Amplification of immunoglobulin transcripts by the non-palindromic adaptor polymerase chain reaction (NPA-PCR). Nucleotide sequence analysis of two human monoclonal antibodies recognizing two cell surface antigens expressed in ovarian, cervix, breast, colon and other carcinomas.

Abstract

We have employed conventional polymerase chain reaction (PCR) and nonpalindromic adaptor PCR (NPA-PCR) for the amplification of the heavy- and light-chain transcripts of the cDNAs of two human monoclonal antibodies (MAb), designated AC6C3 (IgM, lambda) and CR4E8 (IgM, lambda), recognizing two different antigens expressed on the cell surface of human ovarian, cervix, breast, colon, melanoma and other carcinomas. With few exceptions these MAbs do not react with normal tissues. The AC6C3 MAb was developed by fusing regional lymph node lymphocytes from a patient with ovarian carcinoma with cells of the hybrid myeloma line SPAZ4, whereas the CR4E8 MAb was developed by fusing SPAZ4 cells with peripheral blood lymphocytes from a patient with cervical cancer, who was immunized intralymphatically with a viral oncolysate allogeneic tumor vaccine. The AC6C3 MAb immunoprecipitated from lysates of the ovarian tumor cell line SKOV3 a 32 kD polypeptide. The CR4E8 MAb reacted in Western blotting of lysates of the SW756 cervical carcinoma line with a 55 kD band. Cell surface immunofluorescence determinations using the fluorescence activated cell sorter revealed that both MAbs stain ovarian or cervix carcinoma tumor cell lines. We amplified the heavy chain transcripts of these two MAbs by conventional PCR, using mixed 5' end amplification primers, corresponding to the most conserved VH leader sequences and a C mu probe as a 3' end amplification primer. However, the mixed primer approach did not permit the amplification of the lambda-chain transcripts of these two human MAbs. This amplification was successfully carried out using the NPA-PCR that we have previously developed, specifically for the amplification of transcripts with unknown or variable 5' end, such as the T-cell receptors and the immunoglobulins. We have cloned and sequenced the amplified cDNAs. Sequence analysis showed that the V lambda segment of the AC6C3 MAb had 80.29% homology to the human germline Ig lambda-chain V lambda III.1, clone DPL2. The V lambda region of the CR4E8 MAb had 99.28% homology also to the human germline Ig lambda-chain, V lambda III.1, clone DPL23. The AC6C3 MAb lambda-chain employed a J segment with 98% homology to J3. The CR4E8 MAb light chain employed the J(H6-3C4) gene segment. Both MAbs utilized identical VH and JH gene segments and different DH segments. The VH regions of the AC6C3 MAb and of the CR4E8 MAb had, respectively, 98.6% and 98.98% homology to the human Ig germline heavy chain V region DP-7, a member of the VH-1 gene family.(ABSTRACT TRUNCATED AT 400 WORDS)