Does an enzyme other than thrombin contribute to unexpected changes in the levels of the different forms of thrombin activatable fibrinolysis inhibitor in patients with hemophilia A, hemophilia B and von Willebrand disease?

Pro-thrombin activatable fibrinolysis inhibitor (pro-TAFI), also called plasma procarboxypeptidase B or U, is one of the modulators of fibrinolysis in blood. Pro-TAFI is activated by thrombin/thrombomodulin complex or by plasmin to a carboxypeptidase B-like enzyme (TAFI) of 35.8 kD molecular weight. TAFI spontaneously becomes inactive as a result of a temperature-dependent conformational change in the protein (TAFIi). In this study, pro-TAFI, total TAFI antigen and TAFI-TAFIi antigen levels were measured in 32 patients with hemophilia A, 4 patients with hemophilia B, 21 patients with von Willebrand disease (VWD) and 13 healthy controls. A statistically significant decrease in pro-TAFI was found in all groups (10.72+/-4.57 mg/L (p<0.001); 8.00+/-2.35 mg/L (p<0.01) and 8.98+/-2.33 mg/L (p <0.001) for hemophilia A, hemophilia B and VWD, respectively) compared to controls (17.85+4.61 mg/L). A statistically significant increase in TAFI-TAFIi antigen was found in hemophilia A (1.05+/-1.01 mg/L) (p<0.05) and in VWD patients (0.96+/-1.01 mg/L) (p<0.05) compared to controls (0.55+/-0.36 mg/L). There was no difference in total TAFI antigen levels between any group of patients and the controls. Neither did pro-TAFI nor TAFI-TAFIi levels differ within the group of hemophilia A patients in relation to severity (mild, moderate and severe) or among the VWD patients in relation to subtype (type 1, type 2A and type 3). These findings indicate an increased conversion of pro-TAFI to TAFI and/or TAFIi in patients with bleeding disorders. As thrombin generation is seriously impaired in these patients and almost absent in hemophilia A and B and in type 3 VWD, it is possible that plasmin mediates pro-TAFI activation in these patients. Enhanced fibrinolysis via generation of plasmin has previously been reported in hemophilia and VWD. Activation of pro-TAFI by plasmin may be a feedback mechanism that counterbalances increased fibrinolysis in patients with bleeding disorders. The relationship between the TAFI activation pathway and bleeding complications associated with hemophilia A, hemophilia B and VWD requires further investigation.

Effect of linker variation on the stability, potency, and efficacy of carcinoma-reactive BR64-doxorubicin immunoconjugates.

The internalizing anti-Le(y) monoclonal antibody (MAb) BR64 was conjugated to the anticancer drug doxorubicin (DOX) using an acid-labile hydrazone bond to the DOX and either a disulfide or thioether bond to the MAb. The resulting disulfide (BR64-SS-DOX) and thioether (BR64-S-DOX) conjugates were evaluated for stability, potency, and antigen-specific activity in both in vitro and in vivo model systems. The BR64-SS-DOX conjugates demonstrated antigen-specific activity both in vitro and when evaluated against antigen-expressing, DOX-sensitive human carcinoma xenografts. However, the stability and potency of disulfide conjugates were poor, and in vivo activity superior to unconjugated DOX was seen only at doses approaching the maximum tolerated dose. Furthermore, BR64-SS-DOX conjugates were not active against antigen-expressing, DOX-insensitive colon tumor xenografts. In contrast, the BR64-S-DOX conjugates demonstrated good stability both in vitro and in vivo. The increased stability of the BR64-S-DOX conjugates resulted in the delivery of more biologically active DOX to tumors with a concomitant increase in potency and efficacy over that which could be achieved with either unconjugated DOX or BR64-SS-DOX conjugates. Delivery of DOX by BR64-SS-DOX conjugates resulted in complete regressions and cures of both DOX-sensitive lung xenografts and DOX-intensitive colon tumor xenografts. These results demonstrate the importance of linker stability when delivering drugs such as DOX to carcinomas via internalizing antibodies and are likely to have direct relevance to the clinical utility of MAb-directed delivery.

Granulocyte-macrophage colony-stimulating factor plays a role in the functional activity of mast cells.

A peptide homologous to a region of murine granulocyte-macrophage colony-stimulating factor (mGM-CSF), P27-38, which was shown to be a GM-CSF antagonist, inhibited the function of serotonin release from murine mast cells. Peptide P27-38 inhibited immunoglobulin E (IgE)-mediated serotonin release in a dose-dependent manner when induced by either specific antigen or anti-IgE antibody. In contrast, non-receptor-mediated release of serotonin by agents such as compound 48/80 or the calcium ionophore A23187 were not affected by the GM-CSF antagonist. Similar effects were observed with GM-CSF-neutralizing antibodies. The inhibitory effect of P27-38 and the neutralizing antibodies on serotonin release could be reversed by the addition of exogenous GM-CSF to the stimulated mast cells, indicating that the inhibitory activity was probably due to an effect on endogenously produced GM-CSF. These findings suggest that GM-CSF produced by stimulated mast cells is involved in the regulation of their activity in an autocrine manner.

(6-Maleimidocaproyl)hydrazone of doxorubicin--a new derivative for the preparation of immunoconjugates of doxorubicin.

The (6-maleimidocaproyl)hydrazone of doxorubicin was synthesized and conjugated to several mAbs, including chimeric BR96, via a Michael addition reaction to thiol-containing mAbs. DTT reduction of disulfides present in the mAb was a reliable and general method for generating a consistent number of reactive SH groups. The conjugates, after purification by Bio-Beads, were free of unreacted linker and/or doxorubicin. All conjugates released doxorubicin under acidic conditions that mimic the lysosomal environment, while they were relatively stable at neutral pH. BR96 conjugates showed antigen-specific cytotoxicity.

Identification of functional domains in murine granulocyte-macrophage colony-stimulating factor using monoclonal antibodies to synthetic peptides.

Granulocyte-macrophage (GM)-CSF is an important hematopoietic cytokine that regulates proliferation and differentiation of macrophages, neutrophils, and eosinophils. In this study, we generated mAb to five synthetic peptides that correspond to regions along the murine GM-CSF molecule. The ability of anti-peptide mAb to bind to and inhibit biologic activity of murine (m) GM-CSF was determined. mAb with the highest neutralization titers were derived from mice immunized with peptide II, which correspond to amino acids 27 to 38 of mGM-CSF. Immunochemical studies showed that peptide II specifically blocked binding of anti-peptide II mAb to GM-CSF. mAb to two other peptides in the N-terminal half corresponding to residues 7 to 17 and 47 to 58, respectively, of mGM-CSF also inhibited GM-CSF-dependent proliferation and differentiation of murine bone marrow precursors for macrophages and granulocytes. Anti-peptide mAb also inhibited growth of a murine hematopoietic cell line FDCP1 and a murine T cell line HT-2, which was shown to be dependent on GM-CSF for growth in vitro. Biologic activity of both natural and recombinant mGM-CSF was neutralized by anti-peptide mAb. These findings indicate that epitopes in the N-terminal region of mGM-CSF are important for biologic activity, and the epitope defined by peptide II (residues 27 to 38) lies within a particularly important functional domain of the mGM-CSF molecule.

Antigen-specific activity of carcinoma-reactive BR64-doxorubicin conjugates evaluated in vitro and in human tumor xenograft models.

The anticarcinoma antibody BR64 was conjugated to a doxorubicin derivative, doxorubicin 13-[3-(2-pyridyldithio)propionyl]hydrazone, and the resulting conjugates (BR64-DOX) were evaluated for activity and immunological specificity in vitro and in human tumor xenograft models. The BR64-DOX immunoconjugates retained immunoreactivity and cytotoxicity and demonstrated antigen-specific cytotoxicity in vitro. The potency of BR64-DOX immunoconjugates in vitro was related to the drug:monoclonal antibody mole ratio of the conjugates. The antitumor activity of BR64-DOX conjugates was consistently superior to the maximal activity obtained with the parent drug, doxorubicin (DOX), in established human lung and human breast carcinoma xenograft models. The superior antitumor activity of BR64-DOX conjugates was reflected both in tumor growth inhibition and in regressions and cures of established tumors following the administration of tolerated doses of BR64-DOX. The antitumor activity of BR64-DOX conjugates was not the result of synergism between monoclonal antibody BR64 and DOX, because mixtures consisting of monoclonal antibody and optimized DOX were not more active than an equivalent dose of DOX administered alone. The antitumor activity of BR64-DOX conjugates was antigen specific; equivalent doses of nonbinding isotype-matched conjugates were not active against established tumor xenografts.

New hydrazone derivatives of adriamycin and their immunoconjugates--a correlation between acid stability and cytotoxicity.

New N-substituted hydrazine linkers were synthesized and their hydrazone derivatives of adriamycin were prepared. These functionalized adriamycin derivatives were conjugated with a monoclonal antibody, 5E9. The release rate of adriamycin from the hydrazones and from some of the conjugates was studied, and their relationship to the IC50's of the conjugate against 5E9-positive Daudi cells was investigated.

Adriamycin(hydrazone)-antibody conjugates require internalization and intracellular acid hydrolysis for antitumor activity.

Adriamycin hydrazone (ADM-Hzn) immunoconjugates have previously been shown to exhibit antibody-directed antitumor activity in vitro and in vivo. In this report, the biological and biochemical properties of the mAb and linker were investigated. Conjugates prepared with two antibodies 5E9 [anti-(transferrin receptor)] and G28.1 (anti-CD37), (which internalize from the surface of target cells following binding) were more cytotoxic in vitro and had greater antitumor activity against Daudi B lymphoma tumor xenografts than a non-internalizing immunoconjugate prepared with mAb 2H7 (anti-CD20). In addition, the 13-acylhydrazone bond linking the drug to the mAb was labile at pH 5 and released unmodified ADM at a rapid rate (t1/2 = 2.5 h). Immunoconjugates prepared with an oxime linkage at the C-13 position were stable to acid and were not cytotoxic. These findings suggest that internalization of ADM-Hzn immunoconjugates and release of free ADM from the mAb in acidic intracellular compartments were important steps in the mechanism of action of ADM-Hzn immunoconjugates.

Homodimeric forms of bombesin act as potent antagonists of bombesin on Swiss 3T3 cells.

Two Lys3-bombesin dimers were prepared by crosslinking epsilon-amino groups Lys3-bombesin with noncleavable (glutaraldehyde) and cleavable [dimethyl-3,3'-dithiobispropionimidate (DTBP)] crosslinkers. The dimers were purified by HPLC ion-exchange chromatography and were shown to have retained immunoreactivity with an anti-bombesin monoclonal antibody directed against the C-terminal binding region of bombesin. The glutaraldehyde cross-linked bombesin dimer specifically inhibited binding of 125I-GRP to its receptor on Swiss 3T3 cells. Bombesin, at 0.6-60 nM induced mitogenesis in quiescent Swiss 3T3 cells, whereas, incubation of cells with the glutaraldehyde bombesin dimer at concentrations up to 124 nM did not. In competition assays, the bombesin dimer exhibited a dose dependent inhibition of bombesin-induced mitogenic activity and intracellular Ca++ mobilization. The bombesin dimer was 100 to 1000-fold more potent than D-Phe12Leu14-bombesin and D-Phe12bombesin, respectively, in inhibiting bombesin-induced mitogenesis on quiescent Swiss 3T3 cells. Similarly, the DTBP-bombesin dimer was not mitogenic to Swiss 3T3 cells, however, cleavage of the disulfide crosslinker with DTT of cell bound DTBP dimer restored mitogenic activity. Finally, the glutaraldehyde bombesin dimer also inhibited growth of bombesin receptor positive H345 SCLC cells in vitro. These findings suggest that the dimeric forms of bombesin are potent antagonists of bombesin.

Evaluation in vitro of adriamycin immunoconjugates synthesized using an acid-sensitive hydrazone linker.

A novel method for linking Adriamycin (ADM) to monoclonal antibodies is described in which the 13-keto position of the anthracycline is used as the attachment site to the linker arm. A new ADM acylhydrazone derivative, Adriamycin 13-[3-(2-pyridyldithio)propionyl]hydrazone hydrochloride, which contains a pyridyl-protected disulfide, was synthesized and used for conjugation to monoclonal antibodies (MAbs) that were thiolated with N-succinimidyl 3-(pyridyldithiol)propionate or 2-iminothiolane. This resulted in formation of a linker between MAb and drug that contained a disulfide bond. Conjugation conditions were optimized to yield conjugates with high ADM:MAb molar ratios. The final immunoconjugate yields were found to decrease as the ADM:MAb molar ratio of the conjugates increased. Stability studies indicated that ADM was released from the immunoconjugates at mildly acidic pHs ranging from 4.5-6.5. Treatment of immunoconjugates with mild reducing agent dithiothreitol resulted in release of an acylhydrazone derivative of ADM. Flow-cytometric studies showed that the binding activity of various MAbs following conjugation to ADM was preserved at ADM:MAb molar ratios up to 10. Antibody-directed cytotoxicity was demonstrated under several assay conditions using combinations of antigen-positive and antigen-negative cells and binding and nonbinding immunoconjugates. In several experiments, ADM immunoconjugates were more potent than equivalent amounts of unconjugated ADM.

Antitumor activity of adriamycin (hydrazone-linked) immunoconjugates compared with free adriamycin and specificity of tumor cell killing.

Adriamycin (ADM) was chemically coupled to two monoclonal antibodies (MAb) expressed on human B-cell lymphomas. Immunoconjugates were prepared by linking to the MAb an ADM derivative, Adriamycin 13-[3-(2-pyridyldithio)propionyl]hydrazone (ADM-HZN), which releases ADM under mild acidic conditions (see preceding article). The (ADM-HZN) conjugates were tested for antitumor activity on two human B-lymphoma xenografts, Daudi and Ramos, which were growing as solid tumors in athymic mice. The conjugates, injected i.p., significantly inhibited tumor growth when antibody protein doses were greater than or equal to 500 mg/kg (approximately 10 mg/mouse). At these input antibody doses, (ADM-HZN) conjugates were more potent and had greater antitumor activity than free ADM given at an optimized dose and schedule. MAb-conjugated ADM was also tolerated to much higher levels than unconjugated drug. Antitumor activity was not obtained using mixtures of MAb plus free drug or with MAb-drug conjugates that did not bind to the tumor target cell. Thus, the antitumor activity of the immunoconjugate was directed by binding of the MAb portion of the conjugate to target tumor cells.

Immunohistochemical and biochemical characterization of antisera raised to human tumor nucleoli.

Antinucleolar antisera were raised in rabbits, goats and sheep to nucleoli isolated from three human tumor cell lines. The antisera were shown to cross-react by immunofluorescence with human tumor cell lines originating from different organs and with frozen sections from a wide variety of human malignant and non-malignant tissues. Tumor versus normal tissue discrimination by several antisera was significantly improved by treatment of frozen tissues with a buffered glutaraldehyde/Triton X-100 solution prior to immunofluorescent staining. The molecular specificity of these antisera was determined by immunostaining electrotransfer nitrocellulose strips following SDS-PAGE of nucleolar preparations and nuclear extracts. Although different immunostaining patterns were obtained for individual antinucleolar antisera, nucleolar proteins of molecular weight 120, 100, 94, 68, 54, 38, 33, and 32 kDa were the most often recognized by antisera raised in the three different species. G187 antiserum strongly reacted with 100, 94, and 38 kDa proteins from freshly obtained leukemic specimens. The Immunoreactivity of the 100, 94, and 38 kDa proteins was unaffected by glutaraldehyde/Triton X-100 treatment when immunostained with antisera that demonstrated the greatest tumor specificity on sections treated with glutaraldehyde/Triton X-100. These three nucleolar proteins may be more highly associated with nucleoli of malignant cells than with nucleoli of normal cells.

Antibodies to the common leukocyte antigen (T200) inhibit an early phase in the activation of resting human B cells.

T191, a monoclonal antibody reactive with the T200 common leukocyte antigen, profoundly inhibits an early event(s) associated with alpha-immunoglobulin M (alpha IgM)/T cell replacing factor (TRF) or alpha IgM/recombinant interleukin 1 and 2 (rIL 1 and rIL 2)-induced tonsillar B cell proliferation. Kinetic analysis of T191-mediated inhibition indicated that the antibody exerts its effect within 12 to 24 hr of the initiation of cultures and rapidly loses its activity thereafter. Small resting B cells are most sensitive to T191 inhibition, whereas B cells with increasing buoyant density (presumably reflecting stages of increased activation) become progressively T191 insensitive. Analysis of RNA synthesis subsequent to alpha IgM crosslinking of surface immunoglobulin demonstrated that T191 reduced [3H]uridine incorporation by up to 38% during the first 20 hr of culture. In contrast to the effects seen with alpha IgM stimulated B cells, T191 had no inhibitory effect upon phorbol myristate acetate-induced B cell proliferation. The inhibitory effect upon B cell proliferation observed with T191 is not unique among other alpha-T200 antibodies. Four of five previously described alpha-T200 monoclonal antibodies had similar inhibitory effects (82 to 57% maximum inhibition of [3H]thymidine incorporation). However, 13.3, an alpha-T200 monoclonal antibody previously shown to block natural killer (NK) cell-mediated killing was without effect. Likewise, those antibodies capable of inhibiting B cell proliferation failed to block NK-mediated cytolysis. Antibody binding experiments together with proliferation inhibition studies suggest that all of the monoclonal antibodies tested recognized distinct epitopes on the T200 antigen. Both observations are of significance because they demonstrate that the effects seen with anti-T200 antibodies represent an interference with highly specific functional regions on the T200 molecules.

Biochemical analysis and biological activities of immunoaffinity purified natural murine gamma interferon (MuIFN-gamma).

A rapid one-step procedure for the purification of natural murine gamma interferon (MuIFN-gamma) by immunoaffinity chromatography has been developed. Crude MuIFN-gamma was passed through a column containing immobilized anti-MuIFN-gamma monoclonal antibody, and the column was washed with high salt and detergent. Active MuIFN-gamma was subsequently eluted with ammonium thiocyanate/glycerol (yields of 20-40%). Analysis of the immunopurified interferon by sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing conditions revealed biologically active bands of about 19.5, and 14 kd. Under nonreducing conditions, three active bands at 35, 19.5, and 14 kd could be seen. Gel filtration of the immunopurified MuIFN-gamma under reducing or nonreducing conditions showed a single major peak of antiviral activity corresponding to a molecular weight of 43 kd. The protein eluted from each band from the SDS-gel, in addition to possessing antiviral activity, was shown to activate macrophages in three assays: macrophage tumoricidal activity, hydrogen peroxide secretion, and expression of la antigen on the surface of exudate macrophages.

L-Threonine deaminase as a possible antitumor agent.

In vitro studies with L-threonine deaminase purified from sheep liver demonstrated that the enzyme was cytotoxic toward two lines of murine leukemia cells but not toward L cells or normal human fibroblasts. When L-threonine deaminase was injected into mice, it was possible to lower the plasma L-threonine concentration by 90%. The enzyme had a half-life of 1.5 hours in the circulation.