its widespread application. Delivery platforms like nanoparticles and viral vectors offer opportunities for in situ cellular manipulation to treat diseases, with preclinical studies demonstrating functional equivalence to ex vivo-produced cells.

Outstanding Questions

1. Determining the optimal fraction and location of transfected/transduced T cells using LNPs or viral vectors for achieving therapeutic efficacy comparable to ex vivo engineered cells is crucial.
2. Understanding the consequences of off-target CAR expression in different tissues and cell types is essential to mitigate potential toxicity.
3. Investigating how the innate immunogenicity of delivery platforms impacts T cell activity, APC activation, and reactogenicity in serial dosing schedules is necessary.
4. Optimizing the design of nucleic acids to balance immunogenicity and expression kinetics is a key challenge.
5. Evaluating whether established costimulatory domains in ex vivo cells are optimal for in vivo CAR T cell therapy is important.
6. Identifying the optimal route of administration to maximize CAR expression in desired cell types while minimizing off-target expression or innate immune activation is critical.
7. Exploring the therapeutic efficacy of targeting immune lineages with ligands for multiple immune cell types could enhance treatment outcomes.
8. Assessing whether in vivo cell therapy mobilizes endogenous immune cell types to prime a broad adaptive antitumor immune response is essential.
9. Understanding the role of lymphodepletion in removing inhibitory cell types and its impact on in vivo generated CAR T cell function is crucial.
10. Investigating the fate of T cells post transient transfection and potential risks of autoimmunity after CAR mRNA loss is important.
11. Prioritizing disease indications for testing in vivo CAR T cell strategies based on therapeutic feasibility and clinical need is necessary.

In summary, addressing these outstanding questions requires interdisciplinary collaboration among oncologists, immunologists, bioinformaticians, and regulatory scientists. By coming together, we can advance these technologies from promising concepts to clinical realities, ushering in a new era of personalized and effective medicine.

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