Non-AUG translational initiation of a short CAPC transcript generating protein isoform.

CAPC (also known as LRRC26) is a new gene with restricted expression in normal tissues, and with expression in many cancers and cancer cell lines. We have identified and characterized a short-transcript of CAPC (S-CAPC). The nucleotide sequence analysis of CAPC mRNA showed that the transcription for S-CAPC starts at position +610 on the L-CAPC transcript. Interestingly, no translation initiation codon 'AUG' is present in this transcript. To determine if a non-AUG start site is utilized, the S-CAPC sequence was cloned into an expression vector with C-terminal myc and histidine tags, and transfected into 293T cells. Western blot and MALDI-TOF MS analysis on purified S-CAPC gave two distinct peaks at approximately 7.5 kDa. N-terminal amino acid sequencing of the purified 7.5 kDa protein product indicated that translation starts at the codon for cysteine on the S

A flow cytometry method to quantitate internalized immunotoxins shows that taxol synergistically increases cellular immunotoxins uptake.

Tumor microenvironments present significant barriers to penetration by antibodies, immunoconjugates, and other immunotoxins. In this report, we illustrate a novel strategy to increase tumor cell uptake of immunotoxin by combination with Taxol. SS1P is an immunotoxin composed of the Fv portion of a mesothelin-specific antibody fused to a bacterial toxin that is presently undergoing phase II testing in mesothelioma. Using novel flow cytometry and gel filtration methods, we quantified SS1P uptake in individual tumor cells along with levels of shed mesothelin (sMSLN), a barrier of SS1P therapy. The validity of our flow cytometric method was confirmed by the ability to similarly quantitate tumor cell uptake of Herceptin and an immunotoxin targeting HER2/neu. SS1P uptake peaked several hours after SS1P was cleared from the blood, reflecting an intratumor distribution process of SS1P that is independent of blood supply. Using the methods developed, we demonstrated that Taxol could improve SS1P penetration into tumors in parallel with an associated reduction of sMSLN in tumor extracellular fluid. Our findings offer a mechanistic rationale to combine SS1P with Taxol or another cytotoxic drug as a strategy to increase immunotoxin uptake by tumor cells. Further, we suggest one basis to understand why chemotherapy and antibody-based therapies cooperate when combined in cancer treatment.

A recombinant immunotoxin targeting CD22 with low immunogenicity, low nonspecific toxicity, and high antitumor activity in mice.

Recombinant immunotoxins (RITs) are genetically engineered proteins designed to kill cancer cells. The RIT HA22 contains the Fv portion of an anti-CD22 antibody fused to a 38 kDa fragment of Pseudomonas exotoxin A (PE38). As PE38 is a bacterial protein, patients frequently produce antibodies that neutralize its activity, preventing retreatment. We have earlier shown in mice that PE38 contains 7 major B-cell epitopes located in domains II and III of the protein. Here we present a new mutant RIT, HA22-LR-6X, in which we removed most B-cell epitopes by deleting domain II and mutating 6 residues in domain III. HA22-LR-6X is cytotoxic to several lymphoma cell lines, has very low nonspecific toxicity, and retains potent antitumor activity in mice with CA46 lymphomas. To assess its immunogenicity, we immunized 3 MHC-divergent strains of mice with 5 microg doses of HA22-LR-6X, and found that HA22-LR-6X elicited significantly lower antibody responses than HA22 or other mutant RITs with fewer epitopes removed. Furthermore, large (50 microg) doses of HA22-LR-6X induced markedly lower antibody responses than 5 microg of HA22, indicating that high doses can be administered with low immunogenicity. Our experiments show that we have correctly identified and removed B-cell epitopes from PE38, producing a highly active immunotoxin with low immunogenicity and low animal toxicity. Future studies will determine if these properties carry over to humans with cancer.

A binding domain on mesothelin for CA125/MUC16.

Ovarian cancer and malignant mesothelioma frequently express both mesothelin and CA125 (also known as MUC16) at high levels on the cell surface. The interaction between mesothelin and CA125 may facilitate the implantation and peritoneal spread of tumors by cell adhesion, whereas the detailed nature of this interaction is still unknown. Here, we used truncated mutagenesis and alanine replacement techniques to identify a binding site on mesothelin for CA125. We examined the molecular interaction by Western blot overlay assays and further quantitatively analyzed by enzyme-linked immunosorbent assay. We also evaluated the binding on cancer cells by flow cytometry. We identified the region (296-359) consisting of 64 amino acids at the N-terminal of cell surface mesothelin as the minimum fragment for complete binding activity to CA125. We found that substitution of tyrosine 318 with an alanine abolished CA125 binding. Replacement of tryptophan 321 and glutamic acid 324 with alanine could partially decrease binding to CA125, whereas mutation of histidine 354 had no effect. These results indicate that a conformation-sensitive structure of the region (296-359) is required and sufficient for the binding of mesothelin to CA125. In addition, we have shown that a single chain monoclonal antibody (SS1) recognizes this CA125-binding domain and blocks the mesothelin-CA125 interaction on cancer cells. The identified CA125-binding domain significantly inhibits cancer cell adhesion and merits evaluation as a new therapeutic agent for preventing or treating peritoneal malignant tumors.

Conserved regions from Neisseria gonorrhoeae pilin are immunosilent and not immunosuppressive.

PilE is the primary subunit of type IV pili from Neisseria gonorrhoeae and contains a surface-exposed hypervariable region thought to be one feature of pili that has prevented development of a pilin-based vaccine. We have created a three-dimensional structure-based antigen by replacing the hypervariable region of PilE with an aspartate-glutamine linker chosen from the sequence of Pseudomonas aeruginosa PilA. We then characterized murine immune responses to this novel protein to determine if conserved PilE regions could serve as a vaccine candidate. The control PilE protein elicited strong T-cell-dependent B-cell responses that are specific to epitopes in both the hypervariable deletion and control proteins. In contrast, the hypervariable deletion protein was unable to elicit an immune response in mice, suggesting that in the absence of the hypervariable region, the conserved regions of PilE alone are not sufficient for antibody production. Further analysis of these PilE proteins with suppressor cell assays showed that neither suppresses T- or B-cell responses, and flow cytometry experiments suggested that they do not exert suppressor effects by activating T regulatory cells. Our results show that in the murine model, the hypervariable region of PilE is required to activate immune responses to pilin, whereas the conserved regions are unusually nonimmunogenic. In addition, we show that both hypervariable and conserved regions of pilin are not suppressive, suggesting that PilE does not cause the decrease in T-cell populations observed during gonococcal cervicitis.

Type IV pilin structures: insights on shared architecture, fiber assembly, receptor binding and type II secretion.

Type IV pili are long, flexible filaments that extend from the surface of Gram-negative bacteria and are formed by the polymerization of pilin subunits. This review focuses on the structural information available for each pilin subclass, type IVa and type IVb, highlighting the contributions crystal and nuclear magnetic resonance structures have made in understanding pilus function and assembly. In addition, the type II secretion pseudopilus subunit structure and helical assembly is compared to that of the type IV pilus. The pilin subunits adopt an alphabeta-roll fold formed by the hydrophobic packing of the C-terminal half of a long alpha-helix against an antiparallel beta-sheet. The conserved N-terminal half of the same alpha-helix, as well as two sequence- and structurally-variable regions, protrude from this globular head domain. Filament models have a hydrophobic core formed by the signature long alpha-helices, with variable regions at the filament surface.

The pilus-retraction protein PilT: ultrastructure of the biological assembly.

PilT is a biological motor required for the retraction of bacterial type IV pili. Nesseria gonorrhoeae PilT has been purified and its ultrastructure has been examined by freeze-etch electron microscopy, revealing a 115 A outer diameter, 15-35 A inner diameter ring. Aquifex aeolicus PilT crystals were obtained in a primitive hexagonal space group (unit-cell parameters a = b = 107.3, c = 68.5 A) and diffract to a minimum Bragg spacing of 2.8 A when PilT is co-crystallized with adenine nucleotides. Initial phases to 3.5 A resolution have been determined by multiwavelength anomalous dispersion and density modification. Resulting electron-density maps show a hexameric A. aeolicus PilT ring 105 A wide by 55 A high, with an inner cavity that varies in shape and width from 20 to 40 A over the height of the complex. Both PilT ultrastructures are very similar to type II and type IV secretion ATPases in overall shape, size and assembly.