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Purpose: To provide standards for medically integrated dispensing of oral anticancer drugs and supportive care medications., Methods: An Expert Panel was formed, and a systematic review of the literature on patient-centered best practices for the delivery of oral anticancer and supportive care drugs was performed to April 2019 using PubMed and Google Scholar. Available patient-centered standards, including one previously developed by the National Community Oncology Dispensing Association (NCODA), were considered for endorsement. Public comments were solicited and considered in preparation of the final manuscript., Results: A high-quality systematic review that was current to May 2016 was adopted into the evidence base. Five additional primary studies of multifaceted interventions met the inclusion criteria. These studies generally included a multicomponent intervention, often led by an oncology pharmacist, and also included patient education and regular follow-up and monitoring. These interventions resulted in significant improvements to patient quality and safety and demonstrated improvements in adherence and other patient outcomes., Conclusion: The findings of the systematic review were consistent with the NCODA patient-centered standards for patient relationships and education, adherence, safety, collection of data, documentation, and other areas. NCODA standards were adopted and used as basis for these American Society of Clinical Oncology/NCODA standards. Additional information is available at www.asco.org/mid-standards.

The Association of Community Cancer Centers (ACCC), Hematology/Oncology Pharmacy Association (HOPA), National Community Oncology Dispensing Association (NCODA), and Oncology Nursing Society (ONS) partnered together to create a resource for providers, and patients and caregivers on oral chemotherapy agents. The patient education sheets include information on medication names and pronunciation, approved uses, dose and schedule, drug and food interactions, the best practice guidelines for safe handling, administration, and disposal of oral chemotherapy agenzts by patients and caregivers; management strategies for the most common side effects; and pregnancy, sexual activity, and contraception information. Each sheet also has an area to list from which pharmacy the patient will receive the medication. The document and the website also provide the link to the individual product website, prescribing information, and product resources, if available.

A novel class of pyrazolopyrimidine-sulfonamides was discovered as selective dual inhibitors of aurora kinase A (AKA) and cyclin-dependent kinase 1 (CDK1). These inhibitors were originally designed based on an early lead (compound I). SAR development has led to the discovery of potent inhibitors with single digit nM IC(50)s towards both AKA and CDK1. An exemplary compound 1a has demonstrated good efficacy in an HCT116 colon cancer xenograft model., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Bispecific antibodies (BsAbs) target multiple epitopes on the same molecular target or different targets. Although interest in BsAbs has persisted for decades, production of stable and active BsAbs has hindered their clinical evaluation. Here, we describe the production and characterization of tetravalent IgG-like BsAbs that combine the activities of allosteric and competitive inhibitors of the type-I insulin-like growth factor receptor (IGF-1R). The BsAbs, which were engineered for thermal stability, express well, demonstrate favorable biophysical properties, and recognize both epitopes on IGF-1R. Only one BsAb with a unique geometry, denoted BIIB4-5scFv, was capable of engaging all four of its binding arms simultaneously. All the BsAbs (especially BIIB4-5scFv) demonstrated enhanced ligand blocking over the single monoclonal antibodies (mAbs), particularly at high ligand concentrations. The pharmacokinetic profiles of two IgG-like BsAbs were tested in nude mice and shown to be comparable with that of the parental mAbs. The BsAbs, especially BIIB4-5scFv, demonstrated an improved ability to reduce the growth of multiple tumor cell lines and to inhibit ligand-induced IGF-1R signaling in tumor cells over the parental mAbs. BIIB4-5scFv also led to superior tumor growth inhibition over its parental mAbs in vivo. In summary, BsAbs that bridge multiple inhibitory mechanisms against a single target may generally represent a more effective strategy for intervention in oncology or other indications compared with traditional mAb therapy.

Mammalian cells are used for the production of numerous biologics including monoclonal antibodies. Unfortunately, mammalian cells can lose viability at later stages in the cell culture process. In this study, the effects of expressing the anti-apoptosis genes, E1B-19K and Aven, separately and in combination on cell growth, survival, and monoclonal antibody (MAb) production were investigated for a commercial Chinese Hamster Ovary (CHO) mammalian cell line. CHO cells were observed to undergo apoptosis following a model insult, glucose deprivation, and at later stages of batch cell culture. The CHO cell line was then genetically modified to express the anti-apoptotic proteins E1B-19K and/or Aven using an ecdysone-inducible expression system. Stable transfected pools induced to express Aven or E1B-19K alone were found to survive 1-2 days longer than the parent cell line following glucose deprivation while the expression of both genes in concert increased cell survival by 3 days. In spinner flask batch studies, a clonal isolate engineered to express both anti-apoptosis genes exhibited a longer operating lifetime and higher final MAb titer as a result of higher viable cell densities and viabilities. Interestingly, survival was increased in the absence of an inducer, most likely as a result of leaky expression of the anti-apoptosis genes confirmed in subsequent PCR studies. In fed-batch bioreactors, the expression of both anti-apoptosis genes resulted in higher growth rates and cell densities in the exponential phase and significantly higher viable cell densities, viabilities, and extended survival during the post-exponential phase. As a result, the integral of viable cells (IVC) was between 40 and 100% higher for cell lines engineered to express both Aven and E1B-19K in concert, and the operational lifetime of the fed-batch bioreactors was increased from 2 to 5 days. The maximum titers of MAb were also increased by 40-55% for bioreactors containing cells expressing Aven and E1B-19K. These increases in volumetric productivity arose primarily from enhancements in viable cell density over the course of the fed-batch culture period since the specific productivities for the cells expressing anti-apoptosis genes were comparable or slightly lower than the parental hosts. These results demonstrate that expression of anti-apoptosis genes can enhance culture performance and increase MAb titers for mammalian CHO cell cultures especially under conditions such as extended fed-batch bioreactor operation., ((c) 2006 Wiley Periodicals, Inc.)

The lymphotoxin-beta receptor (LT beta R) is a tumor necrosis factor receptor family member critical for the development and maintenance of various lymphoid microenvironments. Herein, we show that agonistic anti-LT beta R monoclonal antibody (mAb) CBE11 inhibited tumor growth in xenograft models and potentiated tumor responses to chemotherapeutic agents. In a syngeneic colon carcinoma tumor model, treatment of the tumor-bearing mice with an agonistic antibody against murine LT beta R caused increased lymphocyte infiltration and necrosis of the tumor. A pattern of differential gene expression predictive of cellular and xenograft response to LT beta R activation was identified in a panel of colon carcinoma cell lines and when applied to a panel of clinical colorectal tumor samples indicated 35% likelihood a tumor response to CBE11. Consistent with this estimate, CBE11 decreased tumor size and/or improved long-term animal survival with two of six independent orthotopic xenografts prepared from surgical colorectal carcinoma samples. Targeting of LT beta R with agonistic mAbs offers a novel approach to the treatment of colorectal and potentially other types of cancers.

Background: CD23 plays a role in the regulation of IgE production and allergy-induced immune and inflammatory responses. A novel anti-CD23 monoclonal antibody, lumiliximab, is a potential therapeutic antibody recently demonstrated to be safe in human beings., Objective: This study investigated the effects of lumiliximab on allergen-induced immune responses from atopic subjects compared with blocking HLA-DR and costimulatory molecules, CD80 and CD86., Methods: Allergen-stimulated PBMCs from atopic subjects were pretreated with lumiliximab or antibodies to CD80, CD86, and HLA-DR. Cultures were analyzed for cell proliferation and IL-1beta, TNF-alpha, and IL-5 cytokine secretion. An allergen-specific T-cell line was developed and analyzed for lymphocyte proliferation in response to allergen with or without lumiliximab. Lumiliximab's effect on CD86 expression was evaluated by flow cytometry in the U937 monocytic cell line., Results: Lumiliximab reduced allergen-induced PBMC proliferation by 50% (n = 6; P = .006). In addition, cultures pretreated with lumiliximab had a reduction in the proinflammatory cytokines IL-1beta (P < .003) and TNF-alpha (P = .05) and the T(H)2 cytokine IL-5 (P = .002). Blocking CD86 resulted in greater reduction in proliferation than lumiliximab (P = .003) but similar effects in cytokine secretion. The anti-CD80 blocking antibody had no effect on cytokine production but did reduce proliferation. Furthermore, the addition of lumiliximab to cytokine stimulated U937 cells reduced surface expression of CD86 (P = .012)., Conclusion: These results indicate that the anti-CD23 mAb, lumiliximab, may be involved in modulating antigen presenting cells and reducing TH2-type immune responses. The use of this antibody may provide clinical benefit for treating allergic diseases.

The gene encoding the rat glycosylation enzyme beta1-4-N-acetylglucosaminyltransferase III (GnTIII) was cloned and coexpressed in a recombinant production Chinese hamster ovary (CHO) cell line expressing a chimeric mouse/human anti-CD20 IgG1 antibody. The new cell lines expressed high levels of antibody and have growth kinetics similar to that of the parent. Relative QPCR showed the cell lines to express varying levels of mRNA. High-performance liquid chromatography (HPLC) analysis showed the enzyme to have added bisecting N-acetylglucosamine (GlcNAc) residues in most (48% to 71%) of the N-linked oligosaccharides isolated from antibody preparations purified from the cell lines. In an ADCC assay the new antibody preparations promoted killing of CD20-positive target cells at approximately 10- to 20-fold lower concentrations than the parent. This activity was blocked using an anti-Fc gamma RIII antibody, supporting the role of Fc gamma RIII binding in this increase. In addition, cell binding assays showed the modified antibody bound better to Fc gamma RIII-expressing cells. The increase in ADCC activity is therefore likely due to an increased affinity of the modified antibody for the Fc gamma RIII receptor.

We have used fluorescence in situ hybridization (FISH) to localize three random Chinese-hamster ovary (CHO) cell chromosomal integration sites and determine gene copy number in their amplified cell lines. Metaphase FISH showed all three to have integrated into different chromosome positions on different chromosomes. All three Geneticin parent cell lines were found to have single integration sites by Southern-blot analysis, and these data were confirmed using both metaphase and interphase FISH. Following amplification, metaphase FISH showed that amplification in two of the cell lines occurred by duplication in the chromosome arm where the integration occurred. However, for one cell line, amplification resulted in the translocation of amplified genes from one marker chromosome to another. Interphase FISH showed an expected increase in gene copy number upon amplification with methotrexate.

Keliximab, a Primatized IgG1 CD4 mAb, was reconfigured to an IgG4 antibody. The gamma4 constant region was further modified by substituting glutamic acid for serine at position 235 in the CH2 domain (IgG4-E), to remove residual binding to Fcgamma receptors, and substitution of serine with proline at position 228 in the hinge region (IgG4-PE) for greater stability. Pharmacokinetic analysis in rats gave a t(1/2) of approximately 4 days for IgG4-E and 9 days for IgG4-PE, consistent with a greater stability of the IgG4-PE molecule. The effects on T cell subsets were assessed in chimpanzees given escalating doses of IgG4-PE: 0.05 mg/kg on Day 16, 1.5 mg/kg dose on Day 43, and 15 mg/kg on Day 85. Receptor modulation was observed at the two highest doses, but no depletion of T cells at any dose. The in vitro and in vivo results demonstrate the potential of this IgG4-PE mAb for use in human trials., (Copyright 2000 Academic Press.)

Several CD4 mAbs have entered the clinic for the treatment of autoimmune diseases or transplant rejection. Most of these mAbs caused CD4 cell depletion, and some were murine mAbs which were further hampered by human anti-mouse Ab responses. To obviate these concerns, a primatized CD4 mAb, clenoliximab, was generated by fusing the V domains of a cynomolgus macaque mAb to human constant regions. The heavy chain constant region is a modified IgG4 containing two single residue substitutions designed to ablate residual Fc receptor binding activity and to stabilize heavy chain dimer formation. This study compares and contrasts the in vitro properties of clenoliximab with its matched IgG1 derivative, keliximab, which shares the same variable regions. Both mAbs show potent inhibition of in vitro T cell responses, lack of binding to complement component C1q, and inability to mediate complement-dependent cytotoxicity. However, clenoliximab shows markedly reduced binding to Fc receptors and therefore does not mediate Ab-dependent cell-mediated cytotoxicity or modulation/loss of CD4 from the surface of T cells, except in the presence of rheumatoid factor or activated monocytes. Thus, clenoliximab retains the key immunomodulatory attributes of keliximab without the liability of strong Fcgamma receptor binding. In initial clinical trials, these properties have translated to a reduced incidence of CD4+ T cell depletion.

CD23, the low affinity receptor for IgE (FcvarepsilonRII), is involved in regulation of IgE synthesis by B-lymphocytes. Five monoclonal antibodies to human CD23 were generated from cynomolgus macaques immunized with purified soluble CD23 (sCD23). Four of the five primate antibodies blocked the binding of IgE complexes to CD23 positive cells and also inhibited the production of IgE in vitro by IL-4 induced human peripheral blood mononuclear cells (PBMC). The variable domains of several primate antibodies were utilized to construct chimeric macaque/human (PRIMATIZED((R))) monoclonal antibodies. PRIMATIZED((R)) p5E8G1, containing human gamma 1 constant region, inhibited IgE production in vitro as efficiently as the parent primate antibody, but the human gamma 4 constant version, PRIMATIZED((R)) p5E8G4, was not as effective in IgE inhibition. An F(ab')(2) of p5E8G1 did not inhibit IgE production but did interfere with IgE inhibition by the intact anti-CD23 antibody in a dose dependent fashion. The murine monoclonal antibody MHM6 recognizes human CD23 at a different epitope than primate antibody 5E8, and inhibits IgE production by IL-4 induced PBMC. As with the F(ab')(2) of p5E8G1, the F(ab')(2) of MHM6 also failed to inhibit IgE production. These data imply that the mechanism by which anti-CD23 antibodies inhibit IgE production requires cross-linking of CD23 to an IgG receptor. These data also imply that neither bivalent cross-linking of CD23 alone or inhibition of CD23 binding to its natural ligands is sufficient to inhibit IgE production.

This chapter has described a bioenergetic analysis of the interaction of sCD4 with an IgG1 and two IgG4 derivatives of an anti-sCD4 MAb. The MAbs have identical VH and VL domains but differ markedly in their CH and CL domains, raising the question of whether their antigen-binding chemistries are altered. We find the sCD4-binding kinetics and thermodynamics of the MAbs are indistinguishable, which indicates rigorously that the molecular details of the binding interactions are the same. We also showed the importance of using multiple biophysical methods to define the binding model before the bioenergetics can be appropriately interpreted. Analysis of the binding thermodynamics and kinetics suggests conformational changes that might be coupled to sCD4 binding by these MAbs are small or absent.

Background: Respiratory syncytial virus (RSV) infection is a major problem in the newborn and aging populations. Fully human monoclonal antibodies with the ability to neutralize RSV could have a major impact on the immunotherapy of the disease. The generation of human antibodies has been difficult because there exists no general way to activate B cells against an antigen of choice in vitro., Materials and Methods: Human spleen cells from individuals exposed to RSV were used to repopulate SCID mice. Hu-SCID mice were boosted with RSV fusion (F)-protein and subsequently developed B cell tumors. The tumors were removed and cultured and subcloned in vitro, using a feeder layer of CD154-expressing T cells. Two of these tumors produced the antibodies designated RF-1 and RF-2. VL genes were isolated by standard PCR techniques, however, it was necessary to use high-temperature reverse transcriptase to clone the VH genes., Results: RF-1 and RF-2 VH genes were both found to be closely related members of the VH2 family. Vk genes originated from the VK III family. RF-1 and RF-2 recombinant antibodies expressed in CHO cells (cRF-1 and cRF-2) were found to have affinities for RSV F-protein of 0.1 nM and 0.07 nM, respectively, and both were able to neutralize several A and B subtypes of RSV., Conclusion: The technique of immortalizing human B lymphocytes, by passage in SCID mice and expression as recombinant antibodies in CHO cells, provides a method by which high-affinity human antibodies can be developed for immunotherapy of viral diseases.

The proto-oncogene product p95Vav (Vav) undergoes rapid phosphorylation on tyrosine following stimulation of the T or B cell antigen receptor, and in response to a variety of other cell surface stimuli. Vav contains, among other, a guanine nucleotide exchange factor domain with homology to the Rho/Rac/CDC42 exchange protein Db1. It has been recently shown that Vav is functionally linked to small GTPases of the Rho family, suggesting that it is an activator of Rho GTPases and may participate in regulation of cytoskeletal organization. The present study shows that cell adhesion to fibronectin triggers rapid phosphorylation of Vav on tyrosine in Vav-transfected CHO cells and in Jurkat T cells. Vav phosphorylation is strongly dependent on adhesion and is mediated by beta 1 integrins. Furthermore, Vav overexpression enhances the adhesion-dependent increase in the rate and extent of phosphorylation on focal adhesion kinase and paxillin, and the formation of stress fibers and lamellipodia. In addition, there is a marked increase in the amount of Vav localized to the triton-insoluble fraction following 1 h of incubation on FN. Finally, Vav increases the growth rate of the cells in an adhesion-dependent manner. Our results strongly implicate Vav as a mediator of integrin signal transduction.

A Primatized anti-CD4 monoclonal antibody (MAb), CE9.1, with V-domain from cynomolgus macaque (showing 92% homology with human consensus sequence V-domains), and a human IgG1 constant region, was characterized in vitro and in vivo in chimpanzees. This MAb binds human CD4 with Kd of 1.0 nM and was also able to bind to human IgG Fc receptors (Fc gamma R). However, despite being of the IgG1 subclass, CE9.1 did not bind to complement component C1q, nor did it mediate complement-dependent cytotoxicity. Examination of T cells from a number of species showed restricted reactivity for CE9.1, recognizing only human and chimpanzee CD4. In both human and chimpanzee MLRs, it had an IC50 of about 10.0 ng/mL. Therefore, a chimpanzee in vivo model was used to characterize CE9.1, CE9.1 caused transient decrease in the number of lymphocytes bearing the CD4 receptor starting at doses of 0.3 mg/kg in an in vivo dose ranging study in one chimpanzee. This effect was reversed within approximately 7 days. In a multiple high-dose study in which 10.0 mg/kg of CE9.1 was administered at intervals of 1-3 months, there was a dramatic loss of CD4 marker with a reciprocal increase in the number of CD3+ CD8- CD4- cells. The CD4 receptor was totally undetectable on these lymphocytes for 1-2 weeks, with a gradual, but complete, reversal within 4 weeks. We interpret these observations as receptor modulation because, although there was apparent loss of CD4+ lymphocytes, an equivalent number of CD3+CD8- T lymphocytes were present in circulation in all four chimpanzees treated with 10.0 mg/kg CE9.1. Even at this high dose, only limited reduction of CD4+ T lymphocytes was observed in these animals. These observations are in sharp contrast to what has been reported in rodents or in human clinical studies using other IgG1 mAbs to human CD4. CD8 counts, although variable, remained unaffected by CE9.1 treatment. No adverse events were observed following administration of CE9.1 to chimpanzees, and there was no detectable host immune responses to the Primatized MAb.

Core-fucosylated glycans, derivatized with 2-aminoacridone, consistently migrate slower than the corresponding oligosaccharides which lack this fucose residue, using the micellar electrophoretic capillary chromatography conditions outlined in this study. alpha-Fucosidase digestion of glycans followed by CE analysis has allowed facile differentiation of these two classes of oligosaccharides and this methodology has been applied to obtain preliminary information on the carbohydrate content from two glycoproteins, a monoclonal IgG antibody and the soluble complement receptor type 1 (sCR1).

Murine monoclonal antibody 2B8 specifically recognizes the CD20 phosphoprotein expressed on the surface of normal B lymphocytes and B-cell lymphomas. The light- and heavy-chain variable regions of 2B8 were cloned, after amplification by the polymerase chain reaction, into a cDNA expression vector that contained human IgG1 heavy chain and human kappa-light chain constant regions. High-level expression of chimeric-2B8 antibody (C2B8) was obtained in Chinese hamster ovary cells. Purified C2B8 exhibited antigen binding affinity and human-tissue reactivity similar to the native murine antibody. In vitro studies showed the ability of C2B8 to bind human C1q, mediate complement-dependent cell lysis of human B-lymphoid cell lines, and lyse human target cells through antibody-dependent cellular cytotoxicity. Infusion of macaque cynomolgus monkeys with doses ranging from 1.6 mg/kg to 6.4 mg/kg resulted in greater than 98% depletion of peripheral blood (PB) B cells and 40% to 70% depletion of lymph node B cells. Recovery of PB B cells usually started at 2 weeks after treatment and required 60 to greater than 90 days to reach normal levels. As much as 95% depletion of B cells in peripheral lymph nodes and bone marrow was observed following weekly injections of 16.8 mg/kg antibody. No toxicity was observed in any of the animals. These results offer the possibility of using an "immunologically active" chimeric anti-CD20 antibody as an alternative approach in the treatment of B-cell lymphoma.

Immunoglobulin variable region genes from non-human primates, cynomolgus macaques, were shown to have 85%-98% homology with human immunoglobulin sequences and yet macaques are phylogenetically distant enough to respond against conserved human antigens. Immunoglobulin genes were isolated from monkeys immunized with human CD4 antigen and a human/monkey chimeric anti-CD4 antibody with 91-92% homology to human immunoglobulin framework regions was cloned and expressed. The antibody has an apparent affinity of 3.2 x 10(-11) M and exhibits potent immunosuppressive properties in vitro.

Tissue-type plasminogen activator (tPA) is a serine protease which cleaves plasminogen to its active form, plasmin. tPA plays a physiologic role in hemostasis, wound healing, and embryogenesis. Therapeutically, recombinant human tPA is used as a thrombolytic in myocardial infarction. Although production of therapeutic quantities of tPA in Chinese hamster ovary (CHO) cells transfected with the human gene for tPA is practical, production costs remain high. One important factor which determines the ultimate cost of tPA (or any other recombinant protein expressed in mammalian cells) is its production level on a per cell basis. We have used postembedding immunocytochemical staining with colloidal gold to study the subcellular localization of tPA in CHO cells expressing recombinant tPA (rCHO) in an effort to understand the factor(s) which might limit secretion. Staining for tPA was evaluated visually and by morphometric analysis and was specific and reproducible. Serially passaged rCHO showed no significant change in staining density over 31 serial passages. Staining density was greatest over dilated cisternae of the rough endoplasmic reticulum and nuclear envelope. Golgi stacks and large acid phosphatase-positive vacuoles (probably lysosomes) were also heavily stained. Staining of lysosomal vacuoles suggested that rCHO might be degrading nascent tPA. Incubation of rCHO with 125I-tPA showed that the cells were not internalizing tPA from the media. These results suggest that rCHO fail to secrete a portion of the tPA they synthesize and that it is degraded in lysosomes. This observation may have important implications on the choice of expression systems for efficient production of large quantities of recombinant proteins.

We have developed a highly modular vector, pDSP1, which contains two independent mammalian transcription cassettes. Each cassette contains SV40 early gene regulatory elements controlling the expression of an easily assayable, selectable Escherichia coli marker gene, either galK or xgprt. The regulatory elements of the galK cassette are bounded by multiple unique and nearly unique restriction sites allowing for the easy removal and replacement of either the regulatory sequences or of the galK gene itself. Expression of the marker genes is monitored by transient transfection into mammalian cells followed by filter enzyme assays. Expression of xgprt serves as an internal control and the relative expression of galK/xgprt is used to quantitate modifications made to the vector. We have used this system to analyze many eukaryotic polyadenylation regions as well as several other eukaryotic gene regulatory elements. We have also removed the galK gene and replaced it with other mammalian genes. The entire galK cassette is contained on a Sal I restriction fragment that can be readily removed and placed into a unique Sal I site in one of our Epstein-Barr virus (EBV), bovine papilloma virus (BPV), or BK defective viral stable expression vectors. We believe that pDSP1 is a powerful vector system for studying eukaryotic gene regulation, and in conjunction with our stable expression vectors, it represents a unified system for exploring expression in mammalian cells both transiently and stably.

A chimaeric recombinant plasminogen activator (rscu-PA- K2) obtained by insertion of the second kringle (K2) of tissue-type plasminogen activator (t-PA) (amino acids 173-262) between residues Asp130 and Ser139 of single chain urokinase-type plasminogen activator (scu-PA) was purified from the conditioned medium of mouse myeloma cells transfected with the previously described plasmid pULB9137 (Piérard et al., J. Biol. Chem. 262, 11771-11778, 1987). Approximately 22 micrograms of purified protein was obtained per liter of conditioned medium with a yield of approximately 25 percent. On sodium dodecylsulfate gel electrophoresis under reducing conditions, rscu-PA- K2 migrated with an apparent Mr of 65,000. Plasmin caused a time- and concentration-dependent conversion to an amidolytically active two chain derivative (rtcu-PA- K2) with a specific activity of 45,000 IU/mg. Both rscu-PA- K2 and rtcu-PA- K2 activated plasminogen directly with Km = 2.0 microM and k2 = 0.00063 s-1 and Km = 100 microM and k2 = 4.1 s-1 respectively. rscu-PA- K2 did not bind extensively to fibrin. It caused concentration-dependent lysis of 125I-fibrin-labeled plasma clots immersed in human plasma with a comparable specific activity and fibrin-specificity as rscu-PA. It is concluded that insertion in scu-PA of the second kringle of t-PA, which is believed to be involved in its fibrin affinity, does not significantly alter the enzymatic properties of scu-PA, but does not confer marked fibrin-affinity to the molecule.

High-level expression of human tissue-type plasminogen activator was accomplished in endothelial cells by a novel approach to dihydrofolate reductase (DHFR) coamplification in DHFR+ cells. A tripartite mammalian expression vector coding for DHFR, neomycin phosphotransferase, and the t-PA gene was introduced into bovine endothelial cells by transfection and selection for G418 resistance. Upon methotrexate selection of these transformants, we obtained endothelial cells that had amplified the plasmid-encoded DHFR and t-PA genes. As a result, cell lines were isolated that efficiently produced t-PA (greater than 4 pg/cell.day). This t-PA was purified and compared with recombinant t-PA produced in Chinese hamster ovary cells. These two t-PA samples differed in carbohydrate composition, and amounts of 530 and 527 amino acid forms but had similar in vitro activity.

The steady-state level attained for any protein in mammalian cells is in part determined by its steady-state level of mRNA. Sequence information in and around the 3' end of an RNA which is involved in specifying and regulating polyadenylation [poly(A)] may have important consequences on mRNA levels, and ultimately on expression of the protein product. In this report we compare the effects on gene expression which result from placing several different poly(A) regions, or no poly(A) region, downstream from a marker gene (galactokinase or galK) that can be readily assayed in mammalian cells. Our results demonstrate that the presence of a poly(A) region is important for efficient gene expression and that the use of the poly(A) region of bovine growth hormone (bGH) reproducibly results in three times higher expression than that of SV40 early or human collagen poly(A) regions. We further demonstrate that changing the promoter region on these chimeric transcription units does not change the effect of the poly(A) region. Neither does changing the assay gene, since comparison of the same poly(A) regions behind another marker gene (xanthine-guanine phosphoribosyl transferase or xgprt) leads to identical differences in expression. When we examine the levels of poly(A)+ RNA that result from each transcription unit, we find that they correlate precisely with the gene expression levels. Apparently the 3' end of an RNA is a determinant of steady-state mRNA levels and, in turn, the subsequent production of the protein product.

To investigate the structure-function relationship in tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA), four hybrid sequences were amplified and overexpressed in a mouse myeloma cell line. The following constructs were made starting from cDNA encoding human t-PA and u-PA: (i) a hybrid in which amino acids (AA) 1-262 of the A-chain of t-PA is fused to AA 139-411 of the B-chain of u-PA; (ii) a hybrid in which the kringle 2 region of t-PA (AA 173-262) is inserted between amino acids 130 and 139 of u-PA; (iii) hybrid #2 having amino acids 1 to 10 deleted and replaced by the finger region of t-PA (AA 1-50); and (iv) a chimera in which the finger region of t-PA is followed by amino acids 10-411 of u-PA and where the lysine residues at positions 135 and 136 of u-PA are replaced by glutamines. These four hybrids were efficiently secreted into the culture medium as single-chain polypeptides of the expected molecular weights and had fully functional catalytic activity. Replacement of the A-chain of u-PA by that of t-PA leads to increased fibrin binding, whereas additions of finger and kringle domains do not. These data suggest that structural domains in serine proteases may not fold and/or function autonomously.

RNase mapping experiments show that the bovine growth hormone (bGH) poly(A) region forms an extensive hairpin loop. Mutants were prepared to change poly(A) region pre-mRNA structure and cleavage site efficiency without altering necessary sequences. An inverted repeat which includes the poly(A) cleavage site was created by insertion of a linker upstream of the poly(A) region to compete with any wild-type secondary structure. RNA mapping analyses show alterations in the nuclease accessibility of this mutant at the natural site of cleavage. This mutant shows a 75% drop in relative reporter gene expression at the steady-state protein and RNA levels. When the linker is inserted as a direct repeat, expression is equivalent to wildtype levels. To show that transcription was not terminated by the inverted repeat, the SV40 late poly(A) region was inserted downstream. These mutants show restored expression and processing at the downstream site. Our experiments reveal that the conformation of the poly(A) site pre-mRNA is important in mediating efficient cleavage-polyadenylation.

The cell cycle of mouse hepatic cells was examined in vivo following partial hepatectomy, by differential chromatid staining in the presence of non-inhibitory concentrations of bromodeoxyuridine (BrdU). Using this technique, distribution curves were obtained for the appearance of metaphase cells in successive generations, and mean cell cycle time (11 hr) was determined. Cell cycle times derived with this technique are several-fold faster than previous reports of regenerating liver which used radionucleotide labelling.