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RetroNectin Reagent for increased transduction of hematopoietic cells RetroNectin reagent
Home › Learning centers › Gene function › T-cell transduction and culture › Adoptive T-cell therapy (ACT)

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Selection guides Virus kits: product finder tool
RetroNectin Reagent for increased transduction of hematopoietic cells RetroNectin reagent
OVERVIEW

Adoptive T-cell therapy (ACT)

  • RetroNectin GMP grade reagent enhances transduction of clinically relevant human cells such as hematopoietic stem cells and T cells
  • Costimulation of T cells with anti-CD3 antibody (OKT3) and RetroNectin GMP grade reagent leads to rapid expansion of T cells with a higher number of naïve cells
Introduction Clinical examples Conclusions References

Introduction  

Traditionally, clinically relevant cell types such as hematopoietic stem cells (HSCs) and T cells have a low transduction efficiency, regardless of the method used to introduce the exogenous DNA. Although there has been demonstrated success in clinical trials using engineered T cells to treat certain types of cancer, favorable outcomes can be limited by the number of cells that acquire and express the exogenous DNA. In one cancer and immune gene therapy technique, adoptive cell therapy (ACT), T cells engineered to express chimeric antigen receptors (CAR) can recognize cancer cells—like those in bone marrow tumors in patients with acute lymphocytic leukemia who are refractory to chemotherapy—and they will eradicate those malignant cells. Although CAR T-cell therapy gives hope to these patients, the typical low transduction efficiency and T-cell expansion rates can compromise the procedure and get in the way of recovery.

To address these bottlenecks, RetroNectin GMP grade reagent improves retroviral- and lentiviral-mediated gene transfer by colocalizing retrovirus particles and target cells. Colocalization is thought to be accomplished by direct binding of viral particles to sequences in the heparin-binding domain and interaction of cellular integrins VLA-4 and/or VLA-5 with the RetroNectin molecule (Hosoi et al. 2014).

RetroNectin reagent’s mechanism of action

Figure 1. RetroNectin reagent is a recombinant human fibronectin fragment that contains three functional domains: the cell-binding domain (C-domain), the heparin-binding domain (H-domain), and the CS-1 sequence. Specifically, virus particles bind RetroNectin reagent via interaction with the H-domain, and target cells bind mainly through the interaction of cell surface integrin receptors VLA-5 and/or VLA-4 with the fibronectin C-domain and CS-1 sites, respectively. By facilitating close proximity, RetroNectin reagent can enhance viral-mediated gene transfer to target cells expressing integrin receptors VLA-4 and/or VLA-5.

The RetroNectin method is being widely used during ex vivo gene therapy to allow efficient transduction of genes into difficult-to-transduce human cell types. In ex vivo gene therapy, cells are taken from a patient, the disease-fighting gene(s) are introduced, and the modified cells are transplanted back into the patient.

Ex vivo gene therapy with RetroNectin reagent

Figure 2. RetroNectin reagent and gene therapy. RetroNectin reagent is also used during expansion culture of T lymphocytes to increase the proportion of naïve T cells. Takara Bio scientists have used RetroNectin reagent for improved T-lymphocyte expansion from peripheral blood mononuclear cells (PBMCs). Using RetroNectin reagent in combination with IL-2 and anti-CD3 antibody improved T-cell expansion in culture (Yu et al. 2008). Moreover, RetroNectin reagent costimulation generated a higher number of T cells with a naïve phenotype than traditional methods. For more data on how RetroNectin reagent can be used to enhance T-cell expansion, read a technical note on this topic.

Clinical examples  

The following section is a curated selection of publications on the use of RetroNectin reagents in clinical settings.

Adoptive cell therapy

T-cell receptor (TCR) gene therapy is an ACT against cancer. In this therapy, a TCR/CAR that recognizes a specific cancer antigen is introduced into lymphocytes from a patient. The modified lymphocytes are cultured in large scale and then infused back into the patient. These cells specifically attack tumor cells expressing the cancer antigen. Here, it is important to have high transduction efficiency for introducing the CAR genes into the lymphocytes.

RetroNectin GMP grade reagent is used to enhance transduction and T-cell expansion for therapies. Dr. Steven Rosenberg at the National Institutes of Health (US), one of the pioneers of ACT, is conducting clinical trials of TCR/CAR gene therapy (Kochenderfer et al. 2012). His group uses RetroNectin GMP grade reagent to transduce patient-derived lymphocytes with TCR/CAR genes that recognize cancer antigens (e.g., MART-1, gp100, or NY-ESO-1) for therapy (Robbins et al. 2011).

T-cell immunotherapy for acute lymphoblastic leukemia

The prognosis for adults with relapsed acute lymphoblastic leukemia is extremely poor, as few therapeutic options are available. Scientists at Memorial Sloan-Kettering Cancer Center reported an immunotherapy strategy for the treatment of five adult patients with acute lymphoblastic leukemia (Brentjens et al. 2013). Each patient's T cells were isolated, altered by the introduction of DNA that would cause the cells to target CD19 and thus attack bone marrow tumor cells, and infused back into the patient's bloodstream. According to the researchers, all patients achieved tumor eradication and complete remission. RetroNectin GMP grade reagent was used during T-cell transduction.

Transduction of CD34+ hematopoietic stem cells

Standardized methodology for transduction of human CD34+ HSCs is critical for the success of gene therapy for a variety of hematopoietic diseases. In 2009, Millington et al. systematically investigated factors involved in ex vivo transduction of Granulocyte-Colony Stimulating Factor (G-CSF)-mobilized peripheral blood CD34+ cells. Their team used a self-inactivating lentiviral vector carrying eGFP to investigate the effect of serum, cytokine combinations, prestimulation time, multiplicity of infection (MOI), transduction duration, and the use of spinoculation and/or RetroNectin reagent on transduction efficiency. The researchers found that transduction using RetroNectin reagent alone resulted in a statistically significant increase in transduction rate (~10% in untreated controls compared to 24.6 ± 1.3% with RetroNectin reagent). RetroNectin reagent in combination with spinoculation resulted in the highest transduction rate (34.1 ± 0.5%), but spinoculation alone had no effect.

Conclusions  

With the RetroNectin method, increasing the efficiency of the virus-cell interaction leads to a higher transduction rate and increases the likelihood of a successful outcome. The RetroNectin method has been utilized in gene therapy clinical trials and over 68 protocols at 44 institutions worldwide. RetroNectin GMP grade reagent for use in ex vivo clinical applications is now available for direct purchase by researchers, without the need for a Material Transfer Agreement (MTA). RetroNectin GMP grade is manufactured as a quality-assured product according to guidelines for Good Manufacturing Practice (GMP) for Investigational Products. Takara Bio owns exclusive worldwide rights to the use of RetroNectin technology.

References  

Brentjens, R. J. et al. CD19-targeted T cells rapidly induce molecular remissions in adults with chemotherapy-refractory acute lymphoblastic leukemia. Sci. Transl. Med. 5, 177ra38 (2013).      

Hosoi, H. et al. Stimulation through very late antigen-4 and -5 improves the multifunctionality and memory formation of CD8+ T cells. Eur. J. Immunol. 44, 1747–58 (2014).               

Kochenderfer, J. N. et al. B-cell depletion and remissions of malignancy along with cytokine-associated toxicity in a clinical trial of anti-CD19 chimeric-antigen-receptor-transduced T cells. Blood 119, 2709–20 (2012).        

Robbins, P. F. et al. Tumor regression in patients with metastatic synovial cell sarcoma and melanoma using genetically engineered lymphocytes reactive with NY-ESO-1. J. Clin. Oncol. 29, 917–24 (2011).

Related Products

Cat. # Product Size License Quantity Details
T210 ◊Anti-CD3 mAb GMP grade 1 mg/1 mL USD $1934.00

License Statement

ID Number  
M69b Before purchasing this product, you must submit a completed copy of the Anti-CD3 mAb (GMP) Order Check Sheet to us. A copy of the ◊Anti-CD3 mAb GMP grade product License Agreement can be found by clicking here.

T210, Anti-CD3 mAB GMP grade, is manufactured and tested in accordance with the following guidelines, "Standards for Manufacturing Control and Quality Control, etc. of Investigational Products (Investigational products GMP)" notified by the Ministry of Health and Welfare Japan ("PFSB Notification No. 0709002, July 9, 2008)" (PFSB: Pharmaceutical and Food Safety Bureau).

Anti-CD3 antibodies are commonly used in T lymphocyte proliferation protocols. Among many available clones, "OKT3" (mouse IgG2a) is the most commonly used. This monoclonal anti-CD3 antibody (clone OKT3) is manufactured as a quality-assured product, according to relevant guidelines for Good Manufacturing Practice (GMP). This product can be used for ex vivo cell culture processing in preclinical research and investigational phase studies.

Notice to purchaser

This product is to be used for research use only, including non-commercial manufacturing for clinical research. This product is not intended for humans or animals in-vivo applications. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

Documents Components You May Also Like Image Data

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Ex vivo T cell expansion from peripheral blood mononuclear cells (PBMC) was performed in the presence of anti-CD3 antibody alone or a combination of anti-CD3 and RetroNectin reagent

Ex vivo T cell expansion from peripheral blood mononuclear cells (PBMC) was performed in the presence of anti-CD3 antibody alone or a combination of anti-CD3 and RetroNectin reagent
Ex vivo T cell expansion from peripheral blood mononuclear cells (PBMC) was performed in the presence of anti-CD3 antibody alone or a combination of anti-CD3 and RetroNectin reagent. After expansion, the percentage of naïve T cells was assessed by expression CD45RA and CCR7.

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T210: Anti-CD3 mAb GMP grade

T210: Anti-CD3 mAb GMP grade
T202 ◊RetroNectin® GMP grade 2.5 mL USD $1704.00

License Statement

ID Number  
M69 This product is covered by the claims of Japanese Patent No. 4406566 and its foreign counterpart patent claims.
M69a Before purchasing this product, you must submit a completed copy of the RetroNectin® (GMP) Ordering Sheet to us. A copy of the ◊RetroNectin® GMP grade product License Agreement can be found by clicking here.

T202, RetroNectin GMP grade, is manufactured and tested in accordance with the following guidelines, "Standards for Manufacturing Control and Quality Control, etc. of Investigational Products (Investigational products GMP)" notified by the Ministry of Health and Welfare Japan ("PFSB Notification No. 0709002, July 9, 2008)" (PFSB: Pharmaceutical and Food Safety Bureau).

RetroNectin reagent is a 63 kD fragment of recombinant human fibronectin fragment (also referred to as rFN-CH-296) that enhances the efficiency of lentiviral- and retroviral-mediated gene transduction. This is particularly important for hematopoietic cells and other hard-to-transfect cell types. Enhanced transduction is thought to result from co-localization of virus particles and target cells. This is accomplished by direct binding of viral particles to sequences in the heparin-binding domain and interaction of target-cell integrins with two other domains in rFN-CH-296. RetroNectin GMP grade is manufactured as a quality-assured product, according to relevant guidelines for Good Manufacturing Practice (GMP). This product can be used for ex vivo clinical applications. Researchers are free to use this product for development of research-based clinical trials. Proper regulatory approval is required before using the GMP-grade product in research-based clinical trials since these trials may require special considerations or more stringent manufacturing standards. This product is not intended for human in vivo applications. It is the end user’s responsibility to ensure that the final product meets the requirements of the application for which it is to be used. Takara Bio Inc., has submitted a Drug Master File (DMF application number 18898) to the Food and Drug Administration for RetroNectin GMP grade.

Notice to purchaser

This product is to be used for research use only, including non-commercial manufacturing for clinical research. This product is not intended for humans or animals in-vivo applications. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

Documents Components You May Also Like Image Data

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Comparison of T cell expansion efficiencies for various commonly used protocols

Comparison of T cell expansion efficiencies for various commonly used protocols

Comparison of T cell expansion efficiencies for various commonly used protocols. Cells co-stimulated using RetroNectin and α-CD3 demonstrated higher-fold expansion as compared to other protocols (Panel A). In addition, the presence of RetroNectin promoted the production of more naïve-like T cells (CCR7+/CD45RA+ phenotype) that have been shown to have better in vivo anti-tumor activity (Panel B).

NOTE: This expansion method requires a license for commercial applications.

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Comparison of retroviral transduction of a K562 cell line using transduction enhancers from different companies

Comparison of retroviral transduction of a K562 cell line using transduction enhancers from different companies

Comparison of retroviral transduction of a K562 cell line using transduction enhancers from different companies. A K562 cell line was transduced with a retroviral vector in the presence of various transduction enhancers. RetroNectin yielded a far higher transduction efficiency relative to each of the other transduction enhancers, including Polybrene, protamine sulfate, and human fibronectin from three other companies.

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Efficient transduction of human PBMCs using RetroNectin, as compared to Polybrene

Efficient transduction of human PBMCs using RetroNectin, as compared to Polybrene

Efficient transduction of human PBMCs using RetroNectin, as compared to Polybrene. Human PBMCs were transduced with varying dilutions of lentivirus encoding ZsGreen1 in the presence of RetroNectin or Polybrene, and the percentages of transduced CD8+ cells were measured by flow cytometry. Transduction efficiencies with Retronectin were significantly higher than those obtained using Polybrene, especially at low MOI (90X dilution).

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T-cell expansion using RetroNectin reagent.

T-cell expansion using RetroNectin reagent.

T-cell expansion using RetroNectin reagent. Peripheral blood T cells were stimulated and expanded in vitro using anti-CD3 (clone OKT3) and IL-2 alone or in combination with RetroNectin reagent (RN), anti-CD28, or anti-4-1BB. Stimulation with RetroNectin reagent yielded higher proportions of cytotoxic CD8+ T cells.


Frequently asked questions about RetroNectin recombinant fibronectin

FAQs about RetroNectin reagent

What is RetroNectin reagent and how does it work? Which cell types can benefit from RetroNectin-assisted viral transduction? Find answers to your questions and more!

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Takara Bio USA, Inc. provides kits, reagents, instruments, and services that help researchers explore questions about gene discovery, regulation, and function. As a member of the Takara Bio Group, Takara Bio USA is part of a company that holds a leadership position in the global market and is committed to improving the human condition through biotechnology. Our mission is to develop high-quality innovative tools and services to accelerate discovery.

FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES (EXCEPT AS SPECIFICALLY NOTED).

Clontech, TaKaRa, cellartis

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  • COVID-19 research
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  • COVID-19 research
  • Viral detection with qPCR
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  • Viral RNA isolation
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  • Real-time PCR
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  • Reverse transcription prior to qPCR
  • High-throughput qPCR solutions
  • RNA extraction and analysis for real-time qPCR
  • Stem cell research
  • Media and supplements
  • Stem cells and stem cell-derived cells
  • Single-cell cloning of edited hiPS cells
  • mRNA and cDNA synthesis
  • In vitro transcription
  • cDNA synthesis kits
  • Reverse transcriptases
  • RACE kits
  • Purified cDNA & genomic DNA
  • Purified total RNA and mRNA
  • PCR
  • Most popular polymerases
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  • GC rich PCR
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  • Nucleic acid purification
  • Plasmid purification kits
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  • RNA purification kits
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  • Tet-inducible expression systems
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  • Free sample: PrimePath Direct Saliva SARS-CoV-2 Detection Kit
  • TALON his-tag purification resin special offer
  • GoStix Plus special offers
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  • Gene function
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  • Viral transduction
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  • APPLICATIONS
  • Molecular diagnostics
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  • Vaccine development
  • Characterizing the viral genome and host response
  • Identifying and cloning vaccine targets
  • Expressing and purifying vaccine targets
  • Immunizing mice and optimizing vaccine targets
  • Pathogen detection
  • Sample prep
  • Detection methods
  • Identification and characterization
  • SARS-CoV-2
  • Antibiotic-resistant bacteria
  • Food crop pathogens
  • Waterborne disease outbreaks
  • Viral-induced cancer
  • Immunotherapy research
  • T-cell therapy
  • Antibody therapeutics
  • T-cell receptor profiling
  • TBI initiatives in cancer therapy
  • Cancer research
  • Sample prep from FFPE tissue
  • Sample prep from plasma
  • Cancer biomarker discovery
  • Cancer biomarker quantification
  • Single cancer cell analysis
  • Cancer genomics and epigenomics
  • HLA typing in cancer
  • Gene editing for cancer therapy/drug discovery
  • Alzheimer's disease research
  • Antibody engineering
  • Sample prep from FFPE tissue
  • Single-cell sequencing
  • Reproductive health technologies
  • Preimplantation genetic testing
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