0
We're unable to sign you in at this time. Please try again in a few minutes.
Retry
We were able to sign you in, but your subscription(s) could not be found. Please try again in a few minutes.
Retry
There may be a problem with your account. Please contact the AMA Service Center to resolve this issue.
Contact the AMA Service Center:
Telephone: 1 (800) 262-2350 or 1 (312) 670-7827  *   Email: subscriptions@jamanetwork.com
Error Message ......
Original Investigation | Association of VA Surgeons

MAGE-A3 With Cell-Penetrating Domain as an Efficient Therapeutic Cancer Vaccine

Ramesh B. Batchu, PhD1,2; Oksana Gruzdyn, BS1,2; Ravindra B. Potti, PhD3,4; Donald W. Weaver, MD1; Scott A. Gruber, MD, PhD, MBA1,2
[+] Author Affiliations
1Laboratory of Surgical Oncology and Developmental Therapeutics, Department of Surgery, Wayne State University, Detroit, Michigan
2John D. Dingell VA Medical Center, Detroit, Michigan
3Department of Biotechnology, Srinidhi Institute of Science and Technology, Hyderabad, India
4Virocan Therapeutics Pvt Ltd, Guntur, India
JAMA Surg. 2014;149(5):451-457. doi:10.1001/jamasurg.2013.4113.
Text Size: A A A
Published online

Importance  In conjunction with chemotherapy, immunotherapy with dendritic cells (DCs) may eliminate minimal disease burden by generating cytotoxic T lymphocytes. Enhanced cytosolic bioavailability of tumor-specific antigens improves access to human leukocyte antigen (HLA) class I molecules for more efficient cytotoxic T lymphocyte generation. Various cell-penetrating domains (CPDs) are known to ferry covalently linked heterologous antigens to the intracellular compartment by traversing the plasma membrane.

Objective  To determine whether generating melanoma antigen family A, 3 (MAGE-A3), a tumor-specific cancer-testis antigen, as a fusion protein with CPD will enhance the cytosolic bioavailability of MAGE-A3.

Design  MAGE-A3 was amplified by polymerase chain reaction using complementary DNA from renal tissue and cloned in frame with a CPD (YARKARRQARR) at the amino-terminal end and hexahistidine at the carboxy-terminal end to generate CPD–MAGE-A3 in a pQE-70 expression vector. Cultures were grown in Escherichia coli BL21 Star (DE3-pLysS) cells followed by nickel–nitrilotriacetic acid affinity purification of recombinant proteins.

Main Outcomes and Measures  Measurement of DC membrane penetration of CPD–MAGE-A3 vs MAGE-A3 and determination of the effect of CPD–MAGE-A3 pulsing on DC phenotypic expression of cell-surface antigens.

Results  Media composition and isopropyl-d-thiogalactosidase induction were optimized to achieve high levels of protein expression followed by purification. Western blot analysis with MAGE-A3 antibodies recognized both MAGE-A3 and CPD–MAGE-A3 proteins, while CPD antibodies recognized only CPD–MAGE-A3. Purified CPD–MAGE-A3 exhibited more efficient DC membrane penetration than did MAGE-A3 alone as confirmed by immunofluorescence analysis. High-level expression of several unique DC markers (CD80, CD83, CD86, and HLA-DR) by flow cytometry was consistent with a mature DC phenotype, indicating that pulsing with CPD–MAGE-A3 did not alter specific cell-surface antigens required for T-cell activation.

Conclusions and Relevance  We have demonstrated for the first time, to our knowledge, that cloning and purification of MAGE-A3 with CPD enhances its cytosolic bioavailability in DCs without altering cell-surface antigens, potentially making it a more potent therapeutic cancer vaccine compared with existing MAGE-A3 protein and peptide vaccines.

Figures in this Article

Sign in

Create a free personal account to sign up for alerts, share articles, and more.

Purchase Options

• Buy this article
• Subscribe to the journal

First Page Preview

View Large
First page PDF preview

Figures

Place holder to copy figure label and caption
Figure 1.
Cloning of Melanoma Antigen Family A, 3 (MAGE-A3) and Cell-Penetrating Domain (CPD)–MAGE-A3 in pDRIVE T Shuttle Vector

Schematic representation of cloning vectors pDRIVE T–MAGE-A3 (A) and pDRIVE T–CPD–MAGE-A3 (B). Primers were designed for MAGE-A3 and CPD–MAGE-A3 without start or stop codons and with 5′ SphI and 3′ BglII ends. C, Polymerase chain reaction was performed with 30 amplification cycles (92°C for 30 seconds, 58°C for 30 seconds, 72°C for 1 minute), and products were purified from agarose gel. This experiment was repeated 3 times, with a representative gel shown. bp indicates base pairs.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 2.
Cloning of Melanoma Antigen Family A, 3 (MAGE-A3) and Cell-Penetrating Domain (CPD)–MAGE-A3 in pQE-70 Expression Vector and Selection of High-Expression Clones

Schematic representation of constructed expression vectors pQE-70–MAGE-A3 (A) and pQE-70–CPD–MAGE-A3 (B). bp indicates base pairs. C and D, We transformed BL21 Star (DE3-pLysS) cells with expression vectors. Three clones were selected, induced with 1mM isopropyl-d-thiogalactosidase at 37°C, and analyzed on 4% to 20% sodium dodecyl sulfate–polyacrylamide gel electrophoresis. This experiment was repeated 3 times, with a representative gel shown. The amount of total protein loaded was approximately 5 μg. The sizes of the recombinant proteins were approximately 35 kDa for MAGE-A3 (C) and 38 kDa for CPD–MAGE-A3 (D).

Graphic Jump Location
Place holder to copy figure label and caption
Figure 3.
Optimization of Culture Broth Medium and Isopropyl-d-thiogalactosidase (IPTG) Induction of Cell-Penetrating Domain (CPD)–Melanoma Antigen Family A, 3 (MAGE-A3) in Escherichia coli BL21 Star (DE3-pLysS) Cells

A, Culture of BL21 harboring CPD–MAGE-A3 was conducted in various bacterial growth media: (1) Superior Broth, (2) Turbo Broth, (3) 2XYT Broth, (4) Power Broth, (5) Hyper Broth, and (6) Luria-Bertani Broth. B, Protein expression analyzed 6 hours after IPTG induction at indicated concentrations. Both experiments were repeated 3 times, with representative gels shown.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 4.
Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis and Western Blot Analysis of Recombinant Melanoma Antigen Family A, 3 (MAGE-A3) and Cell-Penetrating Domain (CPD)–MAGE-A3 Expressed in Escherichia coli BL21 Star (DE3-pLysS) Cells

Control, induced, and purified recombinant proteins of both MAGE-A3 and CPD–MAGE-A3 were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (A) and subjected to Western blot analysis with MAGE-A3 (B) and CPD (C) antibodies as depicted. All experiments were repeated 3 times, with representative gel (A) and Western blots (B and C) shown.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 5.
Immunofluorescence Analysis of Dendritic Cell Membrane Penetration by Melanoma Antigen Family A, 3 (MAGE-A3) and Cell-Penetrating Domain (CPD)–MAGE-A3 Recombinant Proteins

A, Dendritic cells were pulsed for indicated times and then stained with MAGE-A3 antibody followed by visualization with fluorescence microscopy at lower magnification (original magnification ×10). B and C, Higher-magnification images show the presence of CPD–MAGE-A3 in dendritic cell cytosol (original magnification ×40 [B] and ×100 [C]). All experiments were performed in triplicate, with representative fluorescent images shown.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 6.
Flow Cytometric Analysis of Dendritic Cell Markers After Pulsing With Cell-Penetrating Domain (CPD)–Melanoma Antigen Family A, 3 (MAGE-A3)

Dendritic cells were pulsed with 3 µmol of CPD–MAGE-A3 protein, followed by intracellular staining with indicated antibody and analysis by flow cytometry. The axes show fluorescent intensity. This experiment was repeated 3 times, with representative flow diagrams shown. EGFP indicates enhanced green fluorescent protein; FITC, fluorescein isothiocyanate; and PE, phycoerythrin.

Graphic Jump Location

Tables

References

Correspondence

CME
Also Meets CME requirements for:
Browse CME for all U.S. States
Accreditation Information
The American Medical Association is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians. The AMA designates this journal-based CME activity for a maximum of 1 AMA PRA Category 1 CreditTM per course. Physicians should claim only the credit commensurate with the extent of their participation in the activity. Physicians who complete the CME course and score at least 80% correct on the quiz are eligible for AMA PRA Category 1 CreditTM.
Note: You must get at least of the answers correct to pass this quiz.
Please click the checkbox indicating that you have read the full article in order to submit your answers.
Your answers have been saved for later.
You have not filled in all the answers to complete this quiz
The following questions were not answered:
Sorry, you have unsuccessfully completed this CME quiz with a score of
The following questions were not answered correctly:
Commitment to Change (optional):
Indicate what change(s) you will implement in your practice, if any, based on this CME course.
Your quiz results:
The filled radio buttons indicate your responses. The preferred responses are highlighted
For CME Course: A Proposed Model for Initial Assessment and Management of Acute Heart Failure Syndromes
Indicate what changes(s) you will implement in your practice, if any, based on this CME course.
Submit a Comment

Multimedia

Some tools below are only available to our subscribers or users with an online account.

Web of Science® Times Cited: 1

Sign in

Create a free personal account to sign up for alerts, share articles, and more.

Purchase Options

• Buy this article
• Subscribe to the journal

Related Content

Customize your page view by dragging & repositioning the boxes below.

See Also...
Articles Related By Topic
Related Collections
PubMed Articles
Jobs
brightcove.createExperiences();