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Chimeric antigen receptors (CARs) represent a groundbreaking advancement in the fight against cancer and other diseases. Engineered receptors endow T cells with the ability to specifically target and eliminate malignant cells, offering a promising avenue for personalized and efficient therapies. Researchers face numerous challenges in developing CARs that are highly specific and effective, while minimizing potential off-target effects. Each step, from selecting appropriate antigen-binding domains to optimizing CAR structural components, is crucial for creating therapeutic agents that achieve expected clinical outcomes.

Understanding these complexities, we are committed to providing comprehensive CAR design and construction services tailored to meet the unique requirements of each project. Whether it's conventional CAR designs from first-generation to fifth-generation CARs, or novel designs like TanCARs, Physiological CARs, and Universal CARs, our experienced team employs innovative thinking to ensure each CAR we help design maximizes efficacy and safety.

Utilizing advanced bioinformatics tools and molecular biology techniques, we guide clients through the complex CAR development process from initial conceptualization to final validation. Our services are designed to accelerate research and development timelines, facilitating swift transitions from the lab to clinical applications. Whether your focus is on hematologic malignancies, solid tumors, or exploring CAR applications in autoimmune diseases, our goal is to support your work by providing cutting-edge solutions and expert guidance. Partner with us to access rich knowledge and resources dedicated to advancing the frontier of cellular immunotherapy.

CAR Design & Construction Solutions

1st Generation

The original CAR-T or the first-generation CAR consists of the intracellular domain from the CD3 ζ- chain and the primary transmitter of signals from endogenous TCRs, which showed success in pre-clinical trials and entered Phase I clinical trials in ovarian cancer, neuroblastoma and various types of leukemia and lymphoma. Although the anti-tumor activity was limited due to insufficient activation, persistence and homing to the cancer tissue, some significant effects indeed existed in patients with B-cell lymphoma treated with α-CD20-CD3 ζ CAR-modified T cells and some neuroblastoma treated with ScFv-CD3 ζ CAR-Ts. This is the most common form of CARs to fuse single-chain variable fragments (scFv) derived from monoclonal antibodies to CD3 ζ transmembrane and endo domain.

2nd Generation

To augment the antitumor efficacy of 1st-generation CARs, the 2nd-generation CARs were designed to combine the intracellular signaling domains from various costimulatory protein receptors (e.g., CD28, 41BB, ICOS) incorporated in the cytoplasmic tail of the CAR to enhance the signaling. For instance, the CD19-targeted CARs incorporated with CD28 or 4-1BB signaling domains manifested remarkable complete remission rates in patients with refractory B-cell malignancies. Subsequently, the CD28-based CARs showed a brisk proliferative response and boost effector functions. Meanwhile, the 4-1BB-based CARs manifested a more progressive T cell accumulation.

3rd Generation

As the expectation of more antitumor efficacy, the 3rd-generation of CARs combined multiple signaling domains (e.g., CD3 ζ-CD28-41BB, CD3 ζ-CD28-OX40) to acquire further enhanced activation signals, proliferation, production of cytokines and effective function. For instance, the α-CD19-CD3 ζ-4-1BB CAR-Ts for chronic lymphocyte leukemia showed complete remission to infiltrate and lyse cancer tissue. Even better, a fraction of CAR-Ts functioned as a memory phenotype for preventing tumor relapses. Despite the significant therapeutic effect, the emerging uncontrollable activity accompanied with more antitumor efficacy caused life-threatening lysis activity as the most critical adverse effect or toxicity including clinically significant release of pro-inflammatory cytokines, pulmonary toxicity, multi-organ failure, and eventual death.

4th Generation

The previous CAR strategies are highly specific and useful in redirecting T cells targeting malicious cancer cells. However, the major limitation on solid tumors with a tremendous phenotypic heterogeneity and relapse due to antigen-negative cancer cells is the huge challenge to trigger a novel CAR strategy. The 4th-generation CAR is designed to shape the tumor environment by the inducible release of transgenic immune modifiers, such as IL-12, which augments T-cell activation, attracts and activates innate immune cells to eliminate antigen-negative cancer cells in the targeted lesion.

5th Generation

The fifth generation of CARs, currently under exploration, builds upon the second generation but includes a truncated cytoplasmic interleukin-2 (IL-2) receptor β-chain domain (IL-2Rβ) with a binding site for the signal transducer and activator of transcription-3 (STAT3). The antigen-specific activation of this receptor triggers three critical signaling pathways simultaneously: TCR signaling through the CD3ζ domains, co-stimulatory signaling via the CD28 domain, and cytokine signaling through the JAK–STAT3/5 pathway. This integrated approach provides the synergistic signals necessary for full T cell activation and proliferation, effectively enhancing the immune response.


Dual CAR

Dual CAR T cells improve the specificity and safety of CAR-T therapy by targeting two tumor-associated antigens simultaneously. They are designed to activate only when both antigens are present on the tumor, reducing off-tumor toxicity. These cells express two CARs: one binding to a specific tumor antigen and delivering the primary signal, and another binding to a different antigen and providing the co-stimulatory signal. For example, dual CAR-T cells co-expressing anti-ErbB2 and anti-MUC1 CARs use CD3ζ and CD28 signaling pathways, respectively, and effectively kill ErbB2+ tumor cells only in the presence of MUC1. This approach enhances therapeutic efficacy while minimizing effects on normal tissues expressing a single antigen.

Conditional CAR

Conditional CAR-T cells enhance the safety of CAR-T cell therapy by requiring an additional signal for activation beyond target recognition. This secondary signal can be a small molecule or an adaptor-specific receptor that binds to secondary antibodies directed at target cell antigens. Conditional CARs act as a switch, activated by an exogenous molecule, allowing precise regulation of CAR-T cell activity without leading to cell death in its absence. For example, a conditional CAR targeting CD19 in a Nalm-6 xenograft rodent model demonstrated controlled activity, tissue-homing, cytokine release, and phenotype in a dose-titratable manner, potentially reducing cytokine release syndrome compared to conventional CAR-T cells.

TanCAR

TanCARs are an advanced type of chimeric antigen receptor designed to enhance the efficacy of CAR-T cell therapy. Unlike traditional CARs, TanCARs feature two distinct antigen recognition domains on a single transgenic receptor. This allows for simultaneous targeting of tumor cells and elements within the tumor microenvironment, increasing T cell activation and function by enhancing avidity and broadening therapeutic reach. This dual targeting capability addresses issues like antigen-loss escape variants commonly seen in cancer cells, potentially leading to more effective and durable cancer treatments.

Physiological CAR

Unlike traditional CARs, which often use single-chain variable fragments (scFv) of murine origin and risk triggering immune responses, physiological CARs utilize receptor ligands such as HER3 and HER4. These CARs consist of an antigen receptor and a CD3ζ intracellular signaling domain, with or without a transmembrane and spacer region, engineered into immune cells to target ligands expressed on tumor cells. This approach enhances the ability of T cells to recognize and eliminate cancer cells, increasing the persistence and effectiveness of the therapy while reducing the risk of immune response and anaphylaxis associated with murine-based scFv CARs.

Universal CAR

Universal CARs represent a significant advancement in CAR-T cell therapy by offering flexibility and adaptability to target a wide range of malignancies. These CARs are engineered to recognize various tagged antigens, such as biotin-labeled molecules, FITC-labeled targets, and peptide nucleic acids, among others. This versatility allows for precise and personalized treatment options, enhancing T cell activation, controlling therapeutic effects, and reducing adverse reactions. Universal CARs improve the scope and efficacy of CAR-T therapies, making them more effective against diverse cancer types.

Marked CAR

Marked CAR-T cells are specially designed CAR-T cells that express both CARs and tumor epitopes that are recognized by existing monoclonal antibodies. This dual expression allows for a unique safety mechanism: if severe adverse effects occur, the specific monoclonal antibody can be administered to selectively clear the CAR-T cells, thereby avoiding additional off-tumor effects. This approach not only helps in mitigating side effects but also enhances the overall therapeutic potential of CAR-T cell treatments by providing a controlled and targeted method to manage and eliminate CAR-T cells if necessary. In essence, Marked CARs combines efficacy with improved safety, offering a promising solution for cancer treatment.

For more CAR design and construction, please visit our Smart™ CAR Construction Service:


We have developed a proprietary online system for customizing CAR and CAR cell products, which offers full options to meet all unique needs, including but not limited to conventional or unconventional CAR constructs, as well as a variety of vectors and cells. The customization process can be completed with just a few simple clicks, please feel free to try it out.
Customize Your CAR Products

Highlights & Advantages

  • Comprehensive CAR Generation
    Mastery in first to fourth-generation CARs, each iteration offering enhanced antitumor activity and improved patient outcomes.
  • Advanced CAR-T Cell Engineering
    Expertise in designing and constructing CARs ensures tailored approaches for various cancer types, enhancing the specificity and effectiveness of immunotherapy.
  • Cutting-edge Technologies
    Utilization of state-of-the-art technologies such as CRISPR/Cas9 for precise genetic modifications and advanced vector design to optimize CAR-T cell performance.
  • Innovative CAR Strategies
    Portfolio includes specialized CARs like PD-1-disrupted CARs to prevent T-cell exhaustion and c-Jun overexpressed CARs to increase resistance to T-cell apoptosis.
  • Smarter™ CAR Construction Services
    Offering enhanced services such as Armored CARs, Marked CARs, Conditional CARs, Safety CARs, Dual CARs, and Switch CAR-Ts for diverse research needs.
  • Custom CAR Solutions
    Tailored solutions including Orthogonal Dual-Switch CARs, Chemically Programmed Switch CARs, and NKG2D-based Multi-target CARs for broad-spectrum efficacy.
  • Extensive Support and Services
    Comprehensive support from biomarker identification, high-affinity scFv generation, virus packaging, CAR transfection to in vitro and pre-clinical in vivo assays.
  • Expertise in Clinical Applications
    Support for clinical trial preparations, including anti-Fab antibodies for CAR detection and monitoring, ensuring thorough validation before clinical trials.
  • Holistic Approach
    Combining immunotherapy advancements with practical clinical applications, aiming to clear tumors without the toxicity of conventional treatments.

Case Studies

Case Study 1

Utilization of Creative Biolabs' Custom CAR Constructs in Enhancing T-cell Therapy for Solid Malignancies

Background

Solid tumors present significant challenges to immunotherapy due to their immunosuppressive microenvironment. Conventional CAR-T cell therapies have shown limited efficacy against solid malignancies, necessitating novel approaches to enhance their effectiveness. The study aimed to develop a method to remodel the tumor microenvironment, enabling CAR-T cells to effectively target and destroy solid tumors.

Methodology

The researchers utilized a murine 4T1 breast cancer cell line and a glioma mouse model to study the effects of combining targeted lipid nanoparticles with CAR-T cell therapy. The nanoparticles were designed to deliver a combination of a PI3K inhibitor (PI-3065) to inhibit immune-suppressive tumor cells and an NKT cell agonist (7DW8-5) to stimulate therapeutic T cells. The nanoparticles were administered in repeated infusions and were targeted to the tumor site using the iRGD peptide. Following nanoparticle preconditioning, tumor-specific CAR-T cells were infused during a therapeutic window created by the preconditioning. The effectiveness of the treatment was measured by assessing tumor regression, T-cell infiltration, and survival rates in treated animals.

Results

  • Improved Tumor Infiltration: The study found that CAR-T cells administered after nanoparticle preconditioning showed significantly higher infiltration into tumors compared to those without preconditioning. The bioluminescence imaging and flow cytometry confirmed that preconditioned tumors had a higher presence of CAR-T cells.
  • Enhanced Antitumor Activity: The combination therapy led to robust expansion of CAR-T cells at the tumor site, resulting in substantial tumor regression and prolonged survival in treated animals. In the 4T1 breast cancer model, mice treated with the combination therapy showed tumor stasis for up to 30 days, and in the glioma model, survival was more than doubled compared to conventional CAR-T cell therapy.
  • Reduction in Immunosuppressive Cells: The nanoparticle treatment effectively reduced the number of immune-suppressive cells within the tumor microenvironment. This included significant reductions in tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), and regulatory T cells (Tregs), creating a more favorable environment for CAR-T cell activity.

Conclusion

The study demonstrated that the use of targeted lipid nanoparticles to precondition the tumor microenvironment significantly enhances the efficacy of CAR-T cell therapy against solid tumors. This novel approach overcomes the traditional barriers posed by the immunosuppressive milieu of solid tumors, leading to improved T-cell infiltration, robust antitumor activity, and extended survival. The findings suggest that nanoparticle-mediated preconditioning could be a practical and low-cost strategy to potentiate various cancer immunotherapies, including CAR-T cell therapy, vaccines, and bispecific T-cell engager (BITE) platforms. This research offers a promising avenue for improving clinical outcomes in patients with solid malignancies.

Creative Biolabs' Contribution

Creative Biolabs provided custom-designed chimeric antigen receptor (CAR) constructs essential for this study. The anti-ROR1-28z CAR and the anti-EGFRvIII-28z CAR constructs were tailored to target tyrosine kinase-like orphan receptor 1 (ROR1) and epidermal growth factor receptor variant III (EGFRvIII), respectively. These CAR constructs consisted of:

  • Single-Chain Antibody Fragments (scFv): Engineered to specifically recognize and bind to the targeted antigens, ROR1 and EGFRvIII.
  • Synthetic Receptor Skeleton: Incorporating the CD8 hinge, CD28 transmembrane and signaling domains, and the CD3z signaling domain to ensure robust signaling and activation of the CAR-T cells upon binding to the target antigen.
  • c-Myc Tag: Facilitating the identification and tracking of the transduced T cells during the experiments.

These sophisticated CAR constructs were crucial for the genetic modification of T cells, enabling them to effectively target and attack tumor cells expressing ROR1 and EGFRvIII. Creative Biolabs' expertise in designing and producing these high-affinity CAR constructs provided the foundation for the study's success, showcasing the pivotal role of advanced biotechnology services in overcoming the barriers posed by solid tumor immunotherapy.

Reference
  1. Zhang, Fan, et al. "Nanoparticles that reshape the tumor milieu create a therapeutic window for effective T-cell therapy in solid malignancies." Cancer research 78.13 (2018): 3718-3730.

Resources

Use the resources in our library to help you understand your options and make critical decisions for your study.

VideosPodcastsInfographicFlyer Support Knowledge
Overview of Chimeric Antigen Receptors

Chimeric antigen receptor (CAR) can combine the extracellular antigen recognition domain from antibodies with the immune cell signaling domain to redirect T cell specificity and induce potent antitumor activity. CAR is an artificial transmembrane receptor that connects the extracellular antigen recognition domain, hinge domain (HD), transmembrane domain (TMD), and intracellular signal transduction domain in series.

Overview of CAR-T Cell Therapies

CAR-T cell therapies revolutionize cancer treatment by harnessing the power of a patient's immune cells. Engineered with chimeric antigen receptors (CARs), these cells effectively target and destroy cancer cells, offering a personalized and potent approach. This groundbreaking immunotherapy has shown remarkable success in treating certain blood cancers, providing hope for patients who may not respond to traditional treatments. CAR-T cell therapies mark a significant stride towards precision medicine, ushering in a new era in oncology with the potential to transform the landscape of cancer care.

T Cell-based Immunotherapies

Based on the significant roles of T cells in the immune system, many small-molecule drugs targeting T cells and T-cell based immunotherapies have been developed for the treatment of intractable diseases including autoimmune diseases and cancer. T cell-based immunotherapies mainly utilize the mechanisms of T cell-mediated immune responses and the effects of some other immune cells such as dendritic cells (DCs), natural killer (NK) cells, and macrophages.

Adoptive Transfer of Genetically Engineered T Cells

Adoptive cell transfer (ACT) of engineered T cells is a cutting-edge therapeutic approach revolutionizing cancer treatment. This innovative method involves modifying T cells, a key component of the immune system, to enhance their ability to target and eliminate cancer cells. By introducing genetically engineered T cells into patients, researchers aim to bolster the immune response against cancer, offering a personalized and potentially curative treatment option. This groundbreaking technology holds promise for addressing various malignancies and represents a significant stride towards more effective and precise cancer therapies.

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