CAR-T Structure & Generations (1st–5th) | Off-the-Shelf Lentivirus

In the relentless advance of modern medicine, certain names are forever etched in history for defying death. Emily Whitehead, a leukemia patient once told she had “little time left,” is one such name. Through a groundbreaking treatment, she not only reclaimed her life but also propelled CAR-T cell therapy from the confines of the laboratory to the global clinical stage, cementing it as the “ultimate weapon” against hematologic malignancies.

In 2010, five-year-old Emily was diagnosed with acute lymphoblastic leukemia(ALL). While childhood ALL is often viewed as having a silver lining due to its high cure rate, Emily’s case proved exceptionally devastating. After both conventional chemotherapy and a stem cell transplant failed, doctors recommended transitioning to palliative care. With no conventional options left, she became the first pediatric leukemia patient in the world to receive CD19 CAR-T therapy ^[1]. By June 2012, Emily had achieved complete remission and was discharged. Today, having grown into a vibrant young woman, her journey stands as the ultimate living proof of CAR-T therapy’s potential and a powerful testament to medical progress.

Figure 1. Emily’s 20th Birthday (Source: Web, deleted if infringing)

In August 2017, the FDA approved the world’s first CAR-T cell therapy—Novartis’ tisagenlecleucel (Kymriah)—for the treatment of relapsed or refractory B-cell precursor ALL in patients up to 25 years of age. Phase II data demonstrated that within three months of infusion, 83% of the 63 evaluable pediatric patients achieved either complete remission (CR) or CR with incomplete hematologic recovery (CRi) ^[2]. This landmark approval ushered in the CAR-T era. It triggered massive industry shifts: Gilead Sciences acquired Kite Pharma for $11.9 billion to secure its foothold in the lymphoma CAR-T space, while a surge of global institutions mobilized to expand CAR-T applications to a broader spectrum of tumors. From leukemia and lymphoma to multiple myeloma, this “living drug” has offered a lifeline to late-stage patients who have exhausted standard treatment options. As of December 2025, 15 CAR-T products have been approved globally—seven in the United States and eight in China.

Figure 2. Approval Status of CAR-T Products (Compiled by OBiO Tech)

What is CAR-T Therapy?

CAR-T (Chimeric Antigen Receptor T-Cell) immunotherapy represents a groundbreaking form of precision-targeted cancer treatment that has demonstrated remarkable clinical efficacy following continuous optimization. The therapeutic process involves isolating a patient’s autologous T cells, genetically engineering them ex vivo to express chimeric antigen receptors (CARs) capable of specifically recognizing and binding to tumor-associated antigens, expanding these modified cells in vitro, and subsequently reinfusing them into the patient to mediate targeted tumor cell elimination. A pivotal innovation of CAR-T technology is its ability to circumvent major histocompatibility complex (MHC) restriction, enabling direct recognition of surface antigens on target cells.

Figure 3. Schematic Diagram of the CAR-T Cell Therapy Process ^[3]

Introduction to CAR Structure

Serving as the engine of CAR-T cell therapy, the Chimeric Antigen Receptor (CAR) is a sophisticated molecular construct that empowers T cells to identify tumor antigens independently of HLA restriction. By bypassing the limitations of traditional T cell receptors (TCRs), this HLA-independent mechanism enables CAR-modified T cells to target a significantly broader spectrum of malignancies. Structurally, the CAR molecule is meticulously engineered around a modular architecture comprising three critical domains: the extracellular target-binding domain, the transmembrane domain, and the intracellular signaling domain.

Extracellular Domain: Antigen Engagement and Hinge Mechanics

The extracellular region comprises two key structural elements: the antigen recognition domain and the hinge region. The recognition domain utilizes a single-chain variable fragment (scFv)—a synthetic construct linking the variable heavy (VH) and light (VL) chains of a monoclonal antibody. This domain confers strict specificity for tumor-associated antigens (TAAs), translating target recognition into the downstream activation of CAR-expressing T cells.

The function of the hinge region is to enhance the flexibility of the scFv, resolving steric hindrance and positioning the receptor optimally for efficient antigen engagement.

Transmembrane Domain

A hydrophobic α-helix spanning the lipid bilayer that acts as a stable structural anchor. It bridges the extracellular and intracellular regions, ensuring proper receptor localization and facilitating the transduction of activation signals into the T cell. 

Intracellular Domain: Costimulatory and Signal Transduction Domains

The intracellular domain serves as the functional engine of the CAR, comprising the costimulatory domain and the primary signal transduction domain.

Costimulatory Domain (CM): Providing the essential secondary signal for full T cell activation, the CM dictates both the kinetic profile and metabolic programming of the CAR-T cell. The most widely utilized domains are CD28 and 4-1BB, which drive fundamentally distinct cellular fates:

• · CD28: Engineered CARs incorporating CD28 exhibit rapid, potent signaling kinetics. This drives robust effector memory differentiation and shifts cellular metabolism toward aerobic glycolysis, resulting in immediate but potentially short-lived cytotoxic activity.
• · 4-1BB: Conversely, 4-1BB-based CARs deliver attenuated yet sustained signaling. This promotes mitochondrial biogenesis, enhances oxidative respiration, and facilitates fatty acid oxidation. The resulting metabolic reprogramming drives differentiation into central memory T cells, significantly extending *in vivo* persistence and prolonging antitumor durability.

Signal Transduction Domain: Typically derived from the CD3ζ chain of the native T cell receptor (or alternatively the FcεRIγ chain), this module contains Immunoreceptor Tyrosine-Based Activation Motifs (ITAMs). Upon antigen engagement at the tumor cell surface, these ITAMs undergo rapid phosphorylation. This critical switch recruits and activates ZAP-70 kinase ^[4], thereby initiating a robust downstream signaling cascade that orchestrates adaptor protein assembly and triggers definitive T cell effector responses, including clonal proliferation, cytokine secretion, and target cell lysis.

Figure 4. Schematic Diagram of CAR Structure [3]

Generational Evolution of CARs

First-Generation CARs

First-generation CARs established the foundational architecture, comprising an extracellular scFv, a hinge region, a transmembrane domain, and a single intracellular signaling domain—typically the CD3ζ or FcεRI chain. Upon antigen binding, the CD3ζ chain initiates a primary signaling cascade sufficient to trigger baseline T cell activation, mediate target cell lysis, and induce the secretion of cytokines such as IL-2.

Inherent Limitations:Despite demonstrating *in vitro* cytotoxicity, first-generation CARs faced significant biological constraints *in vivo*:

• · Absence of Costimulation: Lacking a costimulatory domain, they failed to deliver the critical secondary signal required for full T cell activation.
• · Metabolic Insufficiency: They were incapable of generating adequate autocrine IL-2 to sustain long-term proliferation, creating a dependency on exogenous cytokine supplementation.
• · Transient Persistence: Consequently, these CAR-T cells exhibited limited *in vivo* expansion, a heightened susceptibility to activation-induced cell death (AICD), and ultimately, restricted antitumor efficacy.

Second-Generation CARs

Building upon the foundational architecture, second-generation CARs incorporated a costimulatory domain—most commonly CD28 or 4-1BB—in tandem with the CD3ζ signaling chain. This dual-signaling design enables T cells to simultaneously integrate antigen-specific stimulation and costimulatory inputs, driving robust IL-2 synthesis and achieving full T cell activation. Consequently, this structural evolution resolves the transient persistence seen in earlier constructs, yielding significant improvements in T cell proliferation, cytotoxic potency, and *in vivo* longevity. Validating this biological advancement, the second-generation architecture has established the definitive clinical paradigm, underpinning every globally approved CAR-T therapy to date.

Third-Generation CARs

Third-generation CARs push the structural boundaries by integrating multiple costimulatory domains—typically combining CD28 and 4-1BB—in tandem with the CD3ζ chain, occasionally incorporating alternative costimulators like OX40. In theory, this multi-signal architecture aims to synergistically activate downstream pathways (JNK, ERK, and NF-κB) to generate a hyper-activated T cell phenotype marked by superior proliferation, extended *in vivo* persistence, and robust cytokine secretion.

However, translational reality has fallen short of these preclinical promises. While third-generation constructs have demonstrated enhanced expansion and potent antitumor activity in B-cell non-Hodgkin lymphoma models, they have consistently struggled to convert this into clinical success. Ultimately, merely stacking additional costimulatory domains has not yielded proportional therapeutic gains and, in many cases, has failed to improve safety profiles—or even exacerbated toxicity—highlighting the limitations of over-engineering T cell signaling pathways.

Fourth-Generation CARs

Also designated as TRUCKs (T cell Redirected Universal Cytokine Killing), fourth-generation CARs are engineered by integrating an NFAT-responsive promoter and a cytokine payload—most notably IL-12—into a second-generation backbone. Upon antigen encounter, this architecture enables the localized, tumor-restricted secretion of IL-12 within the tumor microenvironment (TME). This targeted cytokine release recruits and activates innate immune cells, effectively reversing TME immunosuppression and promoting robust CAR-T infiltration, which is particularly critical for solid tumors. Additionally, these constructs often incorporate an inducible safety switch (such as a suicide gene) to tightly regulate *in vivo* persistence and mitigate severe toxicities like cytokine release syndrome (CRS).

Fifth-Generation CARs

Fifth-generation CARs introduce an intracellular IL-2 receptor β chain (IL-2Rβ) domain fused with a STAT3-binding motif to the conventional second-generation framework. This structural addition creates a self-sustaining signaling loop: upon activation, it harnesses both exogenous and endogenous IL-2 to potently trigger the JAK-STAT pathway. This synergistic signaling significantly amplifies T cell proliferation, enhances effector activation, and promotes prolonged cellular fitness.

The CAR-T landscape is undergoing a paradigm shift. CRISPR-Cas9 gene editing is paving the way for allogeneic “off-the-shelf” universal CAR-T therapies, dramatically reducing manufacturing timelines. In a groundbreaking clinical milestone, allogeneic CAR-T cells have recently demonstrated unprecedented efficacy in treating severe autoimmune diseases, signaling a massive expansion of CAR-T applications beyond oncology. Concurrently, the target landscape is rapidly diversifying from CD19 to BCMA, GPC3, and other solid tumor antigens. Looking ahead, the convergence of advanced gene editing, synthetic biology, and targeted mRNA delivery technologies promises to unlock entirely new therapeutic modalities, positioning cell therapy to redefine treatment across a vast spectrum of human diseases.

Figure 5. The Evolutionary History of CARs [3]

OBiO Tech CAR Lentiviral Products

Backed by more than ten years of specialized focus on virology, OBiO Tech serves as a premier partner for precision CAR vector design and viral manufacturing. To meet the dynamic needs of immuno-oncology research, we offer an extensive off-the-shelf library of CAR lentiviral vectors and tool plasmids for T and NK cell engineering—highlighted by our special promotional offering of CD19 CAR-T lentiviral stocks.

Complementing this is our proprietary target database, covering over 50 targets spanning both blood cancers and solid tumors. By providing highly adaptable vector solutions across a wide spectrum of indications, OBiO Tech effectively de-risks and accelerates the entire preclinical R&D workflow.

CD19 CAR-T Off-the-Shelf Lentiviral Product Vector Information:

References：

1. PMID:28902570

2.https://pps.cpu.edu.cn/cn/article/pdf/preview/e40a4210-f7c7-40e1-be1d-8ad0e0ac4c9c.pdf

3. PMID:39850899

4. PMID:36209106