[The expression of programmed death receptor 1 in non-small cell lung cancer and its clinicopathological features and prognosis showed a connection with epidermal growth factor receptor gene mutations].

Objective: To investigate the relationships between the expression of programmed death 1 (PD-1) and the epidermal growth factor receptor (EGFR) gene mutations in non-small cell lung cancer (NSCLC). The study also attempted to investigate the clinicopathological features and prognosis in NSCLC patients. Methods: The expression of PD-1 protein in 88 cases of NSCLC tumor tissues and adjacent tissues was detected by immunohistochemistry. The mutations of EGFR in NSCLC were detected by Polymerase Chain Reaction-Amplification Refractory Mutation System(PCR-ARMS) method. The expression of PD-1 and patients' clinical characteristics and prognosis were analyzed. Results: PD-1 was positive in 63.6%(56/88) NSCLC tumor tissues, which was significantly higher than that in adjacent normal tissues (21.6%, 19/88) (P<0.05). EGFR gene mutations were found in 43 cases (48.9%), in which 30 cases (69.8%)were PD-1 positive expression. 45 cases had the wild types of EGFR gene, in which 26 cases (57.8%) were PD-1 positive. There were 24 cases of 19Del EGFR mutations, including 20 cases (83.3%) of PD-1 positive expression. 19 patients had 21L858 EGFR mutations, including 10 cases (52.6%) of PD-1 positive expression. The expression of PD-1 in NSCLC was related to patients' smoking status, lymph node metastasis and EGFR gene mutations (P<0.05). The median progression-free survival time of patients with PD-1 positive and negative expression was 7.03 and 18.66 months, respectively (P=0.007). In patients with wild-type EGFR gene, the median progression-free survival time of PD-1 positive and negative expression was 25.21 and 38.24 months, respectively. The difference was statistically significant (P=0.024). The median progression-free survival time in 43 cases of EGFR mutant patients with PD-1 positive and negative expression was 21.23 and 31.44 months. The difference was not statistically significant (P=0.128). Conclusions: PD-1 expresses in both EGFR mutant and wild-type NSCLC, and its expression levelis different with various EGFR mutations. The expression of PD-1 in NSCLC is related to the prognosis of patients, and the prognosis of patients with positive PD-1 expression was poor.

High efficiency reconstitution of a human-mouse chimeric Fab of CAb-1 antibody specific to human colon cancer.

Monoclonal antibody (mAb) has been widely applied in the treatment of human diseases, especially in malignant tumours. However, most antibodies produced in mouse by hybridoma technology might induce severe human anti-mouse reactions. We had reported a murine mAb CAb-1 of therapeutic interest for its specifically binding to a cell surface glycoprotein of human colon cancer. Here, we attempted to generate a reconstituted human-mouse chimeric Fab (cFab) of CAb-1 in vitro to reduce its antigenicity and increase its capacity of penetration. First, the genes of heavy and light chain variable region (VH, VL) of CAb-1 were cloned. Then, the chimeric light chain (cL) and Fd (cFd) were constructed and expressed in Escherichia coli. Finally, the reconstituted cFab was obtained by gradient dialysis of the mixture of cFd and cL. SDS-PAGE and western blot analysis showed the reconstituted cFab with a recovery rate of 70.2% when the initial total concentration of cL and cFd proteins to be 100 microg/ml. The reconstituted cFab maintained the affinity and specificity to colon cancer cells compared with its parental antibody as determined by immunostaining analysis, FACS and ELISA. Our results established a foundation for further application of the cFab in diagnosis and treatment of colon cancer.

Epitope mapping of series of monoclonal antibodies against the hepatocellular carcinoma-associated antigen HAb18G/CD147.

The hepatocellular carcinoma-associated antigen HAb18G/CD147, a member of CD147 family, could promote tumour invasion and metastasis via inducing the secretion of matrix metalloproteinases (MMP). Anti-CD147 monoclonal antibodies (MoAb) have exhibited obvious inhibitory effect on MMP induction. However, none of the epitopes of these MoAb has been reported. We previously prepared five MoAb against HAb18G/CD147, named HAb18, 3B3, 1B3, 5A5 and 4D2. To map the epitopes of these MoAb, a series of truncated fragments of extracellular region of HAb18G/CD147 was expressed in Escherichia coli and the MoAb-binding affinity to these fragments was examined with an enzyme-linked immunosorbent assay and Western blot. The residues (39)LTCSLNDSATEV(50), (36)KILLTCS(42) and (22)AAGTVFTTVEDL(33) were determined to be the epitopes of HAb18, 3B3 and 1B3, respectively, which were further proved by a dot-blot analysis with synthesized peptides and bioinformatics epitope prediction. The binding regions of MoAb 5A5 and 4D2 were located at residues E(120)-R(203). Then we constructed and expressed full-length HAb18G/CD147 and truncated HAb18G/CD147 without residues A(22)-V(50) in COS-7 cells. Gelatin zymography and Boyden chamber assay showed that the COS-7 cells expressing truncated HAb18G/CD147 failed to induce MMP production and enhance the cells' invasive potential, compared with the cells expressing full-length HAb18G/CD147. Taken together with the obviously inhibitory effects of HAb18 on the function of full-length HAb18G/CD147, these findings suggest that residues (22)AAGTVFTTVEDLGSKILLTCSLNDSATEV(50) may play a critical role in the functions of HAb18G/CD147 on MMP secretion and tumour invasion. These key residues can be used as potential drug target in cancer therapy.