Induction of anti-melanoma CTL response using DC transfected with mutated mRNA encoding full-length Melan-A/MART-1 antigen with an A27L amino acid substitution.
Modification of the parental immunodominant Melan-A/MART-1 peptide (MART-1(26-35)) by replacing the alanine with leucine (A27L) enhances its immunogenicity. Because of the reported advantages of RNA over peptides in DC vaccines, we sought to mutate the MART-1 gene to encode a full-length MART-1 antigen with an A27L amino acid substitution. Human DC were transfected with A27L-mutated MART-1 RNA (A27L RNA) or native MART-1 RNA, and then used to stimulate autologous T cells from a series of 8 HLA-A2+ volunteers. After three stimulations, all CTL induced with DC/A27L RNA exhibited more tetramer+ cells, and demonstrated stronger antigen-specific IFNgamma-secreting activity compared to CTL induced with DC/native RNA. A potent MART-1-specific, and predominantly class-I-restricted lysis was detected in most CTL induced with DC/A27L RNA, while native RNA-induced CTL showed minimal and non-specific lysis. HLA-A2+ DC and MART-1 negative/A2+ melanoma cells transfected with the A27L RNA were recognized and killed by MART-1-specific CTL, suggesting that these APC efficiently processed the A27L RNA and presented correct MART-1-specific epitope(s). In summary, introducing an A27L mutation into the MART-1 full-length mRNA sequence enhanced the immunogenicity of the encoded MART-1 Ag. The ease with which such a mutation can be made in RNA presents another potential advantage of using RNA for immunotherapy. Our results support considering this strategy for enhancing the immunogenicity of DC-based RNA vaccines.
Abdel-Wahab
Zeinab
Z
Department of Surgery, Duke University Medical Center, P.O. Box 2624 MSRB, Durham, NC 27710, USA. wahab@duke.edu
Kalady
Matthew F
MF
Emani
Sirisha
S
Onaitis
Mark W
MW
Abdel-Wahab
Omar I
OI
Cisco
Robin
R
Wheless
Lee
L
Cheng
Tsung-Yen
TY
Tyler
Douglas S
DS
Pruitt
Scott K
SK
D019943:Amino Acid Substitution; D000951:Antigens, Neoplasm; D045744:Cell Line, Tumor; D003713:Dendritic Cells / Q000276:immunology*; D006801:Humans; D016130:Immunophenotyping; D007371:Interferon-gamma / Q000096:biosynthesis; D058965:MART-1 Antigen; D008545:Melanoma / Q000276:immunology*; D009154:Mutation; D009363:Neoplasm Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D012333:RNA, Messenger / Q000276:immunology*; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D014162:Transfection
D000951:Antigens, Neoplasm; D058965:MART-1 Antigen; C548667:MLANA protein, human; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D007371:Interferon-gamma