Identification of a panel of tumor-associated antigens from breast carcinoma cell lines, solid tumors and testis cDNA libraries displayed on lambda phage

Abstract

Background: Tumor-associated antigens recognized by humoral effectors of the immune system are a very attractive target for human cancer diagnostics and therapy. Recent advances in molecular techniques have led to molecular definition of immunogenic tumor proteins based on their reactivity with autologous patient sera (SEREX).

Methods: Several high complexity phage-displayed cDNA libraries from breast carcinomas, human testis and breast carcinoma cell lines MCF-7, MDA-MB-468 were constructed. The cDNAs were expressed in the libraries as fusion to bacteriophage lambda protein D. Lambda-displayed libraries were efficiently screened with sera from patients with breast cancer.

Results: A panel of 21 clones representing 18 different antigens, including eight proteins of unknown function, was identified. Three of these antigens (T7-1, T11-3 and T11-9) were found to be overexpressed in tumors as compared to normal breast. A serological analysis of the 21 different antigens revealed a strong cancer-related profile for at least five clones (T6-2, T6-7, T7-1, T9-21 and T9-27).

Conclusions: Preliminary results indicate that patient serum reactivity against five of the antigens is associated with tumor disease. The novel T7-1 antigen, which is overexpressed in breast tumors and recognized specifically by breast cancer patient sera, is potentially useful in cancer diagnosis.

Figure 1

Cloning sites of λKM8 and λKM10 vectors.

Figure 2

Insert length distribution. Forty-eight random clones from T5 library were amplified by PCR using a couple of primers on the sides of insert. Size of inserts was calculated according to their electrophoretic mobility in 3% agarose gel.

Figure 3

Scheme of selection strategy leading to TAA identification. A phage-displayed tumor cDNA library is preincubated with patient serum. TAA-specific antibodies bind to antigens exposed on the phage surface. Abs-phage complex is captured by protein A-coated solid support (ELISA plate or dynebeads). Non-bound phage are washed away. Bound phage are eluted by infection of added bacteria and amplified. Positive clones are isolated by immunoscreening procedure and then picked in ordered array on a bacterial lawn, transferred to nitrocellulose membrane and probed with different positive and negative sera.

Figure 4

Four identified antigen sequences with partial homology to reverse transcriptase homolog. Peptide sequence is reported in single-letter code. Identical amino acids in the selected clones and reverse transcriptase homolog are represented by a dash. These clones were isolated from libraries of different origin. Clones T9-21 and T9-27, isolated from solid tumor library, had significantly high frequency of reactivity with sera from breast cancer patients.

Figure 5

cDNA-PCR analysis of gene expression was done using specific sequence primers. We used SMART cDNAs from 7–10 different tumor samples (patients B84, B85, B87, B89, B90, B91, B92, B93, B95, B96) as template, from single metastasized lymph node indicated as LMB82 (patient B82) and from normal breast, normal testis, lymphocytes from healthy donors. cDNAs were normalized by amplification of β-actin gene. There are agarose gels with ubiquitously-expressed genes in Figure 5A, underexpressed genes in Figure 5B, overexpressed ones in Figure 5C.