CD22 Chimeric Antigen Receptor (CAR): A Comprehensive Guide and Our Service & Product Introduction

CD22 CAR is a type of chimeric antigen receptor immunotherapy designed to target the CD22 protein, which is highly expressed on malignant B cells such as those in acute lymphoblastic leukemia and B-cell lymphomas. By genetically modifying a patient’s T cells to express this receptor, CD22 CAR-T cells can specifically recognize, bind to, and eliminate CD22-positive cancer cells. It serves as an important therapeutic option, particularly for patients who have relapsed or do not respond to CD19 CAR-T treatment, offering an effective second-line targeted strategy for refractory B-cell malignancies.
RGBiotech offers CD22 CAR expression plasmid vectors and customization services that are designed to support researchers in accelerating their CD22-targeted therapy research. Whether you need standard plasmids for basic research or customized vectors for clinical translation, we are committed to providing high-quality products and professional services to meet your needs. Please contact us at admin@rgbiotech.com for more information.

Our CD22 CAR Expression Plasmid Vector Products and Custom Services

RGBiotech provides a comprehensive range of CD22 CAR expression plasmid vector products, covering all generations of CD22 CAR (1st to 5th generation), to meet the diverse needs of researchers in preclinical and clinical research. Our products are designed to facilitate the efficient construction of CD22 CAR-T cells, ADCs, and other CD22-targeted therapies, with high quality, stability, and compatibility. In addition, RGBiotech offers customized plasmid vector construction services to tailor products to specific research requirements.

Product Features

1) Multiple Generations of CD22 CAR: We offer plasmids for 1st to 5th generation CD22 CAR, each with distinct signaling domains to meet different research needs.
a) 1st Generation: Contains only the CD3ζ signaling domain, enabling basic T cell activation and cytotoxicity.
b) 2nd Generation: Adds one co-stimulatory domain (CD28 or 4-1BB), enhancing T cell proliferation, survival, and anti-tumor activity (e.g., scFv-CD8-4-1BB-CD3ζ CAR).
c) 3rd Generation: Contains two co-stimulatory domains (e.g., CD28+4-1BB, CD28+OX40), further improving T cell persistence and anti-tumor efficacy.
d) 4th Generation (Armored CAR): Incorporates additional functional genes (e.g., IL-12, IL-15, PD-1 inhibitor) to enhance T cell activity and overcome the tumor microenvironment.
e) 5th Generation (Switchable CAR): Equipped with a conditional activation system (e.g., small molecule-induced dimerization), allowing precise control of CAR-T cell activity to reduce off-target toxicities.

2) Diverse Vector Backbones: We offer a variety of vector backbones to suit different delivery methods.
a) Non-viral Vectors: Plasmid vectors for transient transfection or stable cell line establishment, with high transfection efficiency in various cell types (e.g., T cells, HEK293 cells).
b) Viral Vectors: Lentiviral vectors (HIV-based, VSV-G pseudotyped), retroviral vectors (γ-retrovirus), and adeno-associated virus (AAV) vectors. These vectors enable efficient transduction of primary T cells and long-term stable expression of CD22 CAR. Lentiviral vectors are particularly suitable for transducing non-dividing cells, while AAV vectors offer low immunogenicity.

3) Flexible Promoter Options: We provide multiple promoter choices to regulate CD22 CAR expression levels and cell-type specificity.
a) Constitutive Promoters: CMV (cytomegalovirus), EF1α (elongation factor 1α), CAG (chicken β-actin promoter + CMV enhancer), ensuring high and stable expression in most cell types.
b) Tissue-Specific Promoters: CD4, CD8, or CD3 promoter, enabling specific expression of CD22 CAR in T cells, reducing off-target expression in non-target cells.
c) Inducible Promoters: Tet-on/Tet-off, NFAT-responsive promoter, allowing conditional expression of CD22 CAR to control T cell activation timing and reduce toxicities.

4) Fluorescent Marker Options: To facilitate the detection and sorting of CAR-expressing cells, our plasmids include various fluorescent markers, such as GFP (green fluorescent protein), RFP (red fluorescent protein), allowing visualization of CAR expression via fluorescence microscopy or flow cytometry.

5) Antibiotic Selection Markers: We offer a range of antibiotic selection markers to facilitate the screening of stable cell lines, such as Puromycin, Hygromycin B, Neomycin (G418), Blasticidin, Zeocin, allowing flexible selection based on cell type and experimental needs.

Product Advantages

Product Applications

Custom CD22 CAR Plasmid Vector Construction Services

Introduction of CD22

CD22, also known as Cluster of Differentiation 22 or SIGLEC-2, is a member of the sialic acid-binding immunoglobulin-like lectin (Siglec) family. It is a transmembrane glycoprotein primarily expressed on B lymphocytes, playing a crucial role in regulating B cell activation, proliferation, and survival. As a key surface marker of B cells, CD22 is highly expressed in most B cell malignancies, making it an ideal target for immunotherapies such as CAR-T cell therapy. Unlike CD19, CD22 expression is relatively stable in some relapsed or refractory B cell tumors, which has driven extensive research on CD22-targeted therapies in recent years.

The human CD22 gene is located on chromosome 19q13.1, with the official gene symbol CD22 (OMIM: 107266; MGI: 88322; HomoloGene: 31052). It spans approximately 15 kb and consists of 14 exons, encoding a 687-amino acid precursor protein. The gene sequence is highly conserved among mammals, with mouse CD22 located on chromosome 7 (19.26 cm, 30,564,827-30,579,767 bp). Alternative splicing of the CD22 gene results in two main isoforms: one full-length isoform containing all 7 extracellular immunoglobulin (Ig) domains and a shorter isoform lacking the second and third N-terminal Ig domains. These isoforms may have distinct functional roles in B cell signaling and immune regulation.

CD22 is a type I transmembrane glycoprotein with a molecular weight of approximately 140 kDa. Its structure can be divided into three main parts: extracellular domain, transmembrane domain, and intracellular domain.
1) Extracellular Domain: Composed of 7 Ig-like domains (one V-type Ig domain and six C2-type Ig domains), with the N-terminal V-type domain serving as the sialic acid-binding site. This domain specifically binds to sialic acid residues linked to galactose via α2,6-linkage, enabling CD22 to interact with ligands on the same cell (cis-binding) or adjacent cells (trans-binding). Common ligands include sialylated glycoproteins on erythrocytes, monocytes, cytokine-activated endothelial cells, T cells, and B cells, as well as soluble IgM and haptoglobin.
2) Transmembrane Domain: A hydrophobic α-helical segment that anchors the protein to the B cell membrane, facilitating signal transduction between the extracellular and intracellular domains.
3) Intracellular Domain: A 141-amino acid cytoplasmic tail containing three immunoreceptor tyrosine-based inhibitory motifs (ITIMs). These ITIMs recruit phosphatases (such as SHP-1 and SHP-2) upon phosphorylation, exerting an inhibitory effect on B cell receptor (BCR) signaling.

CD22 primarily functions as a negative regulator of B cell activation and immune homeostasis, with key roles including:
1) Inhibition of BCR Signaling: By recruiting phosphatases via ITIMs, CD22 dephosphorylates key signaling molecules in the BCR pathway (e.g., Syk, PLCγ2), preventing excessive B cell activation and avoiding autoimmune responses. Loss of CD22 function can lead to hyperactive B cells and increased susceptibility to autoimmune diseases.
2) Regulation of B Cell Trafficking: CD22 is involved in B cell migration and homing to lymphoid tissues (e.g., Peyer's patches in mice), facilitating B cell interactions with other immune cells in the microenvironment.
3) Endocytosis Regulation: CD22 has inherent endocytic activity, which makes it an ideal target for antibody-drug conjugates (ADCs), as binding of antibodies to CD22 triggers internalization, delivering cytotoxic drugs to target cells.
4) Modulation of Microglial Function: In mice, CD22 blockade has been shown to restore homeostatic microglial phagocytosis in aging brains, suggesting potential roles beyond B cell regulation.

CD22 expression is highly restricted to the B cell lineage, with a distinct pattern during B cell development. It is first expressed intracellularly in pro-B and pre-B cells, and as B cells mature, expression shifts to the cell membrane. Highest expression is observed on mature B cells (naive and memory B cells) in peripheral blood, spleen, lymph nodes, and tonsils. Low or no expression is found on plasma cells, hematopoietic stem cells, T cells, natural killer (NK) cells, or other non-hematopoietic tissues (e.g., liver, kidney, heart), ensuring high target specificity for B cell malignancies. Single-cell analysis has shown that CD22 is expressed on mature oligodendrocytes in the central nervous system (CNS) but not on oligodendrocyte precursor cells or neurovascular cells, which may explain the relatively mild neurotoxicity of CD22 CAR-T therapy compared to CD19 CAR-T therapy.

Due to its specific expression on B cells, CD22 is closely associated with B cell-related diseases, particularly hematologic malignancies. It is expressed in more than 90% of B cell acute lymphoblastic leukemia (B-ALL) patients and the majority of B cell non-Hodgkin lymphoma (B-NHL) cases, including diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), and mantle cell lymphoma (MCL). Additionally, CD22 is expressed in hairy cell leukemia (HCL) and chronic lymphocytic leukemia (CLL). CD22 is also involved in autoimmune diseases: loss of CD22 function or abnormal expression can lead to hyperactive B cells, contributing to the development of systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and Sjögren's syndrome. In these diseases, CD22-targeted therapies may help regulate B cell activity and alleviate autoimmune responses.

Introduction of CD22 Chimeric Antigen Receptor (CAR)

A Chimeric Antigen Receptor (CAR) is a recombinant protein that redirects T cells to specifically recognize and kill target cells expressing a specific antigen. A CD22 CAR consists of four core components: an extracellular antigen-binding domain (typically a single-chain variable fragment, scFv, targeting CD22), a hinge region (connecting the extracellular and transmembrane domains), a transmembrane domain (anchoring the CAR to the T cell membrane), and an intracellular signaling domain (activating T cell proliferation, cytotoxicity, and survival). CD22 CAR-T cell therapy involves genetically engineering a patient’s T cells to express CD22 CAR, which then recognize and eliminate CD22-positive tumor cells. It is a promising treatment for relapsed/refractory B cell malignancies, especially for patients who have failed CD19 CAR-T therapy due to CD19 antigen loss.

Current Research Achievements of CD22 CAR

Approved Drugs Targeting CD22

Research Hotspots of CD22 CAR

Research Difficulties & Challenges

Frequently Asked Questions (FAQs)

Q: What is the difference between 2nd and 3rd generation CD22 CAR?
A: The 2nd generation CD22 CAR contains one co-stimulatory domain (CD28 or 4-1BB) in addition to the CD3ζ signaling domain, enhancing T cell proliferation and survival. The 3rd generation CAR contains two co-stimulatory domains (e.g., CD28+4-1BB), which further improves T cell persistence and anti-tumor efficacy. However, some preclinical studies have shown that 2nd generation CARs may have better in vitro and in vivo activity than 3rd generation CARs in certain contexts.

Q: How to reduce the off-target toxicities of CD22 CAR-T cells?
A: Off-target toxicities can be reduced by several strategies: using cell-type specific promoters (e.g., CD3 promoter) to restrict CAR expression to T cells; optimizing the scFv to improve specificity for CD22; developing switchable CARs (e.g., Tet-on/Tet-off) to control CAR activation; and combining with other therapies to reduce the required dose of CAR-T cells. Additionally, the relatively restricted expression of CD22 on B cells helps minimize off-target effects on non-hematopoietic tissues.

Q: Why do some patients relapse after CD22 CAR-T therapy?
A: The main reason for relapse is antigen loss (downregulation or deletion of CD22 expression on tumor cells). Other reasons include T cell exhaustion, tumor microenvironment suppression (e.g., PD-L1 expression, immunosuppressive cytokines), and inadequate CAR-T cell persistence. Using dual-target CARs (e.g., CD19/CD22) or armored CARs (e.g., expressing IL-12) can help reduce relapse rates.

Q: Can CD22 CAR-T therapy be used for solid tumors?
A: Currently, CD22 CAR-T therapy is primarily used for B cell hematologic malignancies. However, recent studies have found low expression of CD22 in some solid tumors (e.g., pancreatic cancer, ovarian cancer). Research is ongoing to explore the potential of CD22 CAR-T therapy in solid tumors, combined with strategies to overcome the solid tumor microenvironment (e.g., immune checkpoint inhibitors, oncolytic viruses).

Q: What is the difference between lentiviral and retroviral vectors for CD22 CAR delivery?
A: Lentiviral vectors can transduce both dividing and non-dividing cells (e.g., primary T cells), integrate into the host genome, and enable long-term stable expression of CD22 CAR. Retroviral vectors (γ-retrovirus) can only transduce dividing cells and have a higher risk of insertional mutagenesis. Lentiviral vectors are generally preferred for CD22 CAR-T cell research due to their broader cell tropism and lower safety risks.

Q: How to improve the delivery efficiency of CD22 CAR plasmids?
A: Transfection efficiency can be improved by: choosing the appropriate transfection reagent (e.g., Lipofectamine 3000, Fugene HD) for different cell types; optimizing the plasmid-to-reagent ratio; ensuring high plasmid purity (low endotoxin levels); and pre-treating cells to ensure good viability (80-90% confluency). For primary T cells, electroporation is often more effective than chemical transfection or using lentiviral/retroviral particles.

Q: How to store CD22 CAR plasmids?
A: Plasmids should be stored at -20°C (short-term, 1-6 months) or -80°C (long-term, more than 6 months) in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) to maintain stability. Avoid repeated freeze-thaw cycles, as this can damage the DNA. For long-term storage, aliquot the plasmids to minimize freeze-thaw cycles.

Q: What should I do if the plasmid has low concentration or purity?
A: Low concentration or purity may be due to inefficient DNA extraction. Re-extract the plasmid using a high-quality DNA extraction kit, ensuring that the bacterial culture is fresh and the lysis step is complete. For low concentration, concentrate the plasmid using ethanol precipitation or a DNA concentration kit. Check the A260/A280 ratio to ensure purity (1.8-2.0); if the ratio is too low, there may be protein contamination, which can be removed by phenol-chloroform extraction.

References

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