Comprehensive Discovery and Quantitation of Protein Heterogeneity via LC-MS/MS Peptide Mapping for Clone Selection of a Therapeutic Protein.

Development of biopharmaceutical production cell lines requires efficient screening methods to select the host cell line and final production clone. This is often complicated by an incomplete understanding of the relationship between protein heterogeneity and function at early stages of product development. LC-MS/MS peptide mapping is well suited to the discovery and quantitation of protein heterogeneity; however, the intense hands-on time required to generate and analyze LC-MS/MS data typically accommodates only smaller sample sets at later stages of clone selection. Here we describe a simple approach to peptide mapping designed for large sample sets that includes higher-throughput sample preparation and automated data analysis. This approach allows for the inclusion of orthogonal protease digestions and multiple replicates of an assay control that encode an assessment of accuracy and precision into the data, significantly simplifying the identification of true-positive annotations in the LC-MS/MS results. This methodology was used to comprehensively identify and quantify glycosylation, degradation, unexpected post-translational modifications, and three types of sequence variants in a previously uncharacterized non-mAb protein therapeutic expressed in approximately 100 clones from three host cell lines. Several product quality risks were identified allowing for a more informed selection of the production clone. Moreover, the variability inherent in this unique sample set provides important structure/function information to support quality attribute identification and criticality assessments, two key components of Quality by Design.

PDGF-D, a new protease-activated growth factor.

Platelet-derived growth factor (PDGF) has been directly implicated in developmental and physiological processes, as well as in human cancer, fibrotic diseases and arteriosclerosis. The PDGF family currently consists of at least three gene products, PDGF-A, PDGF-B and PDGF-C, which selectively signal through two PDGF receptors (PDGFRs) to regulate diverse cellular functions. After two decades of searching, PDGF-A and B were the only ligands identified for PDGFRs. Recently, however, database mining has resulted in the discovery of a third member of the PDGF family, PDGF-C, a functional analogue of PDGF-A that requires proteolytic activation. PDGF-A and PDGF-C selectively activate PDGFR-alpha, whereas PDGF-B activates both PDGFR-alpha and PDGFR-beta. Here we identify and characterize a new member of the PDGF family, PDGF D, which also requires proteolytic activation. Recombinant, purified PDGF-D induces DNA synthesis and growth in cells expressing PDGFRs. In cells expressing individual PDGFRs, PDGF-D binds to and activates PDGFR-beta but not PDGFR-alpha. However, in cells expressing both PDGFRs, PDGF-D activates both receptors. This indicates that PDGFR-alpha activation may result from PDGFR-alpha/beta heterodimerization.

Identification of a novel human fibroblast growth factor and characterization of its role in oncogenesis.

The fibroblast growth factor (FGF) family of signaling molecules has been implicated in normal developmental and physiological processes, as well as in human malignancy. Using a homology-based genomic DNA mining process, we identified a human gene encoding a novel member of the FGF family, that we designate FGF-20. The FGF-20 cDNA was isolated, and its sequence confirmed the gene prediction. FGF-20 is expressed in normal brain, particularly the cerebellum, and in some cancer cell lines. Recombinant FGF-20 protein induces DNA synthesis in a variety of cell types and is recognized by multiple FGF receptors. Ectopic expression of FGF-20 in NIH 3T3 cells renders the cells transformed in vitro and tumorigenic in nude mice. These results underscore the utility of mining genomic DNA databases and reveal FGF-20 to be a novel oncogene that may play a role in human cancer.

The hereditary renal cell carcinoma 3;8 translocation fuses FHIT to a patched-related gene, TRC8.

The 3;8 chromosomal translocation, t(3;8)(p14.2;q24.1), was described in a family with classical features of hereditary renal cell carcinoma. Previous studies demonstrated that the 3p14.2 breakpoint interrupts the fragile histidine triad gene (FHIT) in its 5' noncoding region. However, evidence that FHIT is causally related to renal or other malignancies is controversial. We now show that the 8q24.1 breakpoint region encodes a 664-aa multiple membrane spanning protein, TRC8, with similarity to the hereditary basal cell carcinoma/segment polarity gene, patched. This similarity involves two regions of patched, the putative sterol-sensing domain and the second extracellular loop that participates in the binding of sonic hedgehog. In the 3;8 translocation, TRC8 is fused to FHIT and is disrupted within the sterol-sensing domain. In contrast, the FHIT coding region is maintained and expressed. In a series of sporadic renal carcinomas, an acquired TRC8 mutation was identified. By analogy to patched, TRC8 might function as a signaling receptor and other pathway members, to be defined, are mutation candidates in malignant diseases involving the kidney and thyroid.

Chromosome 3p14 homozygous deletions and sequence analysis of FRA3B.

Loss of heterozygosity (LOH) involving 3p occurs in many carcinomas but is complicated by the identification of four distinct homozygous deletion regions. One putative target, 3p14.2, contains the common fragile site, FRA3B, a hereditary renal carcinoma-associated 3;8 translocation and the candidate tumor suppressor gene, FHIT. Using a approximately 300 kb comsid/lambda contig, we identified homozygous deletions in cervix, breast, lung and colorectal carcinoma cell lines. The smallest deletion (CC19) was shown not to involve FHIT coding exons and no DNA sequence alterations were present in the transcript. We also detected discontinuous deletions as well as deletions in non-tumor DNAs, suggesting that FHIT is not a selective target. Further, we demonstrate that some reported FHIT aberrations represent normal splicing variation. DNA sequence analysis of 110 kb demonstrated that the region is high in A-T content, LINEs and MER repeats, whereas Alu elements are reduced. We note an intriguing similarity in repeat sequence composition between FRA3B and a 152 kb segment from the Fragile-X region. We also identified similarity between a FRA3B segment and a small polydispersed circular DNA. In contrast to the selective loss of a tumor suppressor gene, we propose an alternative hypothesis, that some putative targets including FRA3B may undergo loss as a consequence of genomic instability. This instability is not due to DNA mismatch repair deficiency, but may correlate in part with p53 inactivation.

NotI linking/jumping clones of human chromosome 3: mapping of the TFRC, RAB7 and HAUSP genes to regions rearranged in leukemia and deleted in solid tumors.

By applying the 'recognition mask' strategy to 300 mammalian sequences containing NotI sites we demonstrated that 5' ends of genes are highly enriched in NotI sites. A NotI linking clone NL2-252 (D3S1678) containing transferrin receptor (TFRC) gene was used as an initial point for chromosomal jumping. One of the jumping clones, J21-045 traverses 210 kbp and links NL2-252 to NL26 (D3S1632), a NotI linking clone containing highly polymorphic sequences. The TFRC gene was mapped to 3q29, close to the telomeric marker D3S2344, by linkage analysis, a panel of hybrid cell lines, GeneBridge 4 panel and FISH. Clone NLM-007 (D3S4302) was found to contain ras-homologous gene RAB7. By FISH and a panel of hybrid cell lines this gene was mapped to 3q21. This region is of particular interest due to frequent rearrangements in different types of leukemia. Clone L2-081 (D3S4283) containing new member of ubiquitin-specific proteases (HAUSP gene) was localized in 3p21 inspiring further investigation of involvement of this gene in development of lung and renal carcinomas.

Direct cloning of DNA sequences from the common fragile site region at chromosome band 3p14.2.

Despite several lines of evidence suggesting that common chromosomal fragile sites are biologically important as hot spots for recombination, their structure remains unknown. We showed previously that the plasmid pSV2neo preferentially integrates into bands containing fragile sites in cells transfected under conditions of fragile site induction. Here we report the isolation and characterization of the DNA sequences from two such independent integrations into 3p14.2, a common fragile site (FRA3B). These FRA3B region sequences were shown to lie within a 1330-kb YAC, 850A6, approximately 350 kb telomeric of the breakpoint of t(3;8), a constitutional rearrangement. The two integration sites are 10 kb apart, but each integration is associated with a deletion. We have constructed a partial genomic contig of the integration sites and deleted regions spanning approximately 85 kb. Analysis of the DNA sequences immediately surrounding the plasmid integrations revealed no known coding sequences or repeat structures resembling the (CGG)n motif characteristic of the rare fragile sites. In addition, by Southern blotting analysis, none of the phage clones isolated from the FRA3B region were found to contain CGG repeats. Fluorescence in situ hybridization analysis of genomic clones from this contig to metaphase cells induced to express breaks demonstrated hybridization adjoining the chromosome breaks, and occasionally the hybridization signal spanned the break. The results imply that breakage occurs at variable positions within a large region (at least on the order of 85 kb). Together, these data suggest that the structure of FRA3B differs from that of rare fragile sites.

Distinct 3p21.3 deletions in lung cancer and identification of a new human semaphorin.

Loss of chromosome 3p is a critical event in the pathogenesis of lung cancer. Overlapping homozygous 3p21.3 deletions in lung cancer cell lines involving GNAI2 were characterized and found to involve a region of genomic instability. A new widely expressed Semaphorin, H.SemaIV, was isolated from the GNAI2 deletion region. Reduced H.SemaIV expression allowed identification of additional cell lines with submicroscopic or larger deletions of the locus which occurred in a heterogeneous manner. We also demonstrate the presence of a distinct 3p21.3 homozygous deletion region, adjacent to the DNA mismatch repair gene, hMLH1, and identified deletions in direct tumors. This appears to represent one of the first demonstrations of homozygous deletions affecting 3p in direct lung tumors.

Integrated YAC contig containing the 3p14.2 hereditary renal carcinoma 3;8 translocation breakpoint and the fragile site FRA3B.

An extended YAC contig has been developed for the 3p14 region containing the hereditary renal carcinoma 3;8 translocation breakpoint and the 3p14.2 fragile site FRA3B. This region of chromosome 3 has been implicated by chromosomal translocation, deletion, and loss of heterozygosity in the pathogenesis of several malignant diseases. The contig allows accurate positioning of candidate genes, polymorphic markers, and other 3p rearrangements within this region. The contig, spanning approximately 6 Mb of DNA, contains 51 YACs identified by 27 markers, including a subset of CA repeats located in the 3p14.1-14.2 interval. The order of CA microsatellites, derived from marker content of the YACs, is in agreement with the order previously determined by genetic linkage studies. We find that the protein-tyrosine phosphatase gamma gene, PTPRG, is located minimally 1 Mb proximal to the t(3;8) breakpoint. The more proximal 3p homozygous deletion in the small-cell lung cancer cell line, U2020, is more than 5 Mb from the site of the 3;8 translocation. This integrated physical and genetic map provides a framework for further investigations of malignant diseases associated with proximal 3p loss. In addition, the positioning of separate 3p14.2 aphidicolin-induced breakpoints suggests that FRA3B may represent a region rather than a single site.

Multicolor FISH mapping of YAC clones in 3p14 and identification of a YAC spanning both FRA3B and the t(3;8) associated with hereditary renal cell carcinoma.

Human chromosome band 3p14 contains two tightly linked cytogenetic markers of broad interest, FRA3B and the t(3;8) breakpoint associated with hereditary renal cell carcinoma (RCC). The common fragile site at 3p14.2 (FRA3B) is the most sensitive site on normal human chromosomes to breakage when DNA replication is perturbed by aphidicolin or folate stress. The t(3;8)(p14.2;q24.1) translocation segregates with RCC in a large family and could mark the location of a tumor suppressor gene involved in renal cancers. In studies aimed at positional cloning of FRA3B and the t(3;8) breakpoint, we have used multicolor fluorescence in situ hybridization analysis (FISH) on metaphase spreads and interphase nuclei to order 14 yeast artificial chromosomes (YACs) in 3p14. The YACs used in this study were identified by a group of unordered lambda clones that had been previously localized to the 3p14 region and mapped proximal or distal to the t(3;8) breakpoint. FISH analysis was used to order the YACs and to map them in relation both to the t(3;8) translocation breakpoint and to FRA3B induced on normal chromosomes by treatment with aphidicolin. YACs that closely flanked both the t(3;8) translocation breakpoint and the fragile site were identified. A YAC walk from the closest distal YAC allowed the identification of a 1.3-Mb YAC derived from the CEPH large insert YAC library that spans both the FRA3B and the t(3;8) breakpoint. The order of the YACs and cytogenetic landmarks in 3p14 is cen-(126E1/230B9)-181H6-B15-D20F4-258B7-++ +280D2-70E12-168A8- 403B2-143C5-413C6-468B10-[850A6/t(3;8)/ FRA3B]-74B2.(ABSTRACT TRUNCATED AT 250 WORDS)

Somatic cell hybrid panel and NotI linking clones for physical mapping of human chromosome 3.

To identify by positional cloning the putative tumor-suppressor genes on the short arm of human chromosome 3 that are involved in the initiation or progression of several human malignancies, we have developed human mouse somatic cell hybrids and NotI linking libraries. The somatic cell hybrids contain either the intact human chromosome 3 or its derivatives as the only human genetic material in rodent background. The somatic cell hybrid panel defines five chromosomal regions on the short arm and two chromosomal regions on the long arm of chromosome 3. Two hundred sixty-one NotI linking probes from three independently constructed linking libraries were assigned to the seven chromosomal regions. The somatic cell hybrid panel and the regionally localized NotI linking probes should facilitate the construction of genetic linkage and physical maps to identify various tumor-suppressor and disease-related genes not only on the chromosome 3p, but on the entire chromosome.

Positional cloning of the hereditary renal carcinoma 3;8 chromosome translocation breakpoint.

The chromosome (p14.2;q24.1) translocation t(3;8) has been associated with hereditary renal cancer in one family. Based on cytogenetic analyses and loss-of-heterozygosity experiments, the 3p14 region has been independently implicated as harboring a tumor suppressor gene critical to kidney and lung cancer development. The 3p14.2 region also contains FRA3B, the most sensitive fragile site induced by aphidicolin. A chromosome 3 probe, R7K145, derived from a radiation-reduced hybrid was positioned between the t(3;8) breakpoint and an aphidicolin-induced 3p14 breakpoint. A yeast artificial chromosome (YAC) contig containing R7K145 was developed that crossed the aphidicolin-induced breakpoint on its telomeric side. A subsequent chromosome walk identified a YAC that crossed the 3;8 translocation breakpoint. A lambda sublibrary allowed isolation of clones spanning the rearrangement. Unique and evolutionarily conserved DNA sequences were used to screen a kidney cDNA library. We have identified a gene, referred to as HRCA1 (hereditary renal cancer associated 1), that maps immediately adjacent to the breakpoint. On the basis of its chromosomal position, HRCA1 may be a candidate tumor suppressor gene.

Alu-PCR approach to isolating NotI-linking clones from the 3p14-p21 region frequently deleted in renal cell carcinoma.

In the mammalian genome CpG islands are associated with functional genes and cloning of these islands could be an alternative approach for cloning functional genes. Recently we have developed a new approach for cloning CpG islands and constructing NotI linking libraries. We have initiated the construction of a NotI restriction map for chromosome 3, especially focusing on the rearrangements in the 3p14-p21 region, which are associated with different malignancies. CpG islands from this region are useful for isolation of candidate tumor suppressor genes that map to this region and for isolating NotI-linking clones from 3p14-p21 for mapping purposes. Here we suggest a modification of Alu-PCR as an approach to isolating NotI sites (e.g., CpG islands) from defined regions of the chromosome. Instead of using whole chromosomal DNA for Alu-PCR, we have used representative NotI-linking libraries from hybrid cell lines containing either whole or deleted human chromosome 3 (MCH903.1 and MCH924.4, respectively). This decreases the complexity of the Alu-PCR products 10-100 times compared to the whole human genome. Using this modification, we can isolate NotI-linking clones, which are natural markers on the chromosome, rather than random genomic fragments. Among eight clones selected by this method, seven were from the region deleted in MCH924.4. The results clearly demonstrate the feasibility of Alu-PCR for isolating CpG islands from defined regions of the genome.

New strategy for mapping the human genome based on a novel procedure for construction of jumping libraries.

A novel procedure for construction of jumping libraries is described. The essential features of this procedure are as follows: (1) two diphasmid vectors (lambda SK17 and lambda SK22) are simultaneously used in the library construction to improve representativity, (2) a partial filling-in reaction is used to eliminate cloning of artifactual jumping clones and to obviate the need for a selectable marker. The procedure has been used to construct a representative human NotI jumping library (220,000 independent recombinant clones) from the lymphoblastoid cell line CBMI-Ral-STO, which features a low level of methylation of its resident EBV genomes. A human chromosome 3-specific NotI jumping library (500,000 independent recombinant clones) from the human chromosome 3 x mouse hybrid cell line MCH 903.1 has also been constructed. Of these recombinant clones 50-80% represent jumps to the neighboring cleavable NotI site. With our previously published method for construction of linking libraries this procedure makes a new genome mapping strategy feasible. This strategy includes the determination of tagging sequences adjacent to NotI sites in random linking and jumping clones. Special features of the lambda SK17 and lambda SK22 vectors facilitate such sequencing. The STS (sequence tagged site) information obtained can be assembled by computer into a map representing the linear order of the NotI sites for a chromosome or for the entire genome. The computerized mapping data can be used to retrieve clones near a region of interest. The corresponding clones can be obtained from the panel of original clones, or necessary probes can be made from genomic DNA by PCR.

Involvement of 3p deletions in sporadic and hereditary forms of renal cell carcinoma.

Deletions of the short arm of chromosome 3 and associated allele losses have been reported in the majority of sporadic renal cell carcinomas (RCC). On the basis of the combined cytogenetic and molecular data, it is reasonable to assume that a putative RCC locus, which contributes to tumor development by its loss, is located telomerically of the D3F15S2 site. Using H3E4, a D3F15S2-specific probe, we have isolated a cDNA clone (cl.4-2), and a sequence comparison revealed that the cDNA clone corresponds to the human acyl-peptide hydrolase gene. The gene is fairly universally expressed, but in RCC biopsies its expression is severely reduced, compared to the normal kidney. Cl.4-2 was used for in situ hybridization on metaphase chromosomes prepared from an Epstein-Barr virus (EBV) transformed lymphoblastoid cell line, derived from a t(3;8) (p14.2;q24.1) carrying member of the RCC family described by Cohen et al. in 1979 (N Engl J Med: 301:592-595). Carriers of this translocation regularly develop RCC by middle age. We now report that D3F15S2 is localized on the telomeric side of the constitutional breakpoint, in 3p21. The region of 3p affected by this familial translocation is thus not identical with the region of 3p most frequently deleted in sporadic RCC.

The gene from the short arm of chromosome 3, at D3F15S2, frequently deleted in renal cell carcinoma, encodes acylpeptide hydrolase.

Loss or inactivation of a gene on the short arm of chromosome 3 may contribute to the genesis of renal cell carcinoma. A gene that corresponds to the most frequently lost RFLP site (D3F15S2) is expressed in a variety of human tissues, and at a particularly high level in the kidney. Its expression is markedly reduced in renal cell carcinoma. A database search showed that the gene product is closely related to or identical with acylpeptide hydrolase. The nucleotide identity between the rat acylpeptide hydrolase and the human gene at D3F15S2 is 88%, compatible with normal species differences. It is therefore likely that the human gene product is acylpeptide hydrolase. The renal cell carcinoma is then associated with a decrease of acylpeptide hydrolase activity. The gene may represent a tumor suppressor gene, whose loss contributes to the development of renal cell carcinoma. It might be speculated that it could act e.g. by affecting the activity of a small acetylated growth factor. Alternatively, its decreased expression may merely reflect the impairment of differentiation in RCC, compared to normal kidney. Loss of a linked but irrelevant gene by the 3p deletion is another possibility.

The most frequently lost allelic site in human renal cell carcinoma (D3F15S2) on the short arm of chromosome 3 has homologous sequences on rat chromosome 8.

It has previously been shown that human chromosome 3 has banding homology to rat chromosome 8. We have previously isolated a cDNA from the D3F15S2 region and designated the gene as RIK. In the present study, we localized the homolog of this gene to rat chromosome 8.

Construction of a human chromosome 3 specific NotI linking library using a novel cloning procedure.

Two new diphasmid vectors (lambda SK17 and SK22) and a novel procedure to construct linking libraries are described. A partial filling-in reaction provides counter-selection against false linking clones in the library, and obviates the need for supF selection. The diphasmid vectors, in combination with the novel selection procedure, have been used to construct a chromosome 3 specific NotI linking library from a human chromosome 3/mouse microcell hybrid cell line. The application of the new vectors and the strong biochemical and biological selections resulted in a library of 60,000 NotI linking clones. As practically all of them are real NotI linking clones (no false recombinants) the library represents approximately 3,000 human recombinants (equal to 10-15 genomic equivalents of chromosome 3). Previously published methods for construction of linking libraries are compared with the procedure described in the present paper. The advantages of the new vectors and the novel protocol are discussed.

A gene near the D3F15S2 site on 3p is expressed in normal human kidney but not or only at a severely reduced level in 11 of 15 primary renal cell carcinomas (RCC).

Renal Cell Carcinoma (RCC) has been associated with the loss of heterozygosity at several loci on the short arm of chromosome 3 (3p). We have previously found that one of these loci, D3F15S2 (pH3H2) was lost in 76% of the tumor cells derived from heterozygous donors (Kovacs, G., Erlandsson, R., Boldog, F., Ingvarrsson, S., Müller-Brechlin, R., Klein, G. & Sümegi, J. (1988), Proc. Natl. Acad. Sci., 85, 1571-5). More recently we have identified a putative CpG island in the vicinity of D3F15S2, suggesting that DNA sequences in or around this site may have coding potential (Boldog, F., Erlandsson, R., Klein, G. & Sümegi, J. (1989). Cancer Genet. Cytogenet., 42, 295-306). The screening of a human placenta cDNA library with DNA probes derived from D3F15S2 has led to the isolation of several cDNA clones. They identified a 2.9 Kb long message in human placenta and kidney. In total RNA from 11 of 15 primary RCCs the gene expression was reduced to less than 20% compared to eight normal kidneys. This low level of expression may be due to contaminating normal tissue. In the remaining 4 tumors the expression varied from 24-51% compared to normal kidney. To facilitate reference, the gene was provisionally designated as 'RIK'. It was expressed in the HEK 293, one osteosarcoma (HOS), two carcinoma (COLO320 and QDMT), and three Burkitt lymphoma lines (BL2, BL29 and BL31). It was not expressed in one Burkitt lymphoma (DG75) and two EBV transformed lymphoblastoid cell lines (LCL) (NAD-20 and Cherry).