Alan Garen

Targeting tissue factor on tumor vascular endothelial cells and tumor cells for immunotherapy in mouse models of prostatic cancer

The efficacy and safety of an immunoconjugate (icon) molecule, composed of a mutated mouse factor VII (mfVII) targeting domain and the Fc effector domain of an IgG1 Ig (mfVII/Fc icon), was tested with a severe combined immunodeficient (SCID) mouse model of human prostatic cancer and an immunocompetent mouse model of mouse prostatic cancer. The SCID mice were first injected s.c. with a human prostatic tumor line, forming a skin tumor that produces a high blood titer of prostate-specific antigen and metastasizes to bone. The icon was encoded in a replication-incompetent adenoviral vector that was injected directly into the skin tumor. The tumor cells infected by the vector synthesize and secrete the icon into the blood, and the blood-borne icon binds with high affinity and specificity to mouse tissue factor expressed on endothelial cells lining the lumen of the tumor vasculature and to human tissue factor expressed on the tumor cells. The Fc domain of the icon activates a cytolytic immune attack against cells that bind the icon. The immunotherapy tests in SCID mice demonstrated that intratumoral injections of the adenoviral vector encoding the mfVII/human Fc icon resulted in long-term regression of the injected human prostatic tumor and also of a distant uninjected tumor, without associated toxicity to the mice. Comparable results were obtained with a SCID mouse model of human melanoma. At the end of the experiments the mice appeared to be free of viable tumor cells. This protocol also could be efficacious for treating cancer patients who have vascularized tumors.

Suppressor genes for nonsense mutations. I. The Su-1, Su-2 and Su-3 genes of Escherichia coli.

Three suppressor genes in Escherichia coli , which act on the N1 class of phosphatase nonsense mutations, have been genetically analyzed. The Su-1 gene maps near the histidine region of the chromosome, the Su-3 gene near the tryptophan region, and the Su-2 gene in another (unidentified) region. Each suppressor gene can exist in either an active state, called Su + , or an inactive state, called Su − . The presence of one active suppressor gene, either Su-1 + , Su-2 + or Su-3 + , is a sufficient condition for suppression of N1 phosphatase nonsense mutations. The same phosphatase mutation may respond differently to each suppressor gene, as indicated by differences in specific enzymic activities of the phosphatase molecules produced by the suppressed mutants. It appears that each suppressor gene causes a different structural alteration in the phosphatase molecule. Fine-structure genetic detail within the Su-1 gene has been revealed by analysis of Su − mutations isolated in an Su-1 + strain. There are several closely linked sites in the Su-1 gene at which Su − mutations can occur. The resulting mutants differ in their suppressor phenotypes; some are partially active (leaky mutants) whereas others are inactive. Suppression of a nonsense mutation can be highly efficient, resulting in as much as 55% of the standard amount of phosphatase synthesis. Nevertheless, there is no measurable difference in growth rate between pairs of Su + and Su − strains that are isogenic except for the suppressor mutation. This suggests that the nonsense codon involved with the N1 class of phosphatase mutations (the UAG codon) is not used as a normal chain-terminating codon.

Role of human noncoding RNAs in the control of tumorigenesis

Related studies showed that the protein PSF represses proto-oncogene transcription, and VL30–1 RNA, a mouse noncoding retroelement RNA, binds and releases PSF from a proto-oncogene, activating transcription. Here we show that this mechanism regulates tumorigenesis in human cells, with human RNAs replacing VL30–1 RNA. A library of human RNA fragments was used to isolate, by affinity chromatography, 5 noncoding RNA fragments that bind to human PSF (hPSF), releasing hPSF from a proto-oncogene and activating transcription. Each of the 5 RNA fragments maps to a different human gene. The tumorigenic function of the hPSF-binding RNAs was tested in a human melanoma line and mouse fibroblast line, by determining the effect of the RNAs on formation of colonies in agar and tumors in mice. (i) Expressing in human melanoma cells the RNA fragments individually promoted tumorigenicity. (ii) Expressing in human melanoma cells a shRNA, which causes degradation of the endogenous RNA from which an RNA fragment was derived, suppressed tumorigenicity. (iii) Expressing in mouse NIH/3T3 cells the RNA fragments individually resulted in transformation to tumorigenic cells. (iv) A screen of 9 human tumor lines showed that each line expresses high levels of several hPSF-binding RNAs, relative to the levels in human fibroblast cells. We conclude that human hPSF-binding RNAs drive transformation and tumorigenesis by reversing PSF-mediated repression of proto-oncogene transcription and that dysfunctional regulation of human hPSF-binding RNA expression has a central role in the etiology of human cancer.

Localized defects of blastoderm formation in maternal effect mutants of Drosophila

Abstract In the three maternal effect lethal mutant strains of D. melanogaster described in this report, the homozygous mutant females produce defective eggs that cannot support normal embryonic development. The embryos from these eggs begin to develop for the first 2 hr after fertilization in an apparently normal way, forming a blastula containing a cluster of pole cells at the posterior end and a layer of syncytial blastoderm nuclei. During the subsequent transition from a syncytial to a cellular blastoderm, cell formation in the blastoderm is either partially or totally blocked. In mutant mat(3)1 no blastoderm cells are formed, indicating that there are separate genetic controls for pole cells and blastoderm cells. The other two mutants form an incomplete cellular blastoderm in which certain regions of the blastoderm remain noncellular. The noncellular region in mutant mat(3)3 is on the posterior-dorsal surface, covering about 30% of the total blastoderm. In mutant mat(3)6 blastoderm cells are formed only at the anterior and posterior ends, separated by a noncellular region that covers about 70% of the total blastoderm. The selective effects on blastoderm cell formation in the three mutants emphasize the importance of components present in the egg before fertilization for the transition from a syncytial to a cellular blastoderm. The genes defective in the three mutants are essential only for oogenesis and not for any other period of development, as indicated by a strict dependence of the lethal phenotypes on the maternal genotypes. Heterozygous embryos from the eggs of homozygous mutant females die, whereas homozygous mutant embryos from the eggs of heterozygous females develop into viable adults. One of the mutants, mat(3)3 , has a temperature-sensitive phenotype. Homozygous mat(3)3 females maintained at a restrictive temperature of 29°C show the lethal maternal effect. However, at a permissive temperature of 20°C the females produce viable adult progeny. The temperature-sensitive period in mat(3)3 females occurs during the last 12 hr of oogenesis, consistent with the maternal effect phenotype of the mutant.

Immunotherapy for choroidal neovascularization in a laser-induced mouse model simulating exudative (wet) macular degeneration

Age-related macular degeneration (AMD) is the leading cause of blindness after age 55 in the industrialized world. Severe loss of central vision frequently occurs with the exudative (wet) form of AMD, as a result of the formation of a pathological choroidal neovasculature (CNV) that damages the macular region of the retina. We tested the effect of an immunotherapy procedure, which had been shown to destroy the pathological neovasculature in solid tumors, on the formation of laser-induced CNV in a mouse model simulating exudative AMD in humans. The procedure involves administering an Icon molecule that binds with high affinity and specificity to tissue factor (TF), resulting in the activation of a potent cytolytic immune response against cells expressing TF. The Icon binds selectively to TF on the vascular endothelium of a CNV in the mouse and pig models and also on the CNV of patients with exudative AMD. Here we show that the Icon dramatically reduces the frequency of CNV formation in the mouse model. After laser treatment to induce CNV formation, the mice were injected either with an adenoviral vector encoding the Icon, resulting in synthesis of the Icon by vector-infected mouse cells, or with the Icon protein. The route of injection was i.v. or intraocular. The efficacy of the Icon in preventing formation of laser-induced CNV depends on binding selectively to the CNV. Because the Icon binds selectively to the CNV in exudative AMD as well as to laser-induced CNV, the Icon might also be efficacious for treating patients with exudative AMD.

Amino acid substitutions resulting from suppression of nonsense mutations. II. Glutamine insertion by the Su-2 gene; tyrosine insertion by the Su-3 gene.

The N1 class of phosphatase nonsense mutations is suppressible by either the Su-1, Su-2 or Su-3 suppressor genes of Escherichia coli . It was previously shown that suppression by the Su-1 gene results in the incorporation of a serine residue into the phosphatase molecule at the position specified by a nonsense codon. The present experiments demonstrate that the amino acid incorporated is glutamine when Su-2 is the suppressor gene, and is tyrosine when Su-3 is the suppressor gene. Thus, the same nonsense codon can be translated in three ways, as serine, glutamine or tyrosine, depending on which of the three suppressor genes is responsible for suppression.

Suppressor genes for nonsense mutations: II. The Su-4 and Su-5 suppressor genes of Escherichia coli

Two Escherichia coli suppressor genes, Su-4 and Su-5 , are characterized in the present report. These genes, which suppress the N1 class of phosphatase nonsense mutations, are genetically and physiologically distinct from the previously described suppressor genes for N1 mutations, Su-1 , Su-2 and Su-3 . The Su-4 gene is located in the tryptophan region of the bacterial chromosome, and the Su-5 gene in the galactose region. With the two new suppressor genes it has been possible to detect another class of suppressible phosphatase mutations, the N2 class, which does not respond to the other three suppressor genes. The N2 mutations appear to act as nonsense mutations in an Su − strain, since all unsuppressed N2 mutants fail to produce phosphatase enzymic activity or cross-reacting material. N2 mutations occur preferentially in codons that specify, in the standard phosphatase molecule, either basic or acidic amino acids, in contrast to N1 mutations which occur in codons that specify neutral amino acids. This suggests that the nonsense codon resulting from N2 mutations is different from the UAG nonsense codon resulting from N1 mutations. Other evidence for two different nonsense codons is the demonstration that an N2 codon can be transformed by mutation into an N1 codon. (In another report, the N2 codon has been identified as UAA.) When N1 or N2 mutations are suppressed by the Su-4 + gene, the phosphatase proteins produced appear, from electrophoretic analyses, to contain a neutral amino acid at the position in the molecule specified by the nonsense codon. When the same mutations are suppressed by the Su-5 + gene, there appears to be a bagic amino acid present at the corresponding positions. Thus, each suppressor gene causes a different amino acid to be specified by a nonsense codon. The Su-4 + gene is a moderately effective suppressor of N1 and N2 mutations. The highest level of suppression obtained, as measured by the amount of phosphatase cross-reacting material produced, is 16% of the amount in the standard P + strain. The Su-5 + gene suppresses N1 and N2 mutations less effectively, yielding a maximum of 6% of the standard amount of cross-reacting material. The N2 class of phosphatase mutations is similar in some characteristics, but not in all, to the ochre class of mutations in phage T4.

The immunoconjugate "icon" targets aberrantly expressed endothelial tissue factor causing regression of endometriosis.

Endometriosis is a major cause of chronic pain, infertility, medical and surgical interventions, and health care expenditures. Tissue factor (TF), the primary initiator of coagulation and a modulator of angiogenesis, is not normally expressed by the endothelium; however, prior studies have demonstrated that both blood vessels in solid tumors and choroidal tissue in macular degeneration express endothelial TF. The present study describes the anomalous expression of TF by endothelial cells in endometriotic lesions. The immunoconjugate molecule (Icon), which binds with high affinity and specificity to this aberrant endothelial TF, has been shown to induce a cytolytic immune response that eradicates tumor and choroidal blood vessels. Using an athymic mouse model of endometriosis, we now report that Icon largely destroys endometriotic implants by vascular disruption without apparent toxicity, reduced fertility, or subsequent teratogenic effects. Unlike antiangiogenic treatments that can only target developing angiogenesis, Icon eliminates pre-existing pathological vessels. Thus, Icon could serve as a novel, nontoxic, fertility-preserving, and effective treatment for endometriosis.

Base composition of nonsense codons in Escherichia coli: The N2 codon UAA

Earlier oxperiments showed that the triplet UAG was the nonsense condon produced by the N1 class of alkaline phosphatase nonsense mutations. In the present experiments UAA has been identified as a second nonsense codon produced by the N2 class of alkaline phosphatase nonsense mutations. The composition of each nonsense codon was established on the basis of amino acid substitutions occurring in alkaline phosphatase as a result of mutationally induced alterations of the nonsense codons. The substitution data further indicate that UGA is not a codon for tryptophan in Escherichia coli and might act as a third nonsense codon.

Amino acid substitutions resulting from suppression of nonsense mutations: IV. Leucine insertion by the Su6+ suppressor gene☆

Amino acid substitutions in alkaline phosphatase are shown to result from suppression of nonsense mutations in the phosphatase structural gene. The suppressor gene involved in the experiments was Su-1 , one of several that act on nonsense mutations. With one phosphatase mutant the substitution was serine for tryptophan, and with two other mutants the substitution was serine for glutamine. Thus, the original amino acid that is affected by a nonsense mutation can vary, but the amino acid inserted by the Su-1 suppressor gene is in all cases serine.