Targeting of anti-Thy 1.1 monoclonal antibody conjugated liposomes in Thy 1.1 mice after intravenous administration

125I-labeled liposomes, conjugated to an anti-Thy 1.1 monoclonal antibody (MRCOX7), demonstrated up to 7.4-fold greater lymph node uptake than liposomes conjugated to non-specific monoclonal antibody (R-10) after intravenous injection into Thy 1.1 (AKR-J) mice. Uptake of anti-Thy 1.1-conjugated liposomes by the lymph nodes of AKR-J mice was 3-times greater than their uptake by lymph nodes of Thy 1.2 (AKR-Cu) mice. Lymph node localization of anti-Thy 1.1-liposomes was equal to that of control monoclonal antibody-liposomes in Thy 1.2 mice. Conjugation to either monoclonal antibody substantially increased liposome clearance by the liver, while decreasing liposome uptake in a number of organs outside the reticuloendothelial system. Changes in liposome size and phospholipid composition did not significantly alter these results. Administration of a large predose of unconjugated liposomes prior to injection of MRCOX7-conjugated liposomes increased blood levels and reduced liver uptake of the monoclonal antibody-liposome conjugates, but did not further enhance lymph node uptake. This study demonstrates that targeting of liposomes by conjugation to the appropriate monoclonal antibody, can significantly increase their uptake in lymph nodes which contain high levels of cells expressing the target antigen. However, conjugation to monoclonal antibody also increases clearance of liposomes by the liver. To increase the uptake of monoclonal antibody-conjugated liposomes in target tissue, substantial reduction of their clearance by the reticuloendothelial system will be required.     

Influence of endogenous Thy1.1 cells upon the efficacy of an anti-Thy1.1 antibody-diphtheria toxin conjugate.

The chemical conjugation of antibodies to protein toxins results in cell-specific cytotoxic agents that can be defined in terms of in vitro potency and efficacy; however, it is the in vivo utilities that are largely being pursued in clinical trials. The nature of in vivo target cell depletion by toxin conjugates is largely unknown. The anti-murine Thy1.1 antibody-diphtheria toxin conjugate possesses high in vitro efficacy, and because mice are remarkably resistant to the native toxin, the conjugate possesses in vivo efficacy. When administered intravenously, the conjugate is shown to deplete peripheral blood Thy1.1+ target cells in a concentration-dependent fashion. When the log kill of Thy1.1+ tumor cells was analyzed by the life span extension method, it was determined, however, that the log kill is inversely proportional to the number of target cells. That is, the presence of an endogenous cell population, which is expressing the same surface antigen targeted by the antibody conjugate as on the pathological cell, may drastically lower the clinical efficacy of the immunotoxin. Thus, the greatest potential for antibody-toxin conjugates will be for low target cell burdens and for pathogenic cell populations expressing unique surface antigens. These are important considerations in the design of bioconjugates to insure high in vivo efficacy in elimination of intended target cells.     

Targeting of antibody conjugated,phosphatidylserine-containing liposomes to vascular cell adhesion molecule 1 for controlled thrombogenesis

Phosphatidylserine (PS) membrane exposure plays an important role in blood coagulation, and the development of a liposome formulation containing PS may be of potential therapeutic utility if they can be designed to achieve tumor selective thrombosis. The objective of this study was to develop proof-of-principle data for a thrombogenic PS liposome targeted to vascular cell adhesion molecule 1 (VCAM-1) via the attachment of an anti-VCAM-1 monoclonal antibody (Ab). We have evaluated binding of the anti-VCAM-1 Ab-conjugated PS liposomes to VCAM-1 using two in vitro models, as well as assessing the ability of these liposomes to catalyze blood coagulation reactions. Binding of the Ab-conjugated PS liposomes containing 2 or 14 mol% 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[poly(ethylene glycol) 2000] (DSPE-PEG(2000)) to interleukin 1alpha stimulated human umbilical vein endothelial cells was 8- and 16-fold higher than those without conjugated Ab, respectively, based on the percentage relative increase in cell associated lipid for these liposomes. Binding to VCAM-1-coated ELISA plates produced similar results. The VCAM-1-bound Ab-conjugated PS liposomes were capable of catalyzing blood coagulation reactions upon the exposure of the thrombogenic PS membrane surface. This control of PS surface exposure was achieved using exchangeable PEG-derivatized phosphatidylethanolamines (PE-PEG), with 97% of clotting activity recovered after PE-PEG exchanged out. Our results demonstrate the potential for considering further development of procoagulant liposomes that selectively target thrombogenesis in tumor vasculature.     

Targeting of monoclonal antibody-coated liposomes to sheep red blood cells

A monoclonal mouse anti-sheep red blood cell specific antibody IgG_2b was esterified with palmitic acid which served as a hydrophobic anchor for successfull incorporation into the liposomal membrane. The formation of coated liposomes by dialyzing the mixed antibody/lipid/detergent micelles against phosphate buffer was simplified b by using the same detergent as for the antibody derivatization. No purification step of any intermediate product was necessary. Targeting of the resulting vesicles to sheep red blood cells occured with high efficiency compared with control liposomes. The uptake was fast and specific as demonstrated with sheep and horse red blood cells by the use of radioactively labelled liposomes and by scanning electron microscopy.     

Optical imaging of disseminated leukemia models in mice with near-infrared probe conjugated to a monoclonal antibody

Background

The assessment of anticancer agents to treat leukemia needs to have animal models closer to the human pathology such as implantation in immunodeficient mice of leukemic cells from patient samples. A sensitive and early detection of tumor cells in these orthotopic models is a prerequisite for monitoring engraftment of leukemic cells and their dissemination in mice. Therefore, we developed a fluorescent antibody based strategy to detect leukemic foci in mice bearing patient-derived leukemic cells using fluorescence reflectance imaging (FRI) to determine when to start treatments with novel antitumor agents.

Methods

Two mAbs against the CD44 human myeloid marker or the CD45 human leukocyte marker were labeled with Alexa Fluor 750 and administered to leukemia-bearing mice after having verified the immunoreactivity in vitro. Bioluminescent leukemic cells (HL60-Luc) were used to compare the colocalization of the fluorescent mAb with these cells. The impact of the labeled antibodies on disease progression was further determined. Finally, the fluorescent hCD45 mAb was tested in mice engrafted with human leukemic cells.

Results

The probe labeling did not modify the immunoreactivity of the mAbs. There was a satisfactory correlation between bioluminescence imaging (BLI) and FRI and low doses of mAb were sufficient to detect leukemic foci. However, anti-hCD44 mAb had a strong impact on the tumor proliferation contrary to anti-hCD45 mAb. The use of anti-hCD45 mAb allowed the detection of leukemic patient cells engrafted onto NOD/SCID mice.

Conclusions

A mAb labeled with a near infrared fluorochrome is useful to detect leukemic foci in disseminated models provided that its potential impact on tumor proliferation has been thoroughly documented.     

Selective gene therapy for prostate cancer cells using liposomes conjugated with IgM type monoclonal antibody against prostate-specific membrane antigen

Prostate cancer cells express prostate-specific membrane antigen (PSMA). We developed an IgM type monoclonal antibody against PSMA. The antibody was coupled to poly-L-lysine and thereafter this conjugate was mixed with cationic liposomes containing plasmid DNA. The antibody-liposome complex was tested whether it could deliver the gene of interest selectively to the PSMA positive cells. As assessed by beta-galactosidase reporter gene, the transfection efficiency was 13.2% with anti-PSMA-liposome complex as compared to 4% with control IgM liposome complex. In contrast, no such differences were observed in PSMA negative PC-3, DU145 and T24 cells. Furthermore, in the suicide gene therapy in vitro with thymidine kinase gene plus ganciclovir system, anti-PSMA liposome complex demonstrated a selective growth inhibitory effect on PSMA positive LNCaP cells but not on PSMA negative cell lines.     

Acute toxicity of long-circulating and pH-sensitive liposomes containing cisplatin in mice after intraperitoneal administration

AimsThe objective of this work was to evaluate the acute toxicity of long-circulating and pH-sensitive liposomes containing cisplatin (SpHL-CDDP), after their intraperitoneal administration in male and female mice.Main methodsAfter single administration of free CDDP (5,10,and 20 mg/kg) or SpHL-CDDP (7,12,30,45 and 80 mg/kg), the body weight was recorded and the LD₅₀ was calculated. Blood samples were collected for biochemical and hematological analysis. Kidneys, liver, spleen and bone marrow were removed to histopathological examination.Key findingsMice treated with high doses of free CDDP showed a greater loss of body weight and more delayed recovery time than those treated with SpHL-CDDP. The LD₅₀ values for SpHL-CDDP treatment for male and female mice groups were 2.7 and 3.2 fold higher, respectively, than that obtained for free CDDP. The red and white blood cells counts and quantification of hemoglobin and hematocrit presented no change upon administration of SpHL-CDDP treatment. Free CDDP treatment, however, did lead to an appearance of mild anemia and a reduction in total white blood cell counts. As regards nephrotoxicity, it was observed that free CDDP treatment caused pronounced alterations in the blood urea and creatinine levels of mice. In contrast, these parameters were slightly altered only after SpHL-CDDP treatment at a dose of 30 mg/kg. Microscopic analysis of kidneys from mice treated with SpHL-CDDP showed no morphological alteration. Concerning hepatotoxicity, no histopathological alteration was observed after both treatments.SignificanceThese findings reveal that SpHL-CDDP can eliminate CDDP-induced toxicity and is thus a promising candidate for intraperitoneal chemotherapy.     

The use of monoclonal anti-Thy1 IgG1 for the targeting of liposomes to AKR-A cells in vitro and in vivo

A number of SH-containing proteins or protein derivatives were coupled to small unilamellar liposomes. These were composed of distearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidylethanolamine (DPPE) and cholesterol (1:1, phospholipid/cholesterol molar ratio) and activated (DPPE moiety) with the heterobifunctional reagents N-hydroxysuccinimide ester of iodoacetic acid (hydroxysuccinimide iodoacetate), N-succinimidyl-4-(2-bromoacetylamino)benzoate (SBAB) or N-succinimidyl-3-(2-pyridyldithio)proprionate (SPDP). DPPE was activated with the reagents before or after its incorporation into liposomes. Protein coupling values varied widely depending on the reagent and the protein used, but were highest in the case of SPDP-activated liposomes and SPDP-modified immunoglobulin G (IgG). Monoclonal anti-Thy1 125I-IgG1-bearing liposomes (SPDP- or SBAB-activated) containing quenched carboxyfluorescein were incubated under a variety of conditions with mouse AKR-A cells expressing the cross-reactive Thy 1.1 antigen. The following observations were made; (a) binding of intact liposomes to the cells at 4 degrees C reached plateau values after about 1 h with at least 70% of the liposomes used being capable of associating with the target cells; (b) binding of liposomes to AKR-A cells was much more pronounced than when using another cell line (EL4-Tc); (c) binding to AKR-A cells could be effected with as little as 1.3 molecules (average) of IgG1 per vesicle; (d) binding was inhibited only modestly by the presence of 50% mouse plasma; (e) stability of IgG1-bearing liposomes in terms of entrapped solute and IgG1 retention in the presence of plasma at 37 degrees C was maintained quantitatively for at least 5.5 h, and by 24 h, 54% of the IgG1 was still associated with the liposomes. AKR mice were injected intravenously with 99mTc-labelled AKR-A cells and 2.5 min later with anti-Thy1 125I-IgG1-bearing liposomes containing quenched carboxyfluorescein and 111In-Ca-DTPA or with similar liposomes devoid of IgG1. In parallel experiments, AKR mice received either of the liposome preparations without previous injection of cells. On the basis of patterns of quenched carboxyfluorescein, 111In and 125I-clearance from the circulation, of 99mTc levels in the blood and of values of 111In in the liver and spleen, it appeared that IgG1-bearing liposomes were capable of binding to their target cells in the vasculature. Such binding accelerated the clearance of interacting moieties (i.e., AKR-A cells and liposomes). The present results suggest that targeting of liposomes to circulating in vivo is feasible.     

Target-selective cytotoxicity of methotrexate conjugated with monoclonal anti-MM46 antibody

Summary In studies on antitumor antibody-cytotoxic drug conjugates as potential tumor-selective cytotoxic agents, methotrexate (MTX) was conjugated via its active ester derivative with a murine monoclonal antibody (aMM46) to a mouse mammary tumor antigen (MM antigen) on syngeneic, ascitic C3H/He mouse mammary tumor MM46 cells. The conjugate retained full antibody activity, as assayed by complement-dependent cytolysis. The target-selective cytotoxicity of aMM46-MTX was verified by the observations that this conjugate showed greater cytotoxicity than the corresponding normal mouse immunoglobulin (nIg) conjugate to MM46 cells, neither aMM46 nor nIg being cytotoxic, and that it showed less cytotoxicity to MM antigen negative mouse mammary tumor MM48 cells than to MM46 cells, its cytotoxicity to MM48 cells being similar to that of the nIg conjugate. From the results of assays of cell binding and uptake of ¹³¹I-labeled aMM46 and aMM46-³H-MTX, aMM46 and aMM46-MTX were internalized after their binding to MM46 cell surface antigen. Leupeptin, an inhibitor of the lysosomal endopeptidase cathepsin, decreased the cytotoxicity of aMM46-MTX, supporting the involvement of lysosomal degradation of the conjugate in its action.     

Application of liposomes to generation of monoclonal antibody to glycosphingolipid: production of monoclonal antibody to GgOse4Cer

Liposomes were applied to the immunization with GgOse4Cer and screening for production of monoclonal antibody to GgOse4Cer. Four-week-old and 22-week-old Balb/c mice were immunized with GgOse4Cer and Salmonella minnesota R595 lipopolysaccharides incorporated liposomes which were composed of dipalmitoyl-phosphatidylcholine and cholesterol. Since antibody response to GgOse4Cer was higher in 22-week-old than 4-week-old Balb/c mice after immunization, 22-week-old Balb/c mice were used for the immunization prior to generation of the monoclonal antibodies to GgOse4Cer. The screening of monoclonal antibodies was performed by complement-dependent liposome immune lysis assay using GgOse4Cer-containing liposomes. Six kinds of monoclonal antibodies, AG-1, -2, -3, -4, -5, and -6, of the IgM class were established. The specificities of the monoclonal antibodies obtained were defined by complement-dependent liposome immune lysis assay using various glycosphingolipids incorporated in liposomes and by thin-layer chromatography (TLC) with immunostaining. All of the monoclonal antibodies reacted only with GgOse4Cer in the liposome immune lysis assay. In addition, the monoclonal antibodies reacted only with GgOse4Cer in the TLC immunostaining. However, none of the monoclonal antibodies obtained was capable of removing natural killer activity from C3H/He mice spleen cell suspensions in vitro. Liposomes may be useful in the procedures of immunization and screening for generation of antiserum and monoclonal antibody to GSLs.     

Suppression of overt diabetes in NOD mice by anti-thymocyte serum or anti-Thy 1, 2 antibody

Effects of anti-thymocyte serum (ATS) and anti-Thy 1, 2 monoclonal antibody on the spontaneously occurring diabetes in NOD mice were examined. Spontaneous diabetes in female mice was markedly suppressed by intravenous injection of rabbit anti-mouse thymocyte serum diluted to 1:4 on three consecutive days during the time period from 70 to 100 days after birth; the cumulative incidence of overt diabetes upto 195 days of age was greatly reduced and the onset of diabetes was delayed. Similar effect was observed with anti-Thy 1, 2 antibody treatment. These findings suggest that T lymphocytes play a role in the production of spontaneous diabetes in this mouse strain.     

In vivo influence of the anti-B220 monoclonal antibody administration of the T cell differentiation in mice

B220, a pan-B marker, is known to be also expressed on immature T cells of MRL/1pr or other congenic 1pr mice and minor population of immature thymocytes but not on peripheral T cells. In this study, we investigated in vivo the possibility whether B220 is one kind of premature T or prethymic T precursor cell marker as well as a pan-B cell marker. A monoclonal antibody against B220 glycoprotein was intravenously injected every 2 days into various strains of mice. After the third administration of this antibody, thymocytes decreased remarkably compared with those from the rat IgG-treated group, and spleen cells were also reduced significantly. Further, the number of cells expressing Thy-1, Ly-1, Lyt-2, and asialo GM1 (asGM1) in the spleen were significantly reduced. On the contrary, the number of cells expressing surface IgM (sIgM) or B220 were increased by this treatment, especially after the 8th treatment. Some T-dependent immunological functions including mitogenic responses to lectins and cytotoxic T cell activity were markedly suppressed but mixed lymphocyte reaction (MLR) and natural killer (NK) activity were rather augmented. Thus, B220 may be expressed on some kinds of T cell progenitor. Taken together, in vivo treatment with anti-B220 antibody may influence differentiating stages of some T cells from bone marrow progenitors before or just after their homing into the thymus.     

Survival of athymic (nu/nu) mice after Theiler''s murine encephalomyelitis virus infection by passive administration of neutralizing monoclonal antibody.

Little or no antiviral immune response is mounted in athymic nude mice infected with the Daniels strain of Theiler's murine encephalomyelitis virus. In these athymic mice, increasing levels of infectious virus could be detected in the central nervous system. Seventy-five percent (9 of 12) of the nude mice were moribund or dead by 4 weeks postinfection. In contrast, treatment of Theiler's virus-infected nude mice with a neutralizing monoclonal antibody (H7-2) against the viral protein VP-1 resulted in a dramatic reduction of infectious virus within the central nervous system. All antibody-treated nude animals survived beyond 4 weeks postinfection. Monoclonal antibody titers could be maintained by passive transfer in treated nude mice at levels comparable to those of polyclonal antibody titers found in heterozygous infected nu/+ littermates. Areas of demyelination were detected in the untreated animals as early as 7 days after infection with little or no remyelination present. In approximately one-half of the antibody-treated nude animals, no demyelinating lesions were found. However, the rest of these treated mice were found to have areas of both demyelination and remyelination. Thus, anti-Theiler's murine encephalomyelitis virus antibody against VP-1 can play a dramatic role in the survival of mice, clearance of virus, limiting viral spread, and altering the pattern of disease in the absence of a functional T-cell response.     

NK cell depletion and recovery in SCID mice treated with anti-NK1.1 antibody

The anti-NK1.1 antibody produced by PK136 hybridoma cell line administered subcutaneously to SCID mice effectively decreased the level of peripheral blood NK cells and weight of the spleen for 3-4 days. The antibody treatment did not harm the general state of the animal, and may be practically applied in xenograft experiments.     

Minipig as a potential translatable model for monoclonal antibody pharmacokinetics after intravenous and subcutaneous administration

Subcutaneous (SC) delivery is a common route of administration for therapeutic monoclonal antibodies (mAbs) with pharmacokinetic (PK)/pharmacodynamic (PD) properties requiring long-term or frequent drug administration. An ideal in vivo preclinical model for predicting human PK following SC administration may be one in which the skin and overall physiological characteristics are similar to that of humans. In this study, the PK properties of a series of therapeutic mAbs following intravenous (IV) and SC administration in Göttingen minipigs were compared with data obtained previously from humans. The present studies demonstrated: (1) minipig is predictive of human linear clearance; (2) the SC bioavailabilities in minipigs are weakly correlated with those in human; (3) minipig mAb SC absorption rates are generally higher than those in human and (4) the SC bioavailability appears to correlate with systemic clearance in minipigs. Given the important role of the neonatal Fc-receptor (FcRn) in the PK of mAbs, the in vitro binding affinities of these IgGs against porcine, human and cynomolgus monkey FcRn were tested. The result showed comparable FcRn binding affinities across species. Further, mAbs with higher isoelectric point tended to have faster systemic clearance and lower SC bioavailability in both minipig and human. Taken together, these data lend increased support for the use of the minipig as an alternative predictive model for human IV and SC PK of mAbs.Key words: mAb IgG, neonatal Fc receptor (FcRn), pharmacokinetics, subcutaneous bioavailability, animal model, minipig     

Minipig as a potential translatable model for monoclonal antibody pharmacokinetics after intravenous and subcutaneous administration

Subcutaneous (SC) delivery is a common route of administration for therapeutic monoclonal antibodies (mAbs) with pharmacokinetic (PK)/pharmacodynamic (PD) properties requiring long-term or frequent drug administration. An ideal in vivo preclinical model for predicting human PK following SC administration may be one in which the skin and overall physiological characteristics are similar to that of humans. In this study, the PK properties of a series of therapeutic mAbs following intravenous (IV) and SC administration in Göttingen minipigs were compared with data obtained previously from humans. The present studies demonstrated: (1) minipig is predictive of human linear clearance; (2) the SC bioavailabilities in minipigs are weakly correlated with those in human; (3) minipig mAb SC absorption rates are generally higher than those in human and (4) the SC bioavailability appears to correlate with systemic clearance in minipigs. Given the important role of the neonatal Fc-receptor (FcRn) in the PK of mAbs, the in vitro binding affinities of these IgGs against porcine, human and cynomolgus monkey FcRn were tested. The result showed comparable FcRn binding affinities across species. Further, mAbs with higher isoelectric point tended to have faster systemic clearance and lower SC bioavailability in both minipig and human. Taken together, these data lend increased support for the use of the minipig as an alternative predictive model for human IV and SC PK of mAbs.     

Membrane lipid composition modulates the binding specificity of a monoclonal antibody against liposomes

A hybridoma secreting a monoclonal IgM 'anti-liposome' antibody was produced after injecting a mouse with liposomes containing dipalmitoylphosphatidylcholine, cholesterol, dicetyl phosphate, and lipid A. The antibody was selected by assaying for complement-dependent damage to liposomes lacking lipid A. The monoclonal antibody reacted best with liposomes containing the original immunizing mixture of lipids. Deletion of individual lipid constituents from liposomes diminished the ability of the liposomes to bind (adsorb) the antibody. Binding of the antibody was enhanced by including lipid A or galactosylceramide in the lipid bilayer, or by substituting egg phosphatidylcholine for dimyristoyl- (or dipalmitoyl-) phosphatidylcholine. Sphingomyelin could be substituted for dimyristoylphosphatidylcholine without altering the adsorption of antibody. Although the monoclonal anti-liposome antibody was completely inhibited by phosphocholine, it was probably not a conventional anti-phosphocholine antibody. The antibody apparently had a partial specificity for phosphate, and was inhibited by glycerophosphocholine, glycerophosphate, sodium phosphate, sodium sulfate, and inositol hexaphosphate, but not by choline or inositol.     

A monoclonal antibody for the detection of conjugated forms of abscisic acid in plant tissues

A mouse monoclonal antibody against abscisic acid (ABA) was produced and characterized. It was raised using ABA conjugated to the carrier protein through the carboxyl (Cl) group as immunogen. It did not discriminate between free ABA or its ester derivatives. This antibody, which is the first monoclonal against Cl-conjugated ABA, shows interesting characteristics. It has high affinity (K_a=1.5 × 10⁹ L/mol) and specificity. Compounds structurally similar to ABA, such as phaseic acid, dihydrophaseic acid, and both the 2,trans-isomer and the (R)-enantiomer of ABA, are not reactive. The narrow linear range of the standard curve (0.018–1.8 pmol) ensures great precision of the assay. This monoclonal antibody has been used for the quantification of ABA conjugates in crude aqueous extracts of bean leaves by radioimmuno-assay (RIA). The fractionation of the extracts by high-performance liquid chromatography (HPLC) confirmed the absence of cross-reacting compounds. Because of its affinity and specificity, in combination with antibodies against free ABA, this antibody should be a sound tool for studying the metabolism and immunolocalization of ABA in plant tissues.     

A monoclonal antibody for the detection of conjugated forms of abscisic acid in plant tissues

A mouse monoclonal antibody against abscisic acid (ABA) was produced and characterized. It was raised using ABA conjugated to the carrier protein through the carboxyl (Cl) group as immunogen. It did not discriminate between free ABA or its ester derivatives. This antibody, which is the first monoclonal against Cl-conjugated ABA, shows interesting characteristics. It has high affinity (K_a=1.5 × 10⁹ L/mol) and specificity. Compounds structurally similar to ABA, such as phaseic acid, dihydrophaseic acid, and both the 2,trans-isomer and the (R)-enantiomer of ABA, are not reactive. The narrow linear range of the standard curve (0.018–1.8 pmol) ensures great precision of the assay. This monoclonal antibody has been used for the quantification of ABA conjugates in crude aqueous extracts of bean leaves by radioimmuno-assay (RIA). The fractionation of the extracts by high-performance liquid chromatography (HPLC) confirmed the absence of cross-reacting compounds. Because of its affinity and specificity, in combination with antibodies against free ABA, this antibody should be a sound tool for studying the metabolism and immunolocalization of ABA in plant tissues.