Development of ISB 1442, a CD38 and CD47 bispecific biparatopic antibody innate cell modulator for the treatment of multiple myeloma.

Antibody engineering can tailor the design and activities of therapeutic antibodies for better efficiency or other advantageous clinical properties. Here we report the development of ISB 1442, a fully human bispecific antibody designed to re-establish synthetic immunity in CD38+ hematological malignancies. ISB 1442 consists of two anti-CD38 arms targeting two distinct epitopes that preferentially drive binding to tumor cells and enable avidity-induced blocking of proximal CD47 receptors on the same cell while preventing on-target off-tumor binding on healthy cells. The Fc portion of ISB 1442 is engineered to enhance complement dependent cytotoxicity, antibody dependent cell cytotoxicity and antibody dependent cell phagocytosis. ISB 1442 thus represents a CD47-BsAb combining biparatopic targeting of a tumor associated antigen with engineered enhancement of antibody effector function to overcome potential resistance mechanisms that hamper treatment of myeloma with monospecific anti-CD38 antibodies. ISB 1442 is currently in a Phase I clinical trial in relapsed refractory multiple myeloma.

Conformational plasticity of ligand-bound and ternary GPCR complexes studied by 19F NMR of the ß1-adrenergic receptor.

G-protein-coupled receptors (GPCRs) are allosteric signaling proteins that transmit an extracellular stimulus across the cell membrane. Using 19F NMR and site-specific labelling, we investigate the response of the cytoplasmic region of transmembrane helices 6 and 7 of the ß1-adrenergic receptor to agonist stimulation and coupling to a Gs-protein-mimetic nanobody. Agonist binding shows the receptor in equilibrium between two inactive states and a pre-active form, increasingly populated with higher ligand efficacy. Nanobody coupling leads to a fully active ternary receptor complex present in amounts correlating directly with agonist efficacy, consistent with partial agonism. While for different agonists the helix 6 environment in the active-state ternary complexes resides in a well-defined conformation, showing little conformational mobility, the environment of the highly conserved NPxxY motif on helix 7 remains dynamic adopting diverse, agonist-specific conformations, implying a further role of this region in receptor function. An inactive nanobody-coupled ternary receptor form is also observed.

Thymomas induced by simple alkylating agents in C57BL/Cbi mice: kinetics of the dose response.

Specific-pathogen-free inbred C57BL/Cbi mice (adult virgin females) were given single sublethal doses of N-methyl-N-nitrosourea or N-ethyl-N-nitrosourea and were studied for a lifetime for the development of thymomas. A fatal lymphocytic lymphoma with a "starry sky" pattern due to the presence of macrophages was induced in the thymuses of treated mice within 250 days of treatment. Control and low-dose treatment groups had up to 70% incidence of a histiocytic lymphoma that was usually primary in mesenteric lymph nodes and nearly always occurred later than 250 days after treatment. A "one-hit" linear relationship existed between the time of appearance of induced thymic lymphomas and the log fraction of non-tumor-bearing mice. The absolute latency period of these tumors was constant and independent of dose. The effect of dose was an exponential increase of the total incidence of induced thymic lymphomas. By mathematical analysis, the best estimate of the exponent from the results was 2 or 3, indicating that the development of these induced tumors may be produced by 2 or 3 "events" in the target cell. Possible candidates for these events are premutagenic alkylation of DNA, inactivation of DNA repair, oncovirus activation, regenerative hyperplasia, development of trisomy No 15, and inhibited immunosurveillance.

Synthesis of 1-(2-hydroxyethyl)-1-nitrosourea and comparison of its carcinogenicity with that of 1-ethyl-1-nitrosourea.

1-(2-Hydroxyethyl)-1-nitrosourea (HNU) was prepared by the action of nitrosyl chloride on (2-hydroxyethyl)urea. Attempts to synthesize HNU by an earlier described method were unsuccessful and led to the formation of the cyclized derivative 1-nitroso-2-oxazolidone. In addition, the spectral data that we obtained for HNU differed from those reported earlier. Female C57BL/Cbl mice were treated with single ip doses of HNU to determine its median lethal dose (LD50) and its ability to induce lymphocytic thymic lymphomas in these mice. The results showed that the LD50 was the same as that for 1-ethyl-1-nitrosourea (ENU) and that its was slightly more potent than ENU as a carcinogen in this system.

Lower limb paralysis induced in mice by a temperature-sensitive mutant of Moloney leukemia virus.

A temperature-sensitive mutant of Moloney murine leukemia virus defective in an early function and injected into newborn mice produced lower limb paralysis. Susceptible mice were inbred strains CFW/D, CBA/H, C3H/Bi/Ka, and outbred NIH Swiss stock. Inbred W/Fu rats and C57BL/Ka mice did not develop the paralysis, though the latter were infected with virus; the sera from these mice produced paralysis in susceptible CFW mice.

PTK787/ZK 222584, a novel and potent inhibitor of vascular endothelial growth factor receptor tyrosine kinases, impairs vascular endothelial growth factor-induced responses and tumor growth after oral administration.

PTK787/ZK 222584 (1-[4-chloroanilino]-4-[4-pyridylmethyl] phthalazine succinate) is a potent inhibitor of vascular endothelial growth factor (VEGF) receptor tyrosine kinases, active in the submicromolar range. It also inhibits other class III kinases, such as the platelet-derived growth factor (PDGF) receptor beta tyrosine kinase, c-Kit, and c-Fms, but at higher concentrations. It is not active against kinases from other receptor families, such as epidermal growth factor receptor, fibroblast growth factor receptor-1, c-Met, and Tie-2, or intracellular kinases such as c-Src, c-Abl, and protein kinase C-alpha. PTK787/ZK 222584 inhibits VEGF-induced autophosphorylation of kinase insert domain-containing receptor (KDR), endothelial cell proliferation, migration, and survival in the nanomolar range in cell-based assays. In concentrations up to 1 microM, PTK787/ZK 222584 does not have any cytotoxic or antiproliferative effect on cells that do not express VEGF receptors. After oral dosing (50 mg/kg) to mice, plasma concentrations of PTK787/ZK 222584 remain above 1 microM for more than 8 h. PTK787/ZK 222584 induces dose-dependent inhibition of VEGF and PDGF-induced angiogenesis in a growth factor implant model, as well as a tumor cell-driven angiogenesis model after once-daily oral dosing (25-100 mg/kg). In the same dose range, it also inhibits the growth of several human carcinomas, grown s.c. in nude mice, as well as a murine renal carcinoma and its metastases in a syngeneic, orthotopic model. Histological examination of tumors revealed inhibition of microvessel formation in the interior of the tumor. PTK787/ZK 222584 is very well tolerated and does not impair wound healing. It also does not have any significant effects on circulating blood cells or bone marrow leukocytes as a single agent or impair hematopoetic recovery after concomitant cytotoxic anti-cancer agent challenge. This novel compound has therapeutic potential for the treatment of solid tumors and other diseases where angiogenesis plays an important role.

Protein and Antibody Engineering by Phage Display.

Phage display is an in vitro selection technique that allows for the rapid isolation of proteins with desired properties including increased affinity, specificity, stability, and new enzymatic activity. The power of phage display relies on the phenotype-to-genotype linkage of the protein of interest displayed on the phage surface with the encoding DNA packaged within the phage particle, which allows for selective enrichment of library pools and high-throughput screening of resulting clones. As an in vitro method, the conditions of the binding selection can be tightly controlled. Due to the high-throughput nature, rapidity, and ease of use, phage display is an excellent technological platform for engineering antibody or proteins with enhanced properties. Here, we describe methods for synthesis, selection, and screening of phage libraries with particular emphasis on designing humanizing antibody libraries and combinatorial scanning mutagenesis libraries. We conclude with a brief section on troubleshooting for all stages of the phage display process.

Endoscopic large bowel polypectomy. Adequate treatment of some completely removed, minimally invasive lesions.

Eighteen large bowel adenomas with invasion of the head or the stalk were removed by endoscopic polypectomy or by segmental resection at University Hospital, London, Ontario between 1973 and 1982. All were completely removed by histological criteria. All the patients were traced for an average follow-up period of 4.6 years. None had developed disseminated large bowel cancer. Adding these results to those in the literature, it appears that, provided there is not a high degree of anaplasia of the tumor or lymphatic or venous invasion, endoscopic polypectomy is adequate therapy for such adenomas. An endoscopic recheck of the site of removal after approximately 2 months may be worthwhile as a few local recurrences of benign tumor were reported, although not in the present series. The patients should be followed for life.

2-substituted 3-(aminooxy)propanamines as inhibitors of ornithine decarboxylase: synthesis and biological activity.

1-Amino-3-(aminooxy)-2-propanol (6a) has been synthesized and found to inhibit rat liver ornithine decarboxylase (ODC) with an IC50 in the nanomolar range. Compound 6a served as a basis for the design of new enzyme inhibitors, which led to the identification of 3-(aminooxy)-2-fluoropropanamine (15) as a new powerful enzyme blocker. Compound 15 inhibited ODC at 3 times lower concentrations than 6a and 3-(aminooxy)propanamine (APA), and it was superior to APA as an antiproliferative agent in inhibiting the growth of human T24 bladder carcinoma cells in vitro.

4-Amidinoindan-1-one 2'-amidinohydrazone: a new potent and selective inhibitor of S-Adenosylmethionine decarboxylase.

Two isomeric amidino-2-acetylpyridine amidinohydrazones, 11 and 12, and 4-amidinoindanone amidinohydrazone, 17, have been synthesized and tested for inhibition of S-adenosylmethionine decarboxylase (SAMDC) and diamine oxidase and for antiproliferative activity against T24 human bladder carcinoma cells. Compound 11 inhibited SAMDC with an IC50 of 10 nM and was 140- and > 500-fold more potent than methylglyoxal bis(guanylhydrazone) (MGBG) and 12, respectively. The difference in potency between 11 and 12 was interpreted with the help of molecular modeling and appeared to be associated with two different low-energy conformations of the compounds. Compound 17 which represents a conformationally constrained analogue of 11, was superior to the latter and MGBG with respect to selective inhibition of SAMDC and antiproliferative activity, and is of interest as a potential anticancer agent and a drug for the treatment of protozoal and Pneumocystis carinii infections.

Use of a pharmacophore model for the design of EGF-R tyrosine kinase inhibitors: 4-(phenylamino)pyrazolo[3,4-d]pyrimidines.

In the course of the random screening of a pool of CIBA chemicals, the two pyrazolopyrimidines 1 and 2 have been identified as fairly potent inhibitors of the EGF-R tyrosine kinase. Using a pharmacophore model for ATP-competitive inhibitors interacting with the active site of the EGF-R protein tyrosine kinase (PTK), the class of the pyrazolo[3,4-d]pyrimidines was then optimized in an interactive process leading to a series of 4-(phenylamino)-1H-pyrazolo[3,4-d]-pyrimidines as highly potent inhibitors of the EGF-R tyrosine kinase. The most potent compounds 13, 14, 15, 17, 19, 22, 26, 28, and 30 of this series inhibited the EGF-R PTK with IC50 values below 10 nM. High selectivity toward a panel of nonreceptor tyrosine kinases (c-Src, v-Abl and serine/threonine kinases (PKC alpha, CDK1) was observed. In cells, EGF-stimulated cellular tyrosine phosphorylation was inhibited by compounds 13, 15, 19, 22, and 23 at IC50 values below 50 nM, whereas PDGF-induced tyrosine phosphorylation was not affected by concentrations up to 10 microM, thus indicating high selectivity for the inhibition of the ligand-activated EGF-R signal transduction pathway. Compounds 15 and 19 inhibited proliferation of the EGF-dependent MK cell line with IC50 values below 0.5 microM. In addition, two compounds, 9 and 11, showing satisfactory oral bioavailability in mice after oral administration, exhibited good in vivo efficacy at doses of 12.5 and 50 mg/kg in a nude mouse tumor model using xenografts of the EGF-R overexpressing A431 cell line. From SAR studies, a binding mode for 4-(phenylamino)-1H-pyrazolo[3,4-d]pyrimidines, especially for compound 15, at the ATP-binding site of the EGF-R tyrosine kinase is proposed. 4-(Phenylamino)-1H-pyrazolo[3,4-d]pyrimidines represent a new class of highly potent tyrosine kinase inhibitors which preferentially inhibit the EGF-mediated signal transduction pathway and have the potential for further evaluation as anticancer agents.

Evaluation of desferrithiocin and its synthetic analogues as orally effective iron chelators.

Desferrithiocin, a novel microbial siderophore isolated from cultures of Streptomyces antibioticus DSM 1865, and a number of its derivatives and analogues are evaluated for their ability to promote iron clearance. The compounds have been designed with the objective of identifying the structural features of desferrithiocin which render this ligand an orally effective iron chelator. The desferrithiocin aromatic hydroxyl and the thiazoline ring carboxyl group are shown to be central to desferrithiocin's activity. The ligand's methyl and the aromatic nitrogen play little role in the compound's efficacy. The animal model chosen for this study, the bile duct cannulated rat, provides information regarding both the chelator-induced total iron output and the kinetics of both biliary and urinary iron excretion.

New anilinophthalazines as potent and orally well absorbed inhibitors of the VEGF receptor tyrosine kinases useful as antagonists of tumor-driven angiogenesis.

The sprouting of new blood vessels, or angiogenesis, is necessary for any solid tumor to grow large enough to cause life-threatening disease. Vascular endothelial growth factor (VEGF) is one of the key promoters of tumor induced angiogenesis. VEGF receptors, the tyrosine kinases Flt-1 and KDR, are expressed on vascular endothelial cells and initiate angiogenesis upon activation by VEGF. 1-Anilino-(4-pyridylmethyl)-phthalazines, such as CGP 79787D (or PTK787 / ZK222584), reversibly inhibit Flt-1 and KDR with IC(50) values < 0.1 microM. CGP 79787D also blocks the VEGF-induced receptor autophosphorylation in CHO cells ectopically expressing the KDR receptor (ED(50) = 34 nM). Modification of the 1-anilino moiety afforded derivatives with higher selectivity for the VEGF receptor tyrosine kinases Flt-1 and KDR compared to the related receptor tyrosine kinases PDGF-R and c-Kit. Since these 1-anilino-(4-pyridylmethyl)phthalazines are orally well absorbed, these compounds qualify for further profiling and as candidates for clinical evaluation.

Medical audit of rectal biopsy diagnosis of inflammatory bowel disease.

The records of the rectal biopsy diagnoses of ulcerative colitis and Crohn's disease in the Department of Pathology, St Mark's Hospital, London, were reviewed. The biopsy diagnoses were compared to subsequent resection diagnoses on the same patients, and annual and seasonal variations in the frequency of these and related diagnoses were studied. The accuracy rate for the biopsy diagnosis of ulcerative colitis was about 70% and for Crohn's disease about 40% each time a biopsy was read. The low figure for the accuracy rate for Crohn's disease could be attributed to sampling error inherent in the diagnosis of a disease which is essentially patchy, showing discontinuous pathology. Also, many patients with Crohn's disease have a normal rectum which is biopsied to demonstrate the distinction from ulcerative colitis. In practical terms therefore a 40% accuracy rate in Crohn's disease is probably adequate. The rate of "false-positive" diagnoses was about 5%. There was a seasonal variation in the frequency of these two diagnoses, but no variation attributable to changes in observers, as pathology trainees in the Department change regularly. The frequency of diagnoses of non-specific inflammation and of normal colon did show such non-random variations.

Cytogenetics of murine thymic lymphomas induced by N-methyl-N-nitrosourea: difference in incidence of trisomy 15 in thymic lymphomas induced in neonatal and adult mice.

Chromosome complements of murine thymic lymphomas induced by an alkylating agent N-methyl-N-nitrosourea (MNUA) were analyzed microscopically and karyotypically using the Q-banding technique. The chemical carcinogen was injected intraperitoneally into either neonatal or 7-week-old CFW/D mice. In addition, thymic lymphomas induced in 7-week-old AKR mice and thymic lymphomas developed spontaneously in this strain were also examined. All six lymphomas induced in neonatal CFW/D had hyperdiploid cell lines that accounted for 90% of the cells analyzed. Chromosome analysis of lymphomas induced in adult DFW/D mice showed that only out of nine lymphomas had predominantly hyperdiploid cell lines. The remaining five lymphomas had diploid modal chromosome number although they also carried a variant line characterized by 41 chromosomes. All eight lymphomas induced in adult AKR mice and six out of seven spontaneous AKR lymphomas showed predominantly diploid modal line. The remaining spontaneous lymphoma had a hyperdiploid stem line of 41 chromosomes. Microscopic and karyotypic analysis further identified trisomy 15 as the regular chromosome abnormality in the hyperdiploid cells in lymphomas of each group, whereas cells with diploid chromosome number had no detectable chromosome abnormality. Additional trisomies were also found, but their appearance was restricted to individual tumors. Thus, the incidence of trisomy 15 in lymphomas induced by MNUA in adult CFW/D and AKR mice, as well as in the spontaneous AKR lymphomas, is significantly lower than that in lymphomas induced in neonatal mice by the same carcinogen.

Pharmacological properties of the ornithine decarboxylase inhibitor 3-aminooxy-1-propanamine and several structural analogues.

Analogues of 3-aminooxy-1-propanamine proved to be highly potent and selective inhibitors of ornithine decarboxylase (ODC). The compounds competed with ornithine for the substrate binding site of ODC, but resulted in progressive and apparently irreversible inactivation of the enzyme. Diamine oxidase was inhibited by these compounds to a lesser extent than ODC; the compounds were not metabolized by this enzyme. Several derivatives were growth-inhibitory for human T24 cells and for other mammalian cells, the most active compound being 3-aminooxy-2-fluoro-1-propanamine (AFPA). Growth-arrested cells were largely depleted of putrescine and spermidine. Cellular growth arrest could be antagonized by supplementation with spermidine. Selection for resistance against AFPA led to cells with amplified ODC genes and overexpression of the message. Some of the derivatives were tumoristatic at well-tolerated doses in mice bearing solid T24 tumours. The antiproliferative activity of these compounds appears to be mediated by polyamine depletion.

A twelve-year profile of students' SAT scores, GPAs, and MCAT scores from a small university's premedical program.

PURPOSE: To determine whether significant correlations existed among quantitative and qualitative predictors of students' academic success and quantitative outcomes of such success over a 12-year period in a small university's premedical program. METHOD: A database was assembled from information on the 199 graduates who earned BS degrees in biology from Barry University's School of Natural and Health Sciences from 1980 through 1991. The quantitative variables were year of BS degree, total score on the Scholastic Aptitude Test (SAT), various measures of undergraduate grade-point averages (GPAs), and total score on the Medical College Admission Test (MCAT); and the qualitative variables were minority (54% of the students) or majority status and transfer (about one-third of the students) or nontransfer status. The statistical methods were multiple analysis of variance and stepwise multiple regression. RESULTS: Statistically significant positive correlations were found among SAT total scores, final GPAs, biology GPAs versus nonbiology GPAs, and MCAT total scores. These correlations held for transfer versus nontransfer students and for minority versus majority students. Over the 12-year period there were significant fluctuations in mean MCAT scores. CONCLUSION: The students' SAT scores and GPAs proved to be statistically reliable predictors of MCAT scores, but the minority or majority status and the transfer or nontransfer status of the students were statistically insignificant.

Some mechanisms operative in carcinogenesis a review.

Multiple factors contribute to the development of neoplasia. Sometimes a single agency can bring about a tumour if it has many different effects, but at other times a tumour arises more insidiously due to a succession of events [240,241] which by themselves may be innocent. Alterations in the genome of the cell are at the fore-front of our interest because they can be brought about by most of the carcinogenic agents we know. The cell can repair some such alterations but both forward and destructive mutations do appear. The roles of cell proliferation, cell differentiation, the immune mechanism and carcinogen-activating enzymes are beginning to be understood. The effects of dose, route of administration, and of other agents given at the same time [242-245] must not be lost sight of. Other factors no doubt will be added as we begin to look at the structure and function of cell-surface membranes [246-248], at host susceptibility genetics [26, 249], and at the generation of carcinogens inside the body [250,251]. We are only beginning to understand carcinogenesis. In no single instance do we as yet know how a tumour comes about in full details of molecular biology. It is possible that fully rational treatment of cancer will not be possible until we have such an understanding. Once a tumour becomes independent of carcinogenic factors, it continues to develop in a bizarre fashion which makes its study and treatment by all means other than surgery difficult.

Methylation of DNA in various organs of C57B1 mice by a carcinogenic dose of N-methyl-N-nitrosourea and stabiltty of some methylation products up to 18 hours.

Female 6-8-week-old (57B1 mice were injected i.p. with N-methyl-N-nitrosourea (MNUS) (14C or 3H-methyl-labelled) in saline (80 mg/kg) and DNA was isolated from bone marrow, small bowel, kidneys, liver, lungs, spleen and thymus at various times thereafter up to 18 h. Methylation of DNA was found in all organs examined, and by analyses using column or paper chromatography of DNA hydrolysates, the extent of methylation of DNA purines was determined. Methylated guanine residues (at N-3, N-7 and 0-6 positions) were stable in DNA up to 18 h, but methylated adenines (at N-3 or N-7) were removed from DNA of all organs examined; the overall half-life of methyladenines was about 3 h, but removal appeared to occur in a biphasic manner, with a proportion of methyladenine remaining relatively stable. This relative stability was somewhat more marked in bone marrow than in other organs.

Tissue distribution and mode of DNA methylation in mice by methyl methanesulphonate and N-methyl-N' -nitro-N-nitrosoguanidine: lack of thymic lymphoma induction and low extent of methylation of target tissue DNA at 0-6 of guanine.

The methylating agents methyl methanesulphonate (MMS) and N-methyl N'-nitro-N-nitrosoguanidine (MNNG), administered by single i.p. injection in mice failed to yield thymic lymphoma at doses around 60% of the LD50 values, in contrast to MNUA which gives a high yield of tumours by this route. Comparison of the tissue distribution and mode of DNA methylation by these agents showed a positive correlation with ability to methylate the 0-6 atom of guanine in DNA of the target tissues thymus and bone marrow and tumorigeneis. MMS gave a low yield of this product due to its relatively low Sn1 reactivity but was able to methylate DNA extensively at other sites in the target tissues and other organs examined. MNNG despite its ability to methylate 0-6 of guanine in DNA in vitro to the same relative extent as the potent carcinogen MNUA, methylated DNA of thymus and bone marrow to a very small extent in vivo but was able to methylate DNA in certain other tissues nearer the site of i.p. injection. These findings contrast with the general relatively extensive methylation of 0-6 of guanine in DNA of the target tissues and other organs by N-methyl-N-nitrosourea (MNUA).