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.     

Targeting of anti-Thy 1.1 monoclonal antibody conjugated liposomes in Thy 1.1 mice after intraveneous 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.     

Disulfide-linked antibody-maytansinoid conjugates: optimization of in vivo activity by varying the steric hindrance at carbon atoms adjacent to the disulfide linkage

In this report, we describe the synthesis of a panel of disulfide-linked huC242 (anti-CanAg) antibody maytansinoid conjugates (AMCs), which have varying levels of steric hindrance around the disulfide bond, in order to investigate the relationship between stability to reduction of the disulfide linker and antitumor activity of the conjugate in vivo. The conjugates were first tested for stability to reduction by dithiothreitol in vitro and for plasma stability in CD1 mice. It was found that the conjugates having the more sterically hindered disulfide linkages were more stable to reductive cleavage of the maytansinoid in both settings. When the panel of conjugates was tested for in vivo efficacy in two human colon cancer xenograft models in SCID mice, it was found that the conjugate with intermediate disulfide bond stability having two methyl groups on the maytansinoid side of the disulfide bond and no methyl groups on the linker side of the disulfide bond (huC242-SPDB-DM4) displayed the best efficacy. The ranking of in vivo efficacies of the conjugates was not predicted by their in vitro potencies, since all conjugates were highly active in vitro, including a huC242-SMCC-DM1 conjugate with a noncleavable linkage which showed only marginal activity in vivo. These data suggest that factors in addition to intrinsic conjugate potency and conjugate half-life in plasma influence the magnitude of antitumor activity observed for an AMC in vivo. We provide evidence that bystander killing of neighboring nontargeted tumor cells by diffusible cytotoxic metabolites produced from target cell processing of disulfide-linked antibody-maytansinoid conjugates may be one additional factor contributing to the activity of these conjugates in vivo.     

Antibody-mediated routing of diphtheria toxin in murine cells results in a highly efficacious immunotoxin

The chemical coupling of diphtheria toxin to an antimurine Thy1 antibody resulted in the most efficacious immunotoxin to date. At 1 micrograms/ml the immunotoxin inhibited protein synthesis of a Thy+ AKR murine cell at a rate of 1.4 logs/h, within the order of magnitude of the efficacy of native toxins. This is unusual since murine cells are highly resistant to diphtheria toxin. The conjugate is highly specific; Thy- AKR cells display no intoxication at 1 microgram/ml even after 18 h. The effects of ammonia, acid pulsing of external media, and low temperature reveal some similarities and some differences between intoxication of sensitive cells by toxin and of murine cells by the antibody-toxin conjugate. The differences that result in the high efficacy of the antibody-toxin conjugate appear to result from the antibody-mediated routing. These results imply that murine cells possess an acidic compartment which can mediate toxin cytosolic entry. Unlike the Thy antigen, the toxin receptor on murine cells is unable to route the toxin to this cellular site.     

Blockade of the galactose-binding sites of ricin by its linkage to antibody. Specific cytotoxic effects of the conjugates

A method is described for preparing specific cytotoxic agents by linking intact ricin to antibodies in a manner that produces obstruction of the galactose-binding sites on the B chain of the toxin and so diminishes the capacity of the conjugate to bind non-specifically to cells. The conjugates were synthesised by reacting iodoacetylated ricin with thiolated immunoglobulin and the components of conjugate with reduced galactose-binding capacity were separated by affinity chromatography on Sepharose (a beta-galactosyl matrix) and asialofetuin-Sepharose. Fluorescence-activated cell sorter (FACS) analyses revealed that the fraction of a monoclonal anti-Thy1.1-ricin conjugate that passed through a Sepharose column had markedly diminished capacity to bind non-specifically to Thy1.2-expressing CBA thymocytes and EL4 lymphoma cells. The fraction of conjugate that passed through an asialofetuin-Sepharose column displayed no detectable non-specific binding. Both fractions of conjugate were potent cytotoxic agents for Thy1.1-expressing AKR-A lymphoma cells in tissue culture. They reduced the [3H]leucine incorporation of the cells by 50% at a concentration of 2-5 pM. Comparable inhibition of EL4 cells was only achieved with 3000-7500-fold greater concentrations of conjugate. By contrast, the fraction of anti-Thy1.1-ricin that retained Sepharose-binding capacity showed marked non-specific binding and toxicity to EL4 cells. A conjugate with diminished galactose-binding capacity was also prepared from the W3/25 monoclonal antibody which recognises an antigen upon helper T-lymphocytes in the rat. It elicited powerful and specific toxic effects upon W3/25 antigen-expressing rat T-leukaemia cells. This finding is of particular importance because isolated ricin A-chain disulphide-linked to W3/25 antibody is not cytotoxic. The property of the B-chain in intact ricin conjugates that facilitates delivery of the A-chain to the cytosol thus appears to be independent of galactose recognition. It is concluded that the 'blocked' ricin conjugates combine the advantages of high potency, which is often lacking in antibody-A-chain conjugates, with high specificity, which previously was lacking in intact ricin conjugates.     

Analysis of lymphocyte-target conjugates by flow cytometry. I. Discrimination between killer and non-killer lymphocytes bound to targets and sorting of conjugates containing one or multiple lymphocytes

The use of flow cytometric analysis and sorting techniques for the enumeration and purification of lymphocyte-target conjugates was investigated. Murine cytotoxic T-lymphocytes (CTL) with killer effector function were identified and quantitated during a 3-hour cell-mediated cytotoxicity reaction using multiparameter analysis. Resolution of conjugates containing single and multiple lymphocytes was achieved by two-color fluorescence, and individual conjugate subpopulations were subsequently sorted for further analysis. To measure total and cytotoxic conjugate frequencies, CTL were labelled with FITC-conjugated Thy 1.2 antibody and dead target cells were stained with propidium iodide (PI). Size difference between the CTL and P815 tumor target cells, as measured by Coulter volume and axial light loss, facilitated detection of conjugates which were identified as both large and Thy 1.2-positive. Conjugates containing dead target cells possessed red fluorescence due to PI uptake. The frequency of conjugates containing cytotoxic activity increased with time during the cytotoxicity period and correlated with frequencies obtained in single-cell assays. Analysis of the distribution of single and multiple lymphocyte-bound conjugates was done by co-centrifugation of Hoechst-stained CTL and FITC-labeled P815 target cells. Analysis by two-color fluorescence effectively resolved conjugate populations containing different numbers of CTL and allowed their purification by cell sorting. The purity of the separate populations was confirmed by fluorescence microscopic inspection. The results of these studies demonstrate that flow cytometry can resolve target-bound and free CTL, measure cytolytic efficiency and specifically sort out cytometrically defined subgroups within the effector cell population.     

Isolation of rat bone marrow mast lineage cells using Thy 1.1 and rat stem cell factor.

Recent reports have shown that various marrow-derived cell populations respond vigorously to recombinant rat stem cell factor (rrSCF164), one form of the kit-ligand. In the present study, we isolated cell populations from rat bone marrow using the Thy 1.1 antigen (an antigen that in the rat is differentially expressed on primitive hemopoietic progenitor cells) and fluorescently conjugated rrSCF164 (rrSCF164-PE). We show that rrSCF164 only stimulates cells that are enriched in the brightest Thy 1.1 populations (Thy 1.1bright). Numerous cell lines were generated by serial passage in rrSCF164 containing medium, and the prototypic cell lines have been designated SRT002 and SRT003. Each cell line retains the Thy 1.1bright phenotype and does not respond to interleukins (IL) 1-8, IL-10, granulocyte (G) colony-stimulating factor (CSF), granulocyte macrophage (GM) CSF, M-CSF, or crude preparations of mitogen-stimulated T-cell supernatants. The Thy 1.1bright population of rat marrow was subdivided into a subset that binds rrSCF164-PE (Thy 1.1bright, rrSCF164+). The majority of these cells possess certain characteristics in common with marrow-derived mast cells and the Thy 1.1bright, rrSCF164 responsive cell lines, having similar granule morphology, being metachromatic, and reacting positively with alcian blue. Moreover, rats treated with rrSCF164 displayed significant increases in Thy 1.1bright, rrSCF164+ cells in the bone marrow. These studies show that the combination of Thy 1.1 and rrSCF164 makes possible the isolation of a unique subset of rat bone marrow cells that differentially express the Thy 1.1 antigen and the cell surface receptor c-kit, the majority of which are morphologically similar to marrow-derived mast cells.     

T cell killing of human colon carcinomas by monoclonal-antibody-targeted superantigens

The bacterial superantigen staphylococcal enterotoxin A (SEA) induces T cell activation as well as directing activated T cells to kill major-histocompatibilitycomplex-class-II-expressing tumours such as freshly prepared leukemia cells. We now report that conjugates of SEA and the colon-carcinoma-reactive mAb C215 mediate T-cell-dependent killing of freshly isolated cells obtained from surgical specimens of human colon carcinomas. Cytotoxicity was observed at nanomolar concentrations of conjugate while no or very low effects were seen with the mAb C215 or SEA alone. Tumour-infiltrating lymphocytes (TIL) did not exert any cytotoxicity against conjugate-treated tumour cells immediately after isolation. In vitro culture of TIL with interleukin-2 and SEA resulted in SEA-mAb-conjugate-dependent killing of freshly isolated tumour cells. This suggests that mAb-SEA conjugates may be of potential use to target T lymphocytes, including TIL, against colon carcinoma cells in vivo.     

A flexible peptide spacer increases the efficacy of holoricin anti-T cell immunotoxins

Immunotoxins, constructed by chemically cross-linking an antibody and protein toxin, do not possess the high efficacy of the native toxin. Decreases in toxicity are due in part to the steric constraints imposed on the two macromolecules, which result in both decreased antibody binding and toxin function. In examining the structural features that influence efficacy in holotoxin-antibody conjugates, it was found that the incorporation of a 29-residue polypeptide, derived from the insulin B chain between the antibody and ricin moiety, resulted in an increase in both potency and efficacy. In a murine model system, potency of the peptide spacer conjugate was increased nearly 10-fold; however, when examined by the procedure used to purge bone marrow, the peptide spacer conjugate was not demonstrably more toxic to nontarget cells than the nonspacer conjugate. Thus, in addition to increases of efficacy and potency, this novel immunotoxin demonstrated increased specificity by approximately 10-fold. To test the general utility of peptide spacer inclusion, a T101-ricin conjugate was constructed with the peptide spacer. It yielded a protein synthesis inhibition rate of -0.6 log/h on MOLT-3 cells, greater than 10-fold more efficacious than a previously constructed nonspacer T101-ricin conjugate examined under similar conditions.     

An anti-MUC1 antibody-calicheamicin conjugate for treatment of solid tumors. Choice of linker and overcoming drug resistance

The anti-MUC1 antibody, CTM01, has been chosen to target the potently cytotoxic calicheamicin antitumor antibiotics to solid tumors of epithelial origin that express this antigen. Earlier calicheamicin conjugates relied on the attachment of a hydrazide derivative to the oxidized carbohydrates that occur naturally on antibodies. This produced a "carbohydrate conjugate" capable of releasing active drug by hydrolysis in the lysosomes where the pH is low. Conjugates have now been made that are formed by reacting a calicheamicin derivative containing an activated ester with the lysines of antibodies. This gives an "amide conjugate" that is stable to hydrolysis, leaving the disulfide that is present in all calicheamicin conjugates as the only likely site of drug release from the conjugate. As previously shown for the carbohydrate conjugate, this amide conjugate of CTM01 produces complete regressions of xenograft tumors at doses of 300 microg/kg (calicheamicin equivalents) given three times. This indicates that hydrolytic drug release is not necessary for potent, selective cytotoxicity for calicheamicin conjugates of CTM01. Although the unconjugated calicheamicins are in general less active in cells expressing the multidrug resistance phenotype, both in vitro and in vivo results of studies reported here suggest that the efficacy of the calicheamicins toward such tumors is unexpectedly enhanced by antibody conjugation, especially for the "amide conjugate". These hydrolytically stable conjugates are also active toward cisplatin-resistant ovarian carcinoma cells as well. Such studies indicate that the calicheamicin amide conjugate of CTM01 may have potential for the treatment of MUC1-positive solid tumors, including some types of resistant tumors.     

Lipopolyamine treatment increases the efficacy of intoxication with saporin and an anticancer saporin conjugate

Saporin is a type I ribosome-inactivating protein that is often appended with a cell-binding domain to specifically target and kill cancer cells. Urokinase plasminogen activator (uPA)-saporin, for example, is an anticancer toxin that consists of a chemical conjugate between the human uPA and native saporin. Both saporin and uPA-saporin enter the target cell by endocytosis and must then escape the endomembrane system to reach the cytosolic ribosomes. The latter process may represent a rate-limiting step for intoxication and would therefore directly affect toxin potency. In the present study, we document two treatments (shock with dimethylsulfoxide and lipopolyamine coadministration) that generate substantial cellular sensitization to saporin/uPA-saporin. With the use of lysosome-endosome X (LEX)1 and LEX2 mutant cell lines, an endosomal trafficking step preceding cargo delivery to the late endosomes was identified as a major site for the dimethylsulfoxide-facilitated entry of saporin into the cytosol. Dimethylsulfoxide and lipopolyamines are known to disrupt the integrity of endosome membranes, so these reagents could facilitate the rapid movement of toxin from permeabilized endosomes to the cytosol. However, the same pattern of toxin sensitization was not observed for dimethylsulfoxide- or lipopolyamine-treated cells exposed to diphtheria toxin, ricin, or the catalytic A chain of ricin. The sensitization effects were thus specific for saporin, suggesting a novel mechanism of saporin translocation by endosome disruption. Lipopolyamines have been developed as in vivo gene therapy vectors; thus, lipopolyamine coadministration with uPA-saporin or other saporin conjugates could represent a new approach for anticancer toxin treatments.     

Changes in the populations of null, NK1.1+, and Thy1lo lymphocytes in the bone marrow of tumor-bearing mice: effect of indomethacin treatment.

Mouse bone marrow produces many "null" lymphocytes which lack B and T lineage markers (B220-Thy1-). A subset of these cells expresses the natural killer (NK) cell marker, NK1.1. In addition, some rapidly renewed bone marrow lymphocytes express low intensities of Thy1 (Thy1lo). In view of their possible implication in tumor-host interactions these various cell populations have now been examined in mice injected with either the nonmetastatic Ehrlich ascites (EA) tumor or the Lewis lung carcinoma (LLc), a highly metastatic solid tumor. In each case, the number of null lymphocytes, as defined by a lack of radioautographic labeling of either B220 glycoprotein or Thy1, increased markedly in both the bone marrow and spleen. Treatment with the prostaglandin inhibitor, indomethacin, enhanced the increase in null cells in the bone marrow and spleen of LLc-bearing mice. The number of null small lymphocytes expressing NK1.1, as detected by combined radioautographic and immunoperoxidase techniques, increased almost 30-fold in LLc-bearing mice. The number of Thy1lo small lymphocytes increased in parallel with null cells during EA tumor growth. The findings accord with the hypothesis that the null lymphocyte population produced in mouse bone marrow includes newly formed NK lineage cells which sequentially express NK1.1 and Thy1lo. The present work demonstrates that the populations of null, NK1.1+, and Thy1lo lymphocytes in mouse bone marrow expand rapidly during the early growth of transplanted tumors, the initial increase in null lymphocytes apparently being curtailed by prostaglandin production. The results suggest that the production of null lymphocytes in mouse bone marrow is responsive to tumor development, possibly providing cells to be involved in tumor-host interactions.     

Memory T cells originate from adoptively transferred effectors and reconstituting host cells after sequential lymphodepletion and adoptive immunotherapy

Adoptive transfer of tumor-specific effector T cells induces regression of advanced tumors and induces a long term memory response; however, the origin of this response has not been clearly defined. In this study Thy1.2+ mice bearing advanced MCA-205 tumors were treated with sublethal total body irradiation, followed by adoptive transfer of congenic Thy1.1+ T cells that had been sensitized to tumor in vivo and then activated ex vivo with anti-CD3, IL-2, and IL-7. Splenocytes were recovered >140 days after the initial therapy, and the L-selectinlow memory cell subset was separated into host Thy1.2+ and transferred Thy1.1+ cells and restimulated ex vivo. Both adoptively transferred Thy1.1+ cells as well as reconstituted host Thy1.2+ cells could specifically eliminate MCA-205 pulmonary metastases. Interestingly, hosts with partial responses followed by tumor recurrence nevertheless harbored memory cells that could be isolated and numerically amplified ex vivo to regenerate potent effector function. Memory cells were recovered after adoptive transfer into lymphodepleted nontumor-bearing hosts, indicating that they were not dependent on continued Ag exposure. These experiments establish that rapid ex vivo expansion of tumor Ag-primed T cells does not abrogate their capacity to become long-lived memory cells. Moreover, immune-mediated tumor regression coincident with lymphoid reconstitution produces another wave of host memory cells. These data suggest an approach to rescuing antitumor immune function even in hosts with long-standing progressive tumor through restorative ex vivo activation.     

Heterogeneity of natural killer cell subsets in NK-1.1+ and NK-1.1- inbred mouse strains and their progeny.

The 4LO3311 monoclonal antibody, a new NK-specific reagent recently produced in our laboratory, reacts with spleen cells of 11 mouse strains, most of which do not express the NK-1.1 alloantigen recognized by the PK136 mAb. Among positive strains, the susceptibility of spleen cells to the complement-dependent NK-inhibiting activity of the 4LO3311 mAb was variable but independent of the initial NK cell activity level of cells tested. This property was furthermore not modified after poly(I:C) stimulation. The susceptibility of spleen cells to the in vitro 4LO3311 mAb plus complement treatment is however influenced by the absolute number of 4LO3311+ cells as well as by the density of the corresponding alloantigen at the cell surface. Moreover, it was established that the strain-related variations observed also depended upon the relative size of the 4LO3311 cell subset within the lytic NK cell population. Indeed, when C3H (NK-1.1-4LO3311+) mice were inoculated with the 4LO3311 mAb, the lytic activity of their spleen cells was almost unaltered but 4LO3311-reactive cells were no longer detected in the spleen of treated animals and remaining NK cells were totally resistant to the in vitro 4LO3311 mAb plus complement treatment. These findings indicate that the 4LO3311 mAb identifies a subset rather than all NK cells, even in a NK-1.1- strain. Since a NK-1.1-unreactive cell subset was identified in NZB (NK-1.1+4LO3311-) mice inoculated with the PK136 mAb, the NK-1.1+ cell population is not necessarily responsible for all the splenic NK cell activity in all NK-1.1+ strains. In B6C3F1 hybrid mice, a relatively large subset of NK-1.1-4LO3311- cells was found in addition to those expressing the NK-1.1, the 4LO3311 alloantigen, or both. According to these results, NK cell heterogeneity should thus be taken as an evolving concept whose resolution appears more and more complex with the identification of new NK-specific reagents.     

Development of a thyroid hormone receptor targeting conjugate

Molecular conjugates of hormone receptor-ligands with molecular probes or functional domains are finding diverse applications in chemical biology. Whereas many examples of hormone conjugates that target steroid hormone receptors have been reported, practical ligand conjugates that target the nuclear thyroid hormone receptor (TRbeta) are lacking. TR-targeting conjugate scaffolds based on the ligands GC-1 and NH-2 and the natural ligand triiodothyronine (T3) were synthesized and evaluated in vitro and in cellular assays. Whereas the T3 or GC-1 based conjugates did not bind TRbeta with high affinity, the NH-2 inspired fluorescein-conjugate JZ01 showed low nanomolar affinity for TRbeta and could be used as a nonradiometric probe for ligand binding. A related analogue JZ07 was a potent TR antagonist that is 13-fold selective for TRbeta over TRalpha. JZ01 localizes in the nuclei of TRbeta expressing cells and may serve as a prototype for other TR-targeting conjugates.     

Toxic ligand conjugates as tools in the study of receptor-ligand interactions

We have constructed hybrid proteins in which the toxic A chains of ricin or diptheria toxin have been linked to either asialofetuin, fetuin, or epidermal growth factor (EGF). Both ASF-RTA and ASF-DTA are potent toxins on cultured rat hepatocytes, cells that display the asialoglycoprotein receptor. Toxicity of these two compounds is restricted to hepatocytes and can be blocked by asialoglycoproteins but not the native glycoproteins or asialoagalactoglycoprotein derivatives, indicating that the toxicity of the conjugates is mediated by the hepatic asialoglycoprotein receptor. The EGF-RTA conjugate is an extremely potent toxin on cells that can bind the hormone, but is only poorly effective on cells that are unable to bind EGF. The EGF-DTA conjugate, in contrast, is unable to kill 3T3 cells and is at least two orders of magnitude less effective than EGF-RTA on A431 cells, a cell line with 1-2 X 10(6) EGF receptors per cell. However, when EGF-RTA and EGF-DTA were tested on primary liver hepatocyte cultures, which were susceptible to both ASF-RTA and ASF-DTA, both EGF conjugates were potent toxins. Sensitivity of the hepatocyte cultures to ricin toxicity increases slightly during a 52-hr culture period. In contrast, sensitivity to EGF-RTA and ASF-RTA decline dramatically during this period. Receptors for both ligands remain plentiful on the cell surface during this time.     

Characterization of thymic progenitors in adult mouse bone marrow

Thymic cellularity is maintained throughout life by progenitor cells originating in the bone marrow. In this study, we describe adult mouse bone cells that exhibit several features characteristic of prothymocytes. These include 1) rapid thymic engraftment kinetics following i.v. transplantation, 2) dramatic expansion of thymic progeny, and 3) limited production of hemopoietic progeny other than thymocytes. The adult mouse bone marrow population that is depleted of cells expressing any of a panel of lineage-specific Ags, stem cell Ag-1 positive, and not expressing the Thy1.1 Ag (Thy1.1(-)) (Thy1.1(-) progenitors) can repopulate the thymus 9 days more rapidly than can hemopoietic stem cells, a rate of thymic repopulation approaching that observed with transplanted thymocytes. Additionally, Thy1.1(-) progenitors expand prolifically to generate thymocyte progeny comparable in absolute numbers to those observed from parallel hemopoietic stem cell transplants, and provide a source of progenitors that spans multiple waves of thymic seeding. Nevertheless, the Thy1.1(-) population yields relatively few B cells and rare myeloid progeny posttransplant. These observations describe the phenotype of an adult mouse bone marrow population highly enriched for rapidly engrafting, long-term thymocyte progenitors. Furthermore, they note disparity in B and T cell expansion from this lymphoid progenitor population and suggest that it contains the progenitor primarily responsible for seeding the thymus throughout life.     

In vitro and in vivo gene transfer by an optimized alpha-cyclodextrin conjugate with polyamidoamine dendrimer

The purpose of the present study is to optimize the structure of the polyamidoamine starburst dendrimer (dendrimer) conjugate with alpha-cyclodextrin (alpha-CDE conjugate) as a nonviral vector. alpha-CDE conjugates of dendrimer (generation 3, G3) with various average degrees of substitution (DS) of alpha-CyD of 1.1, 2.4, and 5.4 were prepared. alpha-CDE conjugates formed the complexes with pDNA, resulting in a change of the particle sizes of pDNA complexes, but the distinction of physicochemical properties among their vector/pDNA complexes was only very slight. The membrane-disruptive ability of alpha-CDE conjugates on liposomes encapsulating calcein and their cytotoxicity to NIH3T3 and HepG2 increased with an increase in the DS value of alpha-CyD. In vitro gene transfer activity of alpha-CDE conjugates in both NIH3T3 and HepG2 cells augmented as the charge ratio (vector/pDNA) increased, and the activity of alpha-CDE conjugate (DS 2.4) was the highest at higher charge ratios among dendrimer (G3), the three alpha-CDE conjugates, and TransFast. After intravenous administration of pDNA complexes in mice, alpha-CDE conjugate (DS 2.4) delivered pDNA more efficiently in spleen, liver, and kidney, compared with dendrimer and other alpha-CDE conjugates (DS 1.1 and 5.4). The potential use of alpha-CDE conjugate (G3, DS 2.4) could be expected as a nonviral vector in vitro and in vivo, and these data may be useful for design of alpha-CyD conjugates with other nonviral vectors.     

Development of a sensitive screening method for selecting monoclonal antibodies to be internalized by cells

Antibody–drug conjugates (ADCs), drugs developed by conjugation of an anticancer agent to a monoclonal antibody (mAb), have lately attracted attention in cancer therapy because ADCs can directly bind cancer cells and kill them. Although mAbs for ADCs must be internalized by the target cells, few methods are available for screening mAbs for their ability to be internalized by cells. We have developed a recombinant protein, termed DT3C, which consists of diphtheria toxin (DT) lacking the receptor-binding domain but containing the C1, C2, and C3 domains of Streptococcus protein G (3C). When a mAb–DT3C conjugate, which functions in vitro like an ADC, reduces the viability of cancer cells, the mAb being tested must have been internalized by the target cells. DT3C can thus be a tool to identify efficiently and easily mAbs that can be internalized by cells, thereby enhancing the development of promising ADCs.     

NK1.1+ thymocytes. Adult murine CD4-, CD8- thymocytes contain an NK1.1+, CD3+, CD5hi, CD44hi, TCR-V beta 8+ subset.

CD4-, CD8- thymocytes were purified from thymi obtained from normal C57BL/6 mice. By flow cytometry analysis, 5 to 10% of these double negative (DN) thymocytes were found to express NK1.1 on their surface. The NK1.1+ DN thymocytes were demonstrated, by two-color fluorescence, to be CD3lo, CD5hi, CD44hi, J11d-, B220-, MEL 14-, IL2R- with 60% expressing TCR-V beta 8 as determined by the mAb F23.1. In contrast, splenic and peripheral blood NK cells were NK1.1+, CD3-, CD5-, TCR-V beta 8- with 40 to 60% being MEL 14+. Unlike peripheral NK cells, fresh DN thymocytes enriched for NK1.1+ cells were unable to kill YAC-1, the classical murine NK cell target. However, these cells were able to mediate anti-CD3 redirected lysis even when they were assayed immediately after purification, i.e., with no culture or stimulation. These data demonstrate that adult murine thymocytes contain NK1.1+ cells which are distinct, both by function and phenotype, from peripheral NK cells. These data also raise the issue of a possible NK/T bipotential progenitor cell.