Progress towards the Replacement of the Rabbit Blood Sugar Test for the Quantitative Determination of the Biological Activity of Insulins (USP <121>) with an In Vitro Assay.

For the quantification of insulin activity, United States Pharmacopeia (USP) general chapter <121> continues to require the rabbit blood sugar test. For new insulin or insulin analogue compounds, those quantitative data are expected for stability or comparability studies. At Sanofi, many rabbits were used to fulfil the authority's requirements to obtain quantitative insulin bioactivity data until the in vivo test was replaced. In order to demonstrate comparability between the in vivo and in vitro test systems, this study was designed to demonstrate equivalency. The measurement of insulin lispro and insulin glargine drug substance and drug product batches, including stress samples (diluted or after temperature stress of 30 min at 80 °C), revealed a clear correlation between the in vitro and in vivo test results. The recovery of quantitative in vitro in-cell Western (ICW) results compared to the in vivo test results was within the predefined acceptance limits of 80% to 125%. Thus, the in vitro ICW cell-based bioassay leads to results that are equivalent to the rabbit blood sugar test per USP <121>, and it is highly suitable for insulin activity quantification. For future development compounds, the in vitro in-cell Western cell-based assay can replace the rabbit blood sugar test required by USP <121>.

Claudin-18 splice variant 2 is a pan-cancer target suitable for therapeutic antibody development.

PURPOSE: Antibody-based cancer therapies have emerged as the most promising therapeutics in oncology. The purpose of this study was to discover novel targets for therapeutic antibodies in solid cancer. EXPERIMENTAL DESIGN: We combined data mining and wet-bench experiments to identify strictly gastrocyte lineage-specific cell surface molecules and to validate them as therapeutic antibody targets. RESULTS: We identified isoform 2 of the tight junction molecule claudin-18 (CLDN18.2) as a highly selective cell lineage marker. Its expression in normal tissues is strictly confined to differentiated epithelial cells of the gastric mucosa, but it is absent from the gastric stem cell zone. CLDN18.2 is retained on malignant transformation and is expressed in a significant proportion of primary gastric cancers and the metastases thereof. In addition to its orthotopic expression, we found frequent ectopic activation of CLDN18.2 in pancreatic, esophageal, ovarian, and lung tumors, correlating with distinct histologic subtypes. The activation of CLDN18.2 depends on the binding of the transcription factor cyclic AMP-responsive element binding protein to its unmethylated consensus site. Most importantly, we were able to raise monoclonal antibodies that bind to CLDN18.2 but not to its lung-specific splice variant and recognize the antigen on the surface of cancer cells. CONCLUSIONS: Its highly restricted expression pattern in normal tissues, its frequent ectopic activation in a diversity of human cancers, and the ability to specifically target this molecule at the cell surface of tumor cells qualify CLDN18.2 as a novel, highly attractive pan-cancer target for the antibody therapy of epithelial tumors.

Identification of tumor-associated autoantigens with SEREX.

Serological analysis of tumor antigens by recombinant cDNA expression cloning (SEREX) allows the systematic cloning of tumor antigens recognized by the spontaneous autoantibody repertoire of cancer patients. For SEREX, cDNA expression libraries are constructed from fresh tumor specimens, packaged into lambda-phage vectors, and expressed recombinantly in Escherichia coli. Recombinant proteins expressed during the lytic infection of bacteria are transferred onto nitrocellulose membranes to be probed with diluted autologous patient serum for identification of clones reactive with high-titered IgG antibodies. This chapter describes the SEREX technology in detail.

GBP-5 splicing variants: New guanylate-binding proteins with tumor-associated expression and antigenicity.

We have identified a new gene, gbp-5, with high homology to the guanylate binding proteins (GBP) belonging to the GTPase superfamily including the ras gene. gbp-5 is transcribed at least into three splicing variants (gbp-5a, -5b, and -5ta) leading to two different proteins (GBP-5a/b, GBP-5ta). GBP-5ta is C-terminally truncated by 97aa and has therefore lost its isoprenylation site. Although RT-PCR results indicated expression of GBP-5 members in selected normal tissues, western blotting using two newly generated antibodies revealed that expression of both proteins is restricted to peripheral blood monocytes with GBP-5ta at lower levels. In contrast, cutaneous T-cell lymphoma (CTCL) tumor tissues (seven of seven) were positive solely for GBP-5ta, and four of four CTCL cell lines expressed both proteins. Eight of nine melanoma cell lines expressed GBP-5a/b and four of nine additionally low levels of GBP-5ta. SEREX retesting using CTCL sera indicated a higher immunogenicity for GBP-5ta (nine of 16) than for GBP-5a/b (two of 11). Treatment of CTCL cell lines with interferon-gamma did not alter protein expression of GBP-5ta or GBP-5a/b. The restricted expression pattern of both GBP-5ta and GBP-5a/b and the pivotal role of many known members of the GTP-binding proteins in proliferation and differentiation suggest possible cancer-related functions of gbp-5.

Tissue expression and sero-reactivity of tumor-specific antigens in colorectal cancer.

The expression of 14 individual and two groups of tumor antigens was characterized for colorectal carcinoma by RT-PCR using 26 colorectal carcinoma specimens, eight cell lines, six samples of patients with inflammatory bowl diseases, and nine specimens from different locations of an individual patient with a metastasized rectal carcinoma. The most frequently detected mRNAs were MAGE-A1 (58%), GAGE-3-7 (54%), and cTAGE-5a (31%). At medium frequencies (12-19%) we found cTAGE-1, MAGE-A2, se57-1, RAGE-4, and GAGE-1,2,8, while other tumor antigens were expressed rarely (<9%). 85% of the samples were positive for at least one of the most frequently expressed antigens. Using a secondary SEREX approach and sera of eight colorectal cancer patients we found reactive antibodies against recombinant cTAGE-1 (2 sera), se57-1 (2), truncated GAGE (1), and MAGE-A1 (1). We conclude that certain cancer-germline genes can be detected in colorectal cancer and might therefore be promising targets for immunotherapy.

cTAGE: a cutaneous T cell lymphoma associated antigen family with tumor-specific splicing.

cTAGE-1 is a cutaneous-T-cell-lymphoma-specific tumor antigen recently identified by serologic identification of antigens by recombinant expression cloning. This study was aimed at identifying and characterizing related genes. Rapid amplification of cDNA ends and DNA screening led to five new members of the cTAGE gene family belonging to four different genes, two of which were differentially spliced (cTAGE-1/2 and cTAGE-5). Expression analysis using reverse transcription polymerase chain reaction revealed that cTAGE-1, cTAGE-1B, and cTAGE-5A expression was restricted to testis and tumor tissues, whereas the other cTAGE members were found in two to eight other normal tissues (of 27 tissues tested). Tumor-specific protein expression of cTAGE-5 was confirmed by Western blotting. Sero-reactivity against cTAGE-1, cTAGE-4, cTAGE-5A, and cTAGE-5B was found only in tumor patients (cutaneous T cell lymphoma and melanoma). The immunogenic epitope of cTAGE-1 was determined by using epitope mapping and sera of two cutaneous T cell lymphoma patients. Moreover, cTAGE-1, cTAGE-4, cTAGE-5A, and cTAGE-5B could be detected in most types of tumor tissues and cell lines at variable frequencies, including those of cutaneous T cell lymphoma, melanoma, head and neck squamous cell carcinoma, breast carcinoma, and colon carcinoma. We conclude that cTAGE-1 and cTAGE-5 are new cancer germline antigens and that tumor-specific splicing of cTAGE genes may lead to further candidate proteins for specific immunotherapy of cutaneous T cell lymphoma and other malignancies.

Tumor-specific antigens in cutaneous T-cell lymphoma: expression and sero-reactivity.

Cutaneous T-cell lymphoma (CTCL) is a heterogeneous group of extra-nodal non-Hodgkin lymphomas with primary manifestation in the skin with poor treatment options in the advanced stages. As basis for future immune-therapeutic strategies we have investigated the possible expression of tumor-specific targets in CTCL focusing mainly on so-called cancer-germline genes. cDNAs derived from 20 CTCL tissues and 4 CTCL cell lines were tested with 15 gene-specific and 4 gene family-specific primers by RT-PCR and confirmative Northern blotting. The most frequently detected mRNAs were LAGE-1 (55% with only partial coexpression of the splicing variants), cTAGE-1 (35%), MAGE-A9 (27%) and the GAGE-3-7 group (35%). Furthermore, we could detect NY-ESO-1 (21%) and a MAGE-A subgroup (15%), whereas sub-specification of the latter proved absence of MAGE-A1, -A2, -A3, -A6 and -A12. SCP-1 was found in only one specimen and a several antigens could not been detected in any tumor tissue or cell line (MAGE-B, GAGE-1,2,8 and all 4 RAGE genes). 90% of all CTCL samples were positive for at least 1 of the frequent mRNAs in RT-PCR (LAGE-1, NY-ESO-1, cTAGE-1, MAGE-A9, or GAGE-3to7). Using a secondary SEREX approach we could detect sero-reactivity in sera of CTCL patients against recombinant cTAGE-1 (10/29), GAGE (3/19), MAGE-A1 (1/18), -A3 (1/18), -A6 (2/18) and -A9 (4/18) protein, but not against LAGE-1a, MAGE-A4b or MAGE-A12 protein (n = 19). We conclude, that certain cancer-germline genes can be detected frequently in CTCL and are able to elicit a systemic immune response. These candidate genes might therefore be promising targets for immunotherapeutic interventions in CTCL.

Tumor-associated antigens as possible targets for immune therapy in head and neck cancer: comparative mRNA expression analysis of RAGE and GAGE genes.

Specific immune therapy targeting residual areas of cancer cells may emerge as a powerful treatment strategy for head and neck squamous cell carcinoma (HNSCC). In order to define possible targets for immune therapy, we evaluated the frequency of two groups of tumor antigens-the RAGE and GAGE families-by means of reverse transcriptase polymerase chain reaction using primary HNSCCs (n = 28), mucosa specimens as normal controls (n = 10) and HNSCC cell lines (n = 6). By means of specific primer selection we could differentiate between RAGE-1, -2, -3 and -4, as well as between two groups of GAGE genes (GAGE-1,2,7 vs GAGE-3,4,5,6,8). While all mucosa tissues (from smokers and non-smokers) were negative for both antigen families, 24/28 investigated tumors were positive for up to 5 tumor antigens. Among the RAGE genes, RAGE-1-positive tumors were the most abundant (8/28), followed by RAGE-2 (7/28) and RAGE-4 (6/28). Differences in the locations of HNSCCs were reflected by different RAGE family members being expressed most frequently: larynx, RAGE-1; oropharynx, RAGE-2; and hypopharynx, RAGE-4. Primers against GAGE-1,2,7 and GAGE-3,4,5,6,8 revealed 6/27 and 16/27 positive tumors, respectively. This report suggests that RAGE genes and GAGE-3,4,5,6,8 may be promising candidates for specific immune therapy in HNSCC.

mRNA expression of tumor-associated antigens in melanoma tissues and cell lines.

Tumor-associated antigens (TAA) are increasingly used as specific targets for immune therapy of malignant melanoma. The aim of the present study was to provide a basis for selecting the most suitable TAA by analyzing the mRNA expression of a large panel of TAA by RT-PCR and Northern blotting. We have chosen primers differentiating four groups of TAA (MAGE-A, MAGE-B, and two groups of GAGE-genes) and 13 individual TAA (MAGE-A2 and -A3, RAGE-1, -2, -3, and -4, LAGE-1a and -1b, NY-ESO-1, GAGE-1, SSX-2, SCP-1, and cTAGE-1) based on most recent sequence data. In addition, the RAGE-gene family has been separated into its four members by a novel designed nested PCR, which was confirmed by Northern analysis. Furthermore, the chromosomal organization and relationship between the RAGE-family and MOK was analyzed. RAGE-4 mRNA could be shown for the first time to be present in testis tissue. The most frequently expressed TAA were the MAGE-A and the GAGE-3,-4,-5,-6,-8 group, whereas among individual TAA MAGE-A2, -A3, RAGE-1, -3, and LAGE-1a/b were found within most specimens and are thus promising candidates for immune therapy. In comparison, melanoma metastatic specimens and cell lines show similar profiles of TAA expression, but individual TAA differ notably between both types of samples indicating that results from cell lines are not always applicable to tumor specimen.