Arzoxifene, a new selective estrogen receptor modulator for chemoprevention of experimental breast cancer.

Arzoxifene ([6-hydroxy-3-[4-[2-(1-piperidinyl)-ethoxy]phenoxy]-2-(4-methoxyphenyl)]benzo[b]thiophene) is a selective estrogen receptor modulator (SERM) that is a potent estrogen antagonist in mammary and uterine tissue while acting as an estrogen agonist to maintain bone density and lower serum cholesterol. Arzoxifene is a highly effective agent for prevention of mammary cancer induced in the rat by the carcinogen nitrosomethylurea and is significantly more potent than raloxifene in this regard. Arzoxifene is devoid of the uterotrophic effects of tamoxifen, suggesting that, in contrast to tamoxifen, it is unlikely that the clinical use of arzoxifene will increase the risk of developing endometrial carcinoma.

Reversal of multidrug resistance by the P-glycoprotein modulator, LY335979, from the bench to the clinic.

Multidrug resistance may be conferred by P-glycoprotein (Pgp, ABCB1) or the multidrug resistance associated protein (MRP). These membrane proteins are members of the ATP binding cassette transporter superfamily and are responsible for the removal from the cell of several anticancer agents including doxorubicin. Modulators can inhibit these transporters. LY335979 is among the most potent modulators of Pgp with a Ki of 59 nM. LY335979 is selective for Pgp, and does not modulate MRP-mediated resistance by MRP1 (ABCC1) and MRP2 (ABCC2). LY335979 significantly enhanced the survival of mice implanted with Pgp-expressing murine leukemia (P388/ADR) when administered in combination with either daunorubicin, doxorubicin or etoposide. Coadministration of LY335979 with paclitaxel compared to paclitaxel alone significantly reduced the tumor mass of the Pgp-expressing UCLA-P3.003VLB lung carcinoma in a xenograph model and delayed the development of tumors in mice implanted with the parental drug-sensitive UCLA-P3 tumor. LY335979 was without significant effect on the pharmacokinetics of these anticancer agents. This may be due impart to its poor inhibition of four major cytochrome P450 isozymes important in metabolizing doxorubicin and other oncolytics. The selectivity and potency of this modulator allows the clinical evaluation of the role of Pgp in multidrug resistance. LY335979 is currently in clinical trials.

Reversal of resistance in multidrug resistance protein (MRP1)-overexpressing cells by LY329146.

The benzothiophene LY329146 reverses the drug resistance phenotype in multidrug resistance protein (MRP1)-overexpressing cells when dosed in combination with MRP1-associated oncolytics doxorubicin and vincristine. Additionally, LY329146 inhibited MRP1-mediated uptake of the MRP1 substrate LTC4 into membrane vesicles prepared from MRP1-overexpressing cells.

Selectivity of the multidrug resistance modulator, LY335979, for P-glycoprotein and effect on cytochrome P-450 activities.

Overexpression of ATP-dependent drug efflux pumps, P-glycoprotein (Pgp) or multidrug resistance-associated protein (MRP), confers multidrug resistance to tumor cells. Modulators of multidrug resistance block the action of these pumps, thereby sensitizing cells to oncolytics. A potent Pgp modulator is LY335979, which fully sensitizes Pgp-expressing cells at 0.1 microM in cytotoxicity assays and for which Pgp has an affinity of 59 nM. The present study examines its effect on MRP1-mediated drug resistance and cytochrome P-450 (CYP) activity and its ability to serve as a Pgp substrate. Drug resistance was examined with HL60/ADR and MRP1-transfected HeLa-T5 cells. Drug cytotoxicity was unaffected by 1 microM LY335979; leukotriene C4 uptake into HeLa-T5 membrane vesicles was unaffected. Because the substrate specificity of Pgp and CYP3A overlap, the effect of LY335979 on the 1'-hydroxylation of midazolam by CYP3A in human liver microsomes was examined. The apparent K(i) was 3.8 microM, approximately 60-fold higher than the affinity of Pgp for LY335979. The modulator's effect on Pgp was evaluated with Pgp-overexpressing CEM/vinblastine (VLB)(100) and parental CCRF-CEM cells. Both cell lines accumulated [(3)H]LY335979 equally well and did not efflux [(3)H]LY335979 during a 3-h incubation, indicating that it is not a substrate of Pgp. Equilibrium-binding studies with CEM/VLB(100) plasma membranes and [(3)H]LY335979 showed that Pgp had a K(d) of 73 nM, which is in good agreement with the previously determined K(i) value. Thus, LY335979 is an extremely potent Pgp, and not MRP1 or MRP2, modulator and has a significantly lower affinity for CYP3A than for Pgp.

Pharmacological characterization of LY335979: a potent cyclopropyldibenzosuberane modulator of P-glycoprotein.

The above data indicate that LY335979 displays the following characteristics of an 'ideal modulator' of Pgp-mediated multidrug resistance: high affinity binding to Pgp, high potency for in vitro reversal of drug resistance, high therapeutic index (activity was demonstrated at doses ranging from 1-30 mg/kg) observed in in vivo antitumor efficacy experiments, and a lack of pharmacokinetic interactions that alter the plasma concentration of coadministered oncolytic agents. These desirable features strongly suggest that LY335979 is an exciting new clinical agent to test the hypothesis that inhibition of P-glycoprotein activity will result in reversal of multidrug resistance in human tumors.

Reversal of P-glycoprotein-mediated multidrug resistance by a potent cyclopropyldibenzosuberane modulator, LY335979.

Overexpression of P-glycoprotein (Pgp) by tumors results in multidrug resistance (MDR) to structurally unrelated oncolytics. MDR cells may be sensitized to these oncolytics when treated with a Pgp modulator. The present study evaluates LY335979 as a modulator both in vitro and in vivo. LY335979 (0.1 microM) fully restored sensitivity to vinblastine, doxorubicin (Dox), etoposide, and Taxol in CEM/VLB100 cells. LY335979 modulated Dox cytotoxicity even when LY335979 (0.5 microM) was removed 24 h prior to the cytotoxicity assay. LY335979 blocked [3H]azidopine photoaffinity labeling of the M(r) approximately 170,000 Pgp in CEM/VLB100 plasma membranes and competitively inhibited equilibrium binding of [3H]vinblastine to Pgp (Ki of approximately 0.06 microM). Treatment of mice bearing P388/ADR murine leukemia cells with LY335979 in combination with Dox or etoposide gave a significant increase in life span with no apparent alteration of pharmacokinetics. LY335979 also enhanced the antitumor activity of Taxol in a MDR human non-small cell lung carcinoma nude mouse xenograft model. Thus, LY335979 is an extremely potent, efficacious modulator that apparently lacks pharmacokinetic interactions with coadministered anticancer drugs and is, therefore, an exciting new agent for clinical evaluation for reversal of Pgp-associated MDR.

Novel acid labile COL1 trityl-linked difluoronucleoside immunoconjugates: synthesis, characterization, and biological activity.

LY207702 (1) is a difluorinated purine nucleoside that exhibits impressive antitumor activity in preclinical models. This agent, however, also possesses cardiotoxicity which limits the potential clinical utility of this novel drug candidate. We therefore developed linker chemistry whereby regioselective N6-tritylation of LY207702 (1) allowed this drug to be coupled to epsilon-lysine amino groups of mAb's reactive with human tumor-associated antigens. The resulting immunoconjugates 3 possessed conjugation ratios ranging from 5 to 7 mol of LY207702/mol of mAb, minimal aggregate content (5-10%), and good immunoreactivity. The electronic nature of substituents on the aromatic rings of the trityl group dictated the degree of acid lability of the trityl linker. Increased electronic stabilization of the transient trityl carbocation led to increase in the release rate of free drug, i.e., m-DMT 10a = p-DMT 10b > p-MMT 10d > p-T 10f. Consequently, the more acid labile DMT conjugates 3a and 3b proved to be the most potent cytotoxic agents, and the most stable p-T conjugate 3f exhibited the least antitumor activity when evaluated in vitro and in vivo. p-MeT-linked conjugate 3e, the most stable construct that retained excellent in vivo antitumor activity, was selected for more extensive evaluation. No detectable free drug or metabolite was observed in mouse plasma at a single intravenous dose of p-MeT conjugate 3e, which was consistent with its predicted stability under physiological conditions. This construct did, however, exhibit significant antigen-mediated antitumor activity in vivo. No cardiotoxicity was detected in mice dosed with conjugate 3e (6 mg/kg free drug content per day for 21 days) equivalent to approximately 8 times the total dose required for complete regression of well-established (approximately 1 g) HC1 human colon tumor xenografts in nude mice. Cardiotoxicity was induced in 20% of free drug 1 treated group at the equivalent dose. Cardiomyopathy was, however, observed when the dose of conjugate 3e was increased to 8 mg/kg per day for 21 days. These data suggest that antitumor activity of LY207702 (1) was maintained and its cardiotoxic potential reduced when this agent was administered to human tumor xenograft bearing nude mice as COL1-N6-p-MeT-207702 conjugate 3e.

Dextran modification of a Fab'--beta-lactamase conjugate modulated by variable pretreatment of Fab' with amine-blocking reagents.

The physical and pharmacological properties of proteins can be altered by chemical modification with polymers. Preliminary studies showed that attachment of oxidized dextran to the bacterial protein, beta-lactamase (beta L) effectively reduced in vivo immunogenicity in mice with no loss of enzymatic activity. This report describes a general method for differentially dextran modifying the Fab' component of a Fab'--beta-lactamase conjugate by the use of amine-blocking reagents. Methyl acetimidate (MeAcm) and the N-succinimidyl derivative of (methylsulfonyl)ethyl carbonate (NHS-Msc), reagents which can reversibly block primary amines, were used in model studies to modulate the level of available reactive amines on the F(ab')2 fragments of both the anti-carcinoembryonic antigen antibody, ZCE025, and the antitumor-associated glycoprotein-72 antibody, CC49. MeAcm had little or no effect on immunoreactivity and was maximally effective in modulating dextran attachment, while NHS-Msc was much less effective. A comparison of NHS-Msc and MeAcm is described. Treatment of F(ab')2 with 5-300 mM MeAcm prior to dextran treatment showed a proportional decline in the level of dextran attachment as well as intramolecular cross-linking of the protein by the dextran polymers (6 kDa or 33-mer). A conjugate of beta L coupled to MeAcm-treated ZCE025 Fab' [reduced F(ab')2] was constructed under standard conditions using sulfosuccinimidyl N-[(4-carboxycyclohexyl)methyl]maleimide. After dextran modification, this conjugate maintained good immunoreactivity and enzymatic activity. Biodistribution studies in tumor-bearing nude mice of dextranated and nondextranated conjugate showed comparable overall distribution profiles except that the clearance of the dextranated conjugate from both blood and tumor was delayed about 48-72 h.

Site-specific prodrug activation by antibody-beta-lactamase conjugates: preclinical investigation of the efficacy and toxicity of doxorubicin delivered by antibody directed catalysis.

Antibody directed catalysis (ADC), the catalytic conversion of prodrugs to drugs by enzymes localized at disease targets by appropriate monoclonal antibodies, has shown promise in the treatment of cancer in nude mouse xenograft models. We investigated this concept using antibody enzyme conjugates constructed from beta-lactamase and Fab's reactive with carcinoembryonic antigen, CEA, and tumor associated glycoprotein, TAG-72, to convert prodrugs that are cephalosporin sulfoxide derivatives into oncolytic drugs. Previous work focused on ADC delivery of the potent vinca alkaloid derivative desacetylvinblastine carboxhydrazide (DAVLBHYD). In the current study the ability of the system to deliver doxorubicin was tested in MCF7 breast carcinoma xenografts and OVCAR3 ovarian carcinoma xenografts, and in T380 and LS174T colon tumor xenografts for comparison with previous DAVLBHYD results. ADC enhanced the delivery of doxorubicin in the model systems investigated. Tumor growth suppression was equivalent to or greater than that observed with free doxorubicin at its maximum tolerated dose (MTD). In contrast to the DAVLBHYD results, ADC delivery of doxorubicin did not regress tumors, but did result in a substantial increase in the MTD.

High yield, site-specific coupling of N-terminally modified beta-lactamase to a proteolytically derived single-sulfhydryl murine Fab'.

The preparation of bispecific protein conjugates capable of performing diverse biological functions is an area of active investigation. Such conjugates are routinely prepared using techniques which employ random derivatization of lysine residues, but the overall utility of these methods is limited due to poor yields and heterogeneous conjugates. In this report we describe the development of site-specific linkage methodology for the chemical synthesis of a homogeneous enzyme-antibody Fab' conjugate with coupling efficiencies of at least 72%. The N-terminal threonine residue of beta-lactamase from the P99 strain of Enterobacter cloacae was oxidized to an aldehyde functional group under mild conditions with a 5-fold molar excess of sodium periodate. The murine Fab' with a single sulfhydryl at the hinge region was generated by further digestion of the peptic Fab' fragment with lysyl endopeptidase to remove a decapeptide containing two of the three cysteine residues. Coupling of the two modified proteins was accomplished through a bifunctional coupling reagent containing meleimide and aminooxy functional groups. Synthesis of the linker is described. Yields of 1:1 enzyme-Fab' were at least three times higher than for comparable random derivatization methods. Immunoreactivity and enzymatic activity were unaffected. Biodistribution studies showed a more favorable tumor to blood ratio with the site-specifically linked conjugate.

In vivo expression of P-glycoprotein in a human colon carcinoma xenograft is modulated by therapy with free and monoclonal antibody-conjugated vinca alkaloids.

Well established UCLA-P3 human lung tumor xenografts were significantly regressed by treatment with a monoclonal antibody-vinca immunoconjugate whereas no regressions were observed for the LS174T and SW948 human colon carcinoma xenografts by this therapy. Antibody and complementary DNA probes utilized for detection of the MDR1 gene product and mRNA levels, respectively, revealed that prior to drug treatment the lung tumor had virtually no detectable P-glycoprotein while both colon carcinomas displayed low and heterogeneous expression of this resistance-related protein. It was subsequently determined, however, that the low level of P-glyocoprotein expression observed for one of the colon tumors could be rapidly modulated following therapy with free or MoAb-conjugated vinca. These data indicated that elevated P-glycoprotein levels resulting from drug therapy may play a role in the lack of regression of the human colon xenografts. Most significantly, our results also indicated that the time interval between drug treatment and tissue sampling may be a critical factor to be considered in studies which attempt to correlate P-glycoprotein expression with chemotherapy.

Site-specific prodrug activation by antibody-beta-lactamase conjugates: regression and long-term growth inhibition of human colon carcinoma xenograft models.

Antibody-directed catalysis (ADC) is a two-step method for the delivery of chemotherapeutic agents in which enzyme-antibody conjugate, prelocalized to antigen-bearing tumor cells, catalyzes the site-specific conversion of prodrug to drug. An ADC system consisting of F(ab')-beta-lactamase conjugates and a cephalosporin derivative of the oncolytic agent 4-desacetylvinblastine-3-carboxhydrazide was investigated. The ability of the system to mediate antitumor activity was compared with that of free drug given alone and with covalent drug-antibody conjugates in LS174T and T380 colon carcinoma xenografts in nude mice. Efficacy increased from moderate tumor growth inhibition by using free 4-desacetylvinblastine-3-carboxhydrazide to tumor regression and long-term stabilization with the ADC system. Labile covalent drug-antibody conjugates prepared from the same antibodies were less effective than ADC and required much higher antibody doses. The antigens KS1/4, carcinoembryonic antigen, and tumor-associated glycoprotein-72, TAG-72, present on the model cell lines, were chosen to investigate the effect of differences in subcellular location and expression heterogeneity on the efficacy of ADC delivery. Response was equivalent with the three tumor antigens. Hence, heterogeneous expression and membrane shedding of carcinoembryonic antigen and TAG-72, did not diminish the suitability of these antigens as targets for ADC therapy. In contrast, drug-antibody conjugate efficacy was more sensitive to subcellular location and heterogeneity. Thus, ADC is a highly effective form of immunochemotherapy in preclinical models, with applicability toward a variety of antigen targets.

In vivo antitumor activity of a panel of four monoclonal antibody-vinca alkaloid immunoconjugates which bind to three distinct epitopes of carcinoembryonic antigen.

A panel of four murine monoclonal antibodies apparently directed against three distinct epitopes of carcinoembryonic antigen (CEA) was conjugated via oxidized carbohydrate groups to 4-desacetylvinblastine-3-carboxyhydrazide. The resulting antibody-vinca conjugates were evaluated for antitumor activity against 2-9-day-established LS174T human colorectal carcinoma xenografts. The antibodies (immunoglobulin G, IgG) employed in this study were 11.285.14 (IgG1), 14.95.55 (IgG2a), CEM231 (IgG1), ZCE025 (IgG1). Additive immunofluorescence studies indicated that CEM231 and ZCE025 recognized the same or a closely related epitope(s) on CEA which was distinct from the two epitopes bound by 11.285.14 and 14.95.55. The in vivo antitumor efficacy studies demonstrated that chemoimmunoconjugates prepared from 14.95.55 and ZCE025 were more active than the conjugates constructed from the 11.285.14 and CEM231 antibodies. The 14.95.55 and ZCE025 immunoconjugates were also more efficacious than free drug or drug conjugated to irrelevant murine IgG. The presence of increased carbohydrate content on the light chain of ZCE025 may have been responsible for the ability to construct ZCE025-vinca conjugates with about twice the drug content (approximately 10 mol of vinca/mol of IgG) than was achieved with the other antibodies. The highly conjugated form of ZCE025 demonstrated similar efficacy but was much less toxic than a ZCE025 conjugate containing 5 mol of vinca/mol of IgG. These data indicated that significant differences existed in the ability of monoclonal antibodies to target a cytotoxic agent for effective antitumor activity even when the immunoconjugates recognized the same antigen or even the same or closely related antigen epitope(s). Furthermore, these differences could not have been identified without extensive in vivo evaluation for antitumor efficacy.

Preparation and characterization of a beta-lactamase-Fab' conjugate for the site-specific activation of oncolytic agents.

Antibody-directed catalysis (ADC) is a two-step method for the targeted delivery of chemotherapeutic agents in which enzyme-antibody conjugates, prelocalized to antigen-bearing cells, activate prodrugs designed to be substrates for the enzyme. An enzyme-Fab' conjugate exhibiting both native beta-lactamase activity and immunoreactivity toward carcinoembryonic antigen (CEA) was constructed. Treatment of CEA-expressing LS174T cells with this conjugate imparted beta-lactamase activity to the cells; beta-lactamase activity was not imparted by treatment with unconjugated beta-lactamase and not to CEA negative cells treated with conjugate. Cephalosporin-based prodrugs, and other substrates synthesized as model compounds, were found to have wide variations in their kinetic parameters toward the conjugate, with kcat values ranging from 16 to 3300 s-1 and KM values ranging from 5 to 160 microM. The prodrug derived from desacetylvinblastine-3-carboxylic acid hydrazide (DAVLBHYD) was studied in vitro and found to be 5-fold less cytotoxic to LS174T cells than the parent DAVLBHYD. For antigen-positive cells preincubated with conjugate, however, the prodrug showed the same potency as the parent drug. Thus, the combination of conjugate and prodrug appears to provide antigen-dependent toxicity to tumor cells.

In vivo antitumor activity of a monoclonal antibody-Vinca alkaloid immunoconjugate directed against a solid tumor membrane antigen characterized by heterogeneous expression and noninternalization of antibody-antigen complexes.

It is widely believed that antigen heterogeneity and noninternalization of antigen-antibody complexes will severely limit the antitumor activity of monoclonal antibody-drug conjugates. The B72.3 monoclonal antibody binds to a tumor-associated antigen which is heterogeneously expressed in human carcinomas (J. Schlom, Cancer Res., 46: 3225-3238, 1986). We therefore performed studies to assess the degree of internalization of B72.3 antibody-antigen complexes and the level of in vivo antitumor activity that could be achieved with B72.3 conjugated to 4-desacetyl vinblastine-3-carboxhydrazide. Internalization studies were performed on LS174T colorectal carcinoma and OVCAR-3 ovarian carcinoma cells using iodinated B72.3 as well as an iodinated antibody that binds to the human transferrin receptor, IIB21. These data indicated that, in contrast to HB-21, the B72.3 antigen-antibody complex was not internalized. The B72.3-Vinca alkaloid immunoconjugate demonstrated significant antitumor activity against LS174T xenografts, although complete regressions of established tumors were not achieved. Immunohistochemical analyses indicated that the B72.3 antigen was heterogeneously expressed in the LS174T xenografts and that tumor cells which were not killed by high doses of B72.3-Vinca also expressed the B72.3 antigen. These studies indicated that significant antitumor activity may be achieved by monoclonal antibody-drug conjugates even when antigen heterogeneity and noninternalization of antigen-antibody complexes are encountered. The data also suggested that the formulation of antibody-drug conjugate cocktails to counteract antigen heterogeneity may not be sufficient to eradicate all malignant cells within a solid tumor mass.

In vivo selection of human tumor cells resistant to monoclonal antibody-Vinca alkaloid immunoconjugates.

UCLA-P3 human lung adenocarcinoma cells were grown in nude mice and given repetitive treatments of a monoclonal antibody-Vinca alkaloid immunoconjugate. Although this therapy resulted in a greater than 4-fold reduction in mean tumor mass of the established tumors, some animals experienced a reinitiation of tumor growth after cessation of conjugate treatment. Two such animals were treated again with high doses of monoclonal antibody-Vinca but one of the tumors was no longer regressed by the drug conjugate. The tumor was excised, enzymatically dissociated, and grown in tissue culture. Cultured cells were reimplanted in nude mice and subjected to further therapy with a monoclonal antibody-Vinca conjugate. The resulting tumors were also refractory to the immunoconjugate therapy. This cycle was repeated for a total of three times and resulted in the serial in vivo selection of three conjugate resistant variants. The mechanism responsible for the in vivo resistance of human tumor cells to the monoclonal antibody-Vinca immunoconjugate is unknown but does not appear to involve antigen modulation, altered tumor cell growth rate, or an apparent decrease in tumor targeting in vivo. The resistance was also not accompanied by any detectable elevation in multidrug resistance 1 mRNA or P-glycoprotein expression. Significantly, the resistance pattern was observed only in vivo and was not maintained by cells grown in vitro.

Selective cloning of hybridoma cells for enhanced immunoglobulin production using flow cytometric cell sorting and automated laser nephelometry.

Techniques for selective cloning of murine hybridoma cells by flow cytometric cell sorting and use of automated laser nephelometry to determine the resultant clones' immunoglobulin secretion levels are described. Using a commercially available attachment to a fluorescence-activated cell sorter, individual hybridoma cells were successfully distributed into microtiter wells in an automated manner based on their forward angle light scatter properties and their reaction to fluorescein-conjugated anti-mouse-IgG. The techniques were used to estimate successfully the frequency of immunoglobulin-secreting cells in established cultures. In addition, heterogeneity of cell surface immunoglobulin expression was observed and utilized as a criterion for flow sorting of new hybridoma variants. In these studies, clones derived from high (anti-IgG) intensity sorting regions yielded cultures with enhanced immunoglobulin secretion levels, as determined by automated laser nephelometry. Furthermore, the surface immunoglobulin phenotype of the derived clones was conserved in subsequent progeny. Finally, it was established that inclusion of propidium iodide in the hybridoma cell sorting mixtures improved cloning efficiency by facilitating enhanced discrimination and elimination of nonviable cells. Our results indicate that flow cytometric-assisted single cell deposition provides positive attributes of several traditional hybridoma cloning techniques and, in addition, furnishes a tool for steering the cloning process toward selection of enhanced immunoglobulin producing cultures.

New antitumor monoclonal antibody-vinca conjugates LY203725 and related compounds: design, preparation, and representative in vivo activity.

A method has been developed to allow the direct coupling of the cytotoxic vinca alkaloid 4-desacetylvinblastine-3-carbohydrazide (DAVLB hydrazide) to a variety of murine monoclonal antibodies directed against human solid tumors. Periodate oxidation of carbohydrate residues on the antibodies, followed by reaction with DAVLB hydrazide in aqueous acid affords, in most cases, conjugates with conjugation ratios of 4-6 vincas per antibody in high yield without significantly impairing antigen binding or solubility. The outcome of the conjugation reaction is highly dependent on the concentration of, and time of exposure of the protein to, the oxidant. These conjugates exhibit potent antitumor activity in vivo against a number of human solid tumor-nude mouse xenografts, with efficacy and safety increased over unconjugated DAVLB hydrazide. This antitumor activity is also superior to that of similarly prepared but nontarget tumor binding antibody-DAVLB hydrazide conjugates. MoAb-DAVLB hydrazide conjugates release DAVLB hydrazide in solution in a temperature- and pH-dependent manner. Hydrolytic release of unmodified DAVLB hydrazide from tumor-localized MoAb-DAVLB hydrazide conjugates in vivo may be an important factor in their antitumor activity.

In vivo efficacy of monoclonal antibody-drug conjugates of three different subisotypes which bind the human tumor-associated antigen defined by the KS1/4 monoclonal antibody.

A panel of three hybridomas has been isolated each of which secretes a single species of monoclonal antibody (MoAb) directed against the KS1/4 tumor-associated antigen originally described by Varki et al. (Cancer Res 44: 681, 1984). These MoAbs were designated L1-(IgG2b), L2-(IgG1), and L4-(IgG2a)KS. Binding specificity, immuno-precipitation, and competitive binding analyses indicated that these MoAbs each recognize the same epitope of the KS1/4 antigen. The immunoprecipitation studies indicated that the MoAbs recognized a major antigenic component of 42 kDa and a minor component of 35 kDa. The L-KS antibodies were evaluated as MoAb-drug conjugates against a variety of human tumor targets grown in vivo as nude mouse xenografts. The MoAb-drug conjugates were constructed using protein-A-purified MoAbs conjugated to 4-desacetyl-vinblastine-3-carbohydrazide. Efficacy was determined using various dosing protocols on 2-14 day established tumors of lung, pharynx, colon, and skin origin. Control experiments included the use of dual-flank antigen-positive and negative tumors, free MoAbs, free drug, and mixtures of MoAbs and drug. These studies indicated that significant tumor growth suppression and actual tumor regression could be achieved by the MoAb-vinca conjugates and that this activity was antigen-mediated. The drug conjugates were more efficacious than free drug or free MoAbs administered either singly or in combination with each other.

Tissue distribution and cellular location of the antigens recognized by human monoclonal antibodies 16.88 and 28A32.

Results from a previous study (M. V. Haspel et al., Cancer Res., 45: 3951-3961, 1985) indicated that it was possible to isolate a high proportion of human monoclonal antibodies reactive with cell surface, tumor-associated antigens when the hybridomas were obtained from fusions utilizing peripheral blood lymphocytes from patients immunized with autologous tumor cells. The assignment of membrane reactivity was made from immunoperoxidase studies which used air-dried, nonpermeabilized Cytospin preparations of colon tumor cells. Tumor specificity was assessed by immunohistological assays by using frozen sections of normal and malignant human tissues. We now describe a series of studies using two of these antibodies, 16.88 and 28A32, in which further information was obtained concerning the tumor specificity and cellular location of the target antigens reactive with these monoclonal antibodies. Data were acquired from a variety of experimental techniques which included quantitative and qualitative immunofluorescence on live and permeabilized cells, RBC-rosetting assays, immunoperoxidase studies on Cytospin and frozen tissue sections, and immunoblot procedures. These studies show that the 16.88 and 28A32 human monoclonal antibodies bind to antigens which (a) are located in the cell cytoplasm and are not expressed in detectable levels on the cell surface, and (b) are found in many normal and malignant cell types.