Tumor resistance to anti-mesothelin CAR-T cells caused by binding to shed mesothelin is overcome by targeting a juxtamembrane epitope.

Despite many clinical trials, CAR-T cells are not yet approved for human solid tumor therapy. One popular target is mesothelin (MSLN) which is highly expressed on the surface of about 30% of cancers including mesothelioma and cancers of the ovary, pancreas, and lung. MSLN is shed by proteases that cleave near the C terminus, leaving a short peptide attached to the cell. Most anti-MSLN antibodies bind to shed MSLN, which can prevent their binding to target cells. To overcome this limitation, we developed an antibody (15B6) that binds next to the membrane at the protease-sensitive region, does not bind to shed MSLN, and makes CAR-T cells that have much higher anti-tumor activity than a CAR-T that binds to shed MSLN. We have now humanized the Fv (h15B6), so the CAR-T can be used to treat patients and show that h15B6 CAR-T produces complete regressions in a hard-to-treat pancreatic cancer patient derived xenograft model, whereas CAR-T targeting a shed epitope (SS1) have no anti-tumor activity. In these pancreatic cancers, the h15B6 CAR-T replicates and replaces the cancer cells, whereas there are no CAR-T cells in the tumors receiving SS1 CAR-T. To determine the mechanism accounting for high activity, we used an OVCAR-8 intraperitoneal model to show that poorly active SS1-CAR-T cells are bound to shed MSLN, whereas highly active h15B6 CAR-T do not contain bound MSLN enabling them to bind to and kill cancer cells.

Involvement of the prostate and testis expression (PATE)-like proteins in sperm-oocyte interaction.

BACKGROUND: The prostate and testis expression (PATE)-like family of proteins are expressed mainly in the male genital tract. They are localized in the sperm head and are homologous to SP-10, the acrosomal vesicle protein also named ACRV1. Our aim was to characterize the expression and functional role of three PATE-like proteins in the testis and ejaculated sperm. METHODS: The expression and localization of PATE-like proteins in human testis biopsies (n= 95) and sperm cells were assessed by RT-PCR, immunohistochemistry and immunofluorescence staining (at least 600 sperm cells per specimen). The function of the PATE protein was tested by the hemizona assay and hamster egg penetration test (HEPT). RESULTS: PATE and PATE-M genes and proteins were present almost exclusively in germ cells in the testis: immunoflourescence showed that the percentage of germ cells positive for PATE, PATE-M and PATE-B was 85, 50 and 2%, respectively. PATE and PATE-M proteins were localized in the equatorial segment of the sperm head, while PATE-B protein was localized in the post-acrosomal region. A polyclonal antibody (Ab, at 1:50 and 1:200 dilutions) against the PATE protein did not inhibit sperm-zona binding in the hemizona assay (hemizona index of 89.6 ± 10 and 87 ± 36%, respectively). However, there was inhibition of sperm-oolemma fusion and penetration in the HEPT (penetration index: without Ab 7 ± 3.9; Ab dilution of 1:100, 4 ± 3.5; Ab dilution of 1:20, 0.6 ± 1.2, P < 0.001). CONCLUSIONS: Our data suggest that PATE protein is involved in sperm-oolemma fusion and penetration but not sperm-zona binding.

Partial inactivation of Ankrd26 causes diabetes with enhanced insulin responsiveness of adipose tissue in mice.

AIMS/HYPOTHESIS: ANKRD26 is a newly described gene located at 10p12 in humans, a locus that has been identified with some forms of hereditary obesity. Previous studies have shown that partial inactivation of Ankrd26 in mice causes hyperphagia, obesity and gigantism. Hypothesising that Ankrd26 mutant (MT) mice could develop diabetes, we sought to establish whether the observed phenotype could be (1) solely related to the development of obesity or (2) caused by a direct action of ankyrin repeat domain 26 (ANKRD26) in peripheral tissues. METHODS: To test the hypothesis, we did a full metabolic characterisation of Ankrd26 MT mice that had free access to chow or were placed under two different energy-restricted dietary regimens. RESULTS: Highly obese Ankrd26 MT mice developed an unusual form of diabetes in which white adipose tissue is insulin-sensitive, while other tissues are insulin-resistant. When obese MT mice were placed on a food-restricted diet, their weight and glucose homeostasis returned to normal. In addition, when young MT mice were placed on a pair-feeding diet with normal mice, they maintained normal body weight, but showed better glucose tolerance than normal mice, an increased responsiveness of white adipose tissue to insulin and enhanced phosphorylation of the insulin receptor. CONCLUSIONS/INTERPRETATION: These findings show that the ANKRD26 protein has at least two functions in mice. One is to control the response of white adipose tissue to insulin; the other is to control appetite, which when Ankrd26 is mutated, leads to hyperphagia and diabetes in an obesity-dependent manner.

Interleukin-4 receptor-directed cytotoxin therapy of AIDS-associated Kaposi's sarcoma tumors in xenograft model.

The elusive and enigmatic origin of AIDS-associated Kaposi's sarcoma (AIDS-KS) makes it a complex tumor and therefore difficult to treat. Here we demonstrate that AIDS-KS cells express surface interleukin-4 (IL-4) receptors, and that IL-4 toxin (IL-4(38-37)-PE38KDEL) is specifically cytotoxic to these cells. Intratumoral, intraperitoneal and intravenous administration of IL-4 toxin in nude mice with established subcutaneous AIDS-KS tumors caused considerable anti-tumor activity in a dose-dependent manner, with highest dose producing durable complete responses. Metabolic changes, including cachexia and lymphopenia, induced by KS tumors were prevented by IL-4 toxin treatment. This report establishes IL-4(38-37)-PE38KDEL as an experimental therapeutic agent for the treatment of AIDS-KS.

Treatment of advanced solid tumors with immunotoxin LMB-1: an antibody linked to Pseudomonas exotoxin.

Immunotoxin LMB-1 is composed of monoclonal antibody B3 chemically linked to PE38, a genetically engineered form of Pseudomonas exotoxin. B3 recognizes a carbohydrate antigen (Le(Y)) present on many human solid tumors. LMB-1 has excellent antitumor activity in nude mice bearing Le(Y)-positive tumors. We conducted a phase I study of 38 patients with solid tumors who failed conventional therapy and whose tumors expressed the Le(Y) antigen. Objective antitumor activity was observed in 5 patients, 18 had stable disease, 15 progressed. A complete remission was observed in a patient with metastatic breast cancer to supraclavicular nodes. A greater than 75% tumor reduction and resolution of all clinical symptoms lasting for more than six months was observed in a colon cancer patient with extensive retroperitoneal and cervical metastasis. Three patients (two colon, one breast cancer) had minor responses. The maximum tolerated dose of LMB-1 is 75 microgram/kg given intravenously three times every other day. The major toxicity is vascular leak syndrome manifested by hypoalbuminemia, fluid retention, hypotension and, in one case, pulmonary edema. Although immunotoxins have been evaluated in clinical studies for more than two decades, this is the first report of antitumor activity in epithelial tumors.

Cyclic AMP mediates the concanavalin A agglutinability of mouse fibroblasts.

We have devised a quantitative way to measure the agglutination of cells which utilizes the size discrimination feature of an automatic particle counter. With this method we have studied the agglutinability by concanavalin A of 3T3 cells, a mutant of 3T3 cells (3T3cAMP(tcs)) in which cyclic AMP levels fall when the cells are subjected to temperature change or fresh serum, and L929 cells. We find with 3T3cAMP(tcs) cells that low levels of cyclic AMP correlate with increased agglutinability and that high levels of cyclic AMP correlate with decreased agglutinability. Prior treatment of these cells with a cyclic AMP phosphodiesterase inhibitor or Bt(2)cAMP blocks the increase in agglutinability induced by temperature change. When 3T3 cells are treated with fresh serum, their agglutinability also increases although to a much smaller extent than with 3T3cAMP(tcs) cells. Cells change their agglutinability very rapidly. Treatment of L929 cells for 15 min with 1-methyl-3-isobutyl xanthine at 1 mM decreases their agglutinability to the level of normal 3T3 cells. We conclude that in normal and transformed cells the level of cyclic AMP regulates agglutinability.

Cyclic amp and cell morphology in cultured fibroblasts. Effects on cell shape, microfilament and microtubule distribution, and orientation to substratum.

The change in shape of 3T3 and L929 cells due to Bt2cAMP treatment is accompanied by altered intracellular distribution of microfilaments and microtubules. Bt2cAMP added to cells in low density culture causes (a) microfilaments to accumulate in bundles near the plasma membrane, mainly at the cell periphery, and (b) microtubules to accumulate beneath these microfilament bundles. In narrow cell processes that form characteristically in Bt2cAMP-treated L cells, microtubules accumulate in parallel arrays near the center of these processes. A new simple method for evaluating the relative distance of the cell from its underlying substratum is desribed. In normal medium, 3T3 cells attach to their substratum near the nucleus and at the tips of cell processes, bridging irregularities in the plastic surface. With Bt2cAMP treatment, attachment occurs at the cell edge and at many isolated points under the cytoplasm, and the cells conform more closely to irregularities of the underlying substratum. A model of the mechanism by which cAMP modulates cell shape is presented.

Quantitation of a transformation-sensitive, adhesive cell surface glycoprotein. Decrease of several untransformed permanent cell lines.

We have quantitated the transformation-sensitive, cell surface LETS glycoprotein on many untransformed cell types. By SDS-polyacrylamide gel electrophoresis, this trypsin-sensitive iodinatable glycoprotein comprises 1-3% of total cellular protein of the seven early passage cell types tested. In contrast, it constitutes less than 0.15% of the protein in four of six continuous cell lines. This decrease is reflected in alterations both in [14C]glucosamine labeling and in the immunofluorescent staining of early passage vs. these four permanent cell lines. These results help to clarify previous experiments in which CSP, a purified LETS protein, partially restored a fibroblastic phenotype to cells transformed by tumor viruses. These findings also indicate that a major decrease in this cell surface glycoprotein can occur in the establishment of a continuous cell line without resulting in cellular transformation.

Transit of epidermal growth factor through coated pits of the Golgi system.

Using the direct conjugate of epidermal growth factor (EGF) and horseradish peroxidase, we have followed the entry of EGF into KB (human carcinoma) cells. EGF initially was found bound diffusely to the entire cell surface at 4 degrees C; on warming to 37 degrees C, EGF was found clustered in clathrin-coated pits on the plasma membrane in 1 min or less. Within 1-2 min at 37 degrees C, EGF began to accumulate in receptosomes within the cell and remained there for up to 10 min. At 10-13 min after warming to 37 degrees C, EGF was found in thin reticular membranous elements of the Golgi system, as well as concentrated in the clathrin-coated pits present on these membranes. By 15 min after warming, EGF began to be delivered to lysosomes located near the Golgi system. These findings suggest that clathrin-coated pits in the Golgi reticular system accumulate EGF before delivery to lysosomes.

alpha 2-macroglobulin adsorbed to colloidal gold: a new probe in the study of receptor-mediated endocytosis.

alpha 2-Macroglobulin (alpha 2 M) was adsorbed to colloidal gold and used as a new tool in the study of receptor-mediated endocytosis. alpha 2 M-gold is easy to prepare and is clearly visualized at the electron microscope level. When cells were incubated with alpha 2 M-gold at 0 degrees C, gold was visualized both diffusely over the cell surface and concentrated in coated pits. After cells to which alpha 2 M-gold had been bound at 0 degrees C were warmed, the gold was rapidly internalized into uncoated vesicles, previously termed receptosomes. After 30 min of incubation or longer, gold was found in small lysosomes and, later, in large lysosomes and very small vesicles in the region of the Golgi complex. This pattern of localization is similar to that previously described, using peroxidase-labeled anti-alpha 2 M antibodies. By incubating cells with both alpha 2 M-gold and vesicular stomatitis virus (VSV), we studied the internalization of these two markers simultaneously. VSV and alpha 2 M-gold rapidly clustered in the same coated pits and were internalized in the same receptosomes. Proteins and hormones adsorbed to gold may be useful in the study of receptor-mediated endocytosis.

alpha 2 Macroglobulin binding to the plasma membrane of cultured fibroblasts. Diffuse binding followed by clustering in coated regions.

Using transmission electron microscopy, we have studied the interaction of alpha 2 macroglobulin (alpha 2 M) with the surface of cultured fibroblasts. When cells were incubated for 2 h at 4 degrees C with ferritin-conjugated alpha 2 M, approximately 90% of the alpha 2 M was diffusely distributed on the cell surface, and the other 10% was concentrated in "coated" pits. A pattern of diffuse labeling with some clustering in "coated" pits was also obtained when cells were incubated for 5 min at 4 degrees C with alpha 2 M, fixed with glutaraldehyde, and the alpha 2 M was localized with affinity-purified, peroxidase-labeled antibody to alpha 2 M. Experiments in which cells were fixed with 0.2% paraformaldehyde before incubation with alpha 2 M showed that the native distribution of alpha 2 M receptors was entirely diffuse without significant clustering in "coated" pits. This indicates that some redistribution of the alpha 2 M-receptor complexes into clusters occurred even at 4 degrees C. In experiments with concanavalin A(Con A), we found that some of the Con A clustered in coated regions of the membrane and was internalized in coated vesicles, but much of the Con A was directly internalized in uncoated vesicles or pinosomes. We conclude that unoccupied alpha 2 M receptors are diffusely distributed on the cell surface. When alpha 2 M-receptor complexes are formed, they rapidly cluster in coated regions or pits in the plasma membrane and subsequently are internalized in coated vesicles. Because insulin and epidermal growth factor are internalized in the same structures as alpha 2 M (Maxfield, F.R., J. Schlessinger, Y. Schechter, I. Pastan, and M.C. Willingham. 1978. Cell, 14: 805--810.), we suggest that all peptide hormones, as well as other proteins that enter the cell by receptor-mediated endocytosis, follow this same pathway.

Identification of two lysosomal membrane glycoproteins.

Two murine lysosome-associated membrane proteins, LAMP-1 of 105,000-115,000 D and LAMP-2 of 100,000-110,000 D, have been identified by monoclonal antibodies that bind specifically to lysosomal membranes. Both glycoproteins were distinguished as integral membrane components solubilized by detergent solutions but not by various chaotropic agents. The lysosome localization was demonstrated by indirect immunofluorescent staining, co-localization of the antigen to sites of acridine orange uptake, and immunoelectron microscopy. Antibody binding was predominantly located at the limiting lysosomal membrane, distinctly separated from colloidal gold-labeled alpha-2-macroglobulin accumulated in the lumen during prolonged incubation. LAMP-1 and LAMP-2 also appeared to be present in low concentrations on Golgi trans-elements but were not detected in receptosomes marked by the presence of newly endocytosed alpha-2-macroglobulin, or in other cellular structures. LAMP-1 and LAMP-2 were distinguished as different molecules by two-dimensional gel analysis, 125I-tryptic peptide mapping, and sequential immunoprecipitations of 125I-labeled cell extracts. Both glycoproteins were synthesized as a precursor protein of approximately 90,000 D, and showed a marked heterogeneity of apparent molecular weight expression in different cell lines. LAMP-2 was closely related or identical to the macrophage antigen, MAC-3, as indicated by antibody adsorption and tryptic peptide mapping. It is postulated that these glycoproteins, as major protein constituents of the lysosomal membrane, have important roles in lysosomal structure and function.

Recombinant toxins for cancer treatment.

Recombinant toxins target cell surface receptors and antigens on tumor cells. They kill by mechanisms different from conventional chemotherapy, so that cross resistance to conventional chemotherapeutic agents should not be a problem. Furthermore, they are not mutagens and should not induce secondary malignancies or accelerate progression of benign malignancies. They can be mass-produced cheaply in bacteria as homogeneous proteins. Either growth factor-toxin fusions or antibody-toxin fusions can be chosen, depending on the cellular target.

Cyclic adenosine monophosphate in bacteria.

Both cyclic AMP and a specific inducer acting in concert are required for the synthesis of many inducible enzymes in E. coli. Little enzyme is made in the absence of either. In contrast to the specific inducers which stimulate the synthesis only of the proteins required for their metabolism, cyclic AMP controls the synthesis of many proteins. Glucose and certain other carbohydrates decrease the differential rate of synthesis of inducible enzymes by lowering cyclic AMP concentrations. In the lac operon, cyclic AMP acts at the promoter site to facilitate initiation of transcription. This action requires another protein, the cyclic AMP receptor protein. The nucleotide stimulates tryptophanase synthesis at a translational level. The action of cyclic AMP in E. coli may serve as a model to understand its action on transcriptional and translational processes in eukaryotes.

Actinomycin D: inhibition of phospholipid synthesis in chick embryo cells.

When chick embryo fibroblasts are exposed to actinomycin D for 30 minutes to 3 hours, there is progressive inhibition of phospholipid synthesis, so that by 3 hours the inhibition is 40 percent. All phospholipids are affected. Since this inhibition is twice as great as the inhibition of protein synthesis, some effects of actinomycin D previously ascribed to decreased protein synthesis must be reevaluated.

Radioreceptor assay of adrenocorticotropic hormone: new approach to assay of polypeptide hormones in plasma.

Biologically active iodine-125-labeled adrenocorticotropic hormone (ACTH) binds specifically to ACTH receptors extracted from adrenals. Unlabeled ACTH at 1 picogram per milliliter significantly displaces labeled ACTH from these receptors. This system, which appears to be applicable to all polypeptide hormones, provides a rapid and sensitive method for measurements of biologically active ACTH in dilute whole plasma.

Promoter region of the human Harvey ras proto-oncogene: similarity to the EGF receptor proto-oncogene promoter.

Regulation of transcription of members of the ras gene family undoubtably plays an important role in controlling cellular growth. Examination of this level of regulation requires identification of the promoter regions of the ras proto-oncogenes. Four major transcriptional start sites were detected in the human Harvey ras 1 proto-oncogene. The promoter region contains neither a TATA box nor a CAAT box in their characteristic upstream positions, has an extremely high G+C content (80 percent), and contains multiple GC boxes including seven CCGCCC repeats and three repeats of the inverted complement, GGGCGG. This region has strong promoter activity when placed upstream from the chloramphenicol acetyl transferase gene and transfected into monkey CV1 cells. In these ways the Harvey ras 1 proto-oncogene promoter resembles the promoter of the gene encoding the epidermal growth factor (EGF) receptor. The similarity between the two proto-oncogene promoters may be relevant to the mechanism by which the expression of such "growth control" genes is regulated.

Journey to the center of the cell: role of the receptosome.

Fibroblasts contain a specific internalization pathway that carries hormones as well as some proteins and viruses from the cell surface to the cell interior. Initially, the ligands bind to mobile receptors that are randomly distributed on the cell surface. Next the ligand-receptor complexes are trapped and concentrated in specialized regions of the membrane termed bristle-coated pits. From the pit a smooth-walled vesicle containing the ligand forms and carries the ligand to the cell interior. Because of its role in receptor-mediated endocytosis, this vesicle has been termed a "receptosome."

Modulation of activity of the promoter of the human MDR1 gene by Ras and p53.

Drug resistance in human cancer is associated with overexpression of the multidrug resistance (MDR1) gene, which confers cross-resistance to hydrophobic natural product cytotoxic drugs. Expression of the MDR1 gene can occur de novo in human cancers in the absence of drug treatment. The promoter of the human MDR1 gene was shown to be a target for the c-Ha-Ras-1 oncogene and the p53 tumor suppressor gene products, both of which are associated with tumor progression. The stimulatory effect of c-Ha-Ras-1 was not specific for the MDR1 promoter alone, whereas a mutant p53 specifically stimulated the MDR1 promoter and wild-type p53 exerted specific repression. These results imply that the MDR1 gene could be activated during tumor progression associated with mutations in Ras and p53.

Thyroglobulin: evidence for crystallization and association.

Crystals of thyroglobulin have been obtained from ammonium sulfate solutions and have been examined by electron microscopy as shadowed carbon replicas. Unit size in the crystal is 228 +/- 9 angstroms, which corresponds to a molecular weight of 5,300,000. Data are in accord with the possibility that this unit represents a polymer of thyroglobulin.