Preparation and evaluation of nanoparticles made of chitosan or N-trimethyl chitosan and a cisplatin-alginate complex.

In this work, nanoparticles with a negative or positive surface charge were prepared through electrostatic interaction of an anionic cisplatin-alginate complex with a cationic polyelectrolyte, namely chitosan or N-trimethyl chitosan (substitution degree of 85%). Statistical experimental design allowed the study of the influence of component amounts on the characteristics of nanoparticles. Mean particle diameter ranged from 180 nm to 350 nm. After 24 h, while the cisplatin-alginate complex released almost all the drug in saline-buffered solution at pH 7.4, approximately 40% w/w of total cisplatin was released from negative nanoparticles and roughly 50% w/w from positive ones. The same cumulative amounts of released drug were found after 48 h, with a progressive reduction to lower values up to 6 days. Drug loading of nanoparticles with a positive zeta potential (43 mV-60 mV) ranged from 13% w/w to 21% w/w and particle yield, referred to total polymers, was about 15% w/w (50% w/w if referred to cisplatin-alginate complex). Nanoparticles with a negative zeta potential (-34 mV) were obtained with a yield of 40% w/w and a drug loading of 18% w/w. These nanoparticles were the least active on all cell lines tested, while the cytotoxic activity of the positive nanoparticles was similar to or lower than that of cisplatin, probably depending on the combination of sizes and zeta potential values, on P388 murine and A2780 human cells. On A549 human cells, the nanoparticles with the smallest size and the lowest positive zeta potential were more active than cisplatin and showed a similar capability in inducing apoptosis in A2780 human cells. These results indicate that cisplatin complexes with polycarboxylate polymers can be transformed into cisplatin particulate carriers of high potential interest.

Magnetic resonance imaging at 1.5 T with immunospecific contrast agent in vitro and in vivo in a xenotransplant model.

OBJECT: Demonstrating the feasibility of magnetic resonance imaging (MRI) at 1.5 T of ultrasmall particle iron oxide (USPIO)-antibody bound to tumor cells in vitro and in a murine xenotransplant model. METHODS: Human D430B cells or Raji Burkitt lymphoma cells were incubated in vitro with different amounts of commercially available USPIO-anti-CD20 antibodies and cell pellets were stratified in a test tube. For in vivo studies, D430B cells and Raji lymphoma cells were inoculated subcutaneously in immunodeficient mice. MRI at 1.5 T was performed with T1-weighted three-dimensional fast field echo sequences (17/4.6/13 degrees ) and T2-weighted three-dimensional fast-field echo sequences (50/12/7 degrees ). For in vivo studies MRI was performed before and 24 h after USPIO-anti-CD20 administration. RESULTS: USPIO-anti-CD20-treated D430B cells, showed a dose-dependent decrease in signal intensity (SI) on T2*-weighted images and SI enhancement on T1-weighted images in vitro. Raji cells showed lower SI changes, in accordance to the fivefold lower expression of CD20 on Raji with respect to D430B cells. In vivo 24 h after USPIO-anti-CD20 administration, both tumors showed an inhomogeneous decrease of SI on T2*-weighted images and SI enhancement on T1-weighted images. CONCLUSIONS: MRI at 1.5 T is able to detect USPIO-antibody conjugates targeting a tumor-associated antigen in vitro and in vivo.

An Epstein-Barr virus-infected lymphoblastoid cell line (D430B) that grows in SCID-mice with the morphologic features of a CD30+ anaplastic large cell lymphoma, and is sensitive to anti-CD30 immunotoxins.

BACKGROUND AND OBJECTIVE: In this study we describe a newly established CD30+ Epstein Barr virus (EBV)-infected B cell line derived from an EBV-infected B cell culture (utilized, once irradiated, as a feeder) which showed a B clonal rearrangement and strong CD30 antigen expression. DESIGN AND METHODS: The cells injected into SCID mice were able to grow giving rise to CD30+ solid tumors with the morphologic features of an anaplastic large cell lymphoma (ALCL). Thus we tried to establish a model to investigate the potency of immunoconjugates containing a CD30 monoclonal antibody (Ber-H2) and ribosome-inactivating proteins (saporin, momordin and ricin A-chain) as toxic moieties. RESULTS: We observed a strong cytotoxic activity of the anti-CD30 immunotoxins on the in vitro growth of D430B cells. High levels of anti-tumor activity were also observed in vivo, in the SCID mouse model. INTERPRETATION AND CONCLUSIONS: The antitumor immunotoxin therapy was sccessful in our chosen animal model, the effecacy seeming to be associated with strength of CD30 expression. Our data suggest that immunotoxins should be tested (before use) on the tumor cells of the subject to be treated and that immunotoxins should be directed to different tumor-associated antigens to avoid selection of cell populations with different antigenic mosaics.

IL4 production and increased CD30 expression by a unique CD8+ T-cell subset in B-cell chronic lymphocytic leukaemia.

Phenotypic and functional abnormalities within the residual non-B-cell compartment of B-cell chronic lymphocytic leukaemia (CLL) suggest an interaction between tumour cells and host immune effectors. To explore the possibility of a polarized Th1/Th2 response we have studied CD30 antigen expression and the pattern of cytokine production by purified CLL T cells. Activated T cells from CLL patients showed a significant increase in the expression of CD30 compared to normal controls. Accordingly, high levels of soluble CD30 were detected in supernatants from activated T-cell cultures, as well as in CLL serum samples. Messenger RNA for IL4 was found in both resting and, to a greater extent, in activated CLL T lymphocytes. The latter cells were also capable of releasing IL4. Three-colour immunofluorescence analyses revealed a strong CD30 expression in the CD3+/CD8+/CD28- large granular lymphocyte subset, which is considerably expanded in CLL. Production of IL4, as well as expression and release of CD30 by these T cells, was conclusively demonstrated at the clonal level. These findings document an expansion of a peculiar subset of 'Th2-like' cells in CLL, with an increased IL4 production and CD30 expression and release, that are likely to contribute to both the B-cell accumulation and immune-defects characteristic of this disease.

Targeting of saporin to Hodgkin's lymphoma cells by anti-CD30 and anti-CD25 bispecific antibodies.

CD25 and CD30 represent suitable target molecules for bispecific antibody (bimAb)-driven toxin delivery to lymphoid tumour cells. We describe two new anti-CD30/anti-saporin bimAbs (termed CD30 x sap1 and CD30 x sap2), produced by hybrid hybridomas, which react against non-cross-reactive epitopes of the saporin molecule, and compared their effect with a bimAb reacting with saporin and with CD25 (CD25 x sap1). In a protein synthesis inhibition assay these bimAbs were able to enhance saporin toxicity (IC50 = 8.5 x 10(-9) M in the absence of mAbs) with a similar activity: in the presence of 10(-9) M CD30 x sap1 bimAb the IC50 was 2.75 x 10(-11) M, whereas with CD30 x sap2 bimAb the IC50 was 6.5 x 10(-11) M and CD25 x sap1 bimAb displayed an IC50 of 3 x 10(-11) M (as saporin). The combined use of the two anti-CD30 bimAbs further increased cytotoxicity by 100-fold, resulting in an IC50 of 1.9 x 10(-13) M. A slightly less efficient improvement was obtained by combining the CD25 x sap1 bimAb with the CD30 x sap2 bimAb directed against a different toxin epitope (saporin IC50 to 7 x 10(-13) M). In contrast, no synergistic effect was observed using the combination of the anti-CD25 bimAb with the anti-CD30 bimAb reacting with the same epitope of saporin (IC50 = 4.5 x 10(-11) M). Analysis of FITC-saporin binding to L540 cells by flow cytometry demonstrated that the appropriate combinations of the two anti-CD30/anti-saporin bimAbs or of the anti-CD30/anti-saporin and anti-CD25/anti-saporin bimAbs had a cooperative effect on the binding of the ribosome-inactivating protein (RIP) to the cells, when compared with single bimAbs.

The IL-2 receptor complex: expression and function on normal and leukemic B cells.

The interleukin 2 receptor (IL-2R) is composed of at least three different chains that may be variably expressed on normal and neoplastic lymphocytes. While considerable knowledge has been gained on the expression of the IL-2R alpha chain on human leukemic cells of different origin, less is known of the beta and gamma chains. In view of the highly pleiotropic functions exerted by IL-2, the IL-2-IL-2R interactions may play an important role in various leukemias. In this review we focus on the expression and function of the IL-2R complex on human normal and leukemic B cells. Possible implications in the expansion of the neoplastic clone and in the clinical course of the disease are discussed.

Phenotypic, functional and molecular analysis of CD3- LGL expansions indicates a relationship to two different CD3- normal counterparts.

In this study we have analysed the CD3 and TCR transcript expression in three CD3- large granular lymphocyte (LGL) expansions. These LGL populations show a heterogenous attern of expression for CD2, CD8, CD16, CD56 and CD57 antigens. LGL1 is CD2+CD8-CD16+CD56+CD57+, while LGL2 is CD2-CD8+CD16-CD56-CD57-; LGL3 is similar to LGL1, except for CD8 antigen expression. Functional analysis has revealed a different behaviour of these LGL expansions in a cytotoxicity assay against the NK-sensitive K562 cell line. LGL1 and 3 display a significant NK-like activity, while LGL2 is inefficient against K562 target cells. TCR and CD3 transcript characterization of LGL expansions 1 and 3 showed that they expressed multiple TCR delta transcripts, a non-functional TCR beta transcript, CD3-zeta and -epsilon mRNA, but they lacked CD3 delta transcript and they lacked or expressed at very low levels of CD3 gamma transcript. On the other hand, LGL2 expressed TCR delta, CD3-gamma, -epsilon and -zeta transcripts, while it lacked CD3 delta mRNA. On the basis of these data, LGL1 and 3 seem to be closely related to peripheral blood mature natural killer (NK) cells, whereas LGL2 displays a pattern of TCR and CD3 expression similar to that found in CD1-2-3-4-8-16-thymocytes.

Cellular mechanisms of exogenous peptide binding to HLA class II molecules in B cells.

We have investigated the ability of APC Class II molecules to bind and release exogenous peptides, two phenomena that are still poorly understood. In order to investigate the half-life of the complex of an exogenous peptide with DR molecules we have evaluated the uptake and release of the radiolabeled peptide 17-29-Tyr of influenza virus matrix protein (MA 17-29-Y) by a B-EBV cell line at different times and under different conditions. We have found that the kinetics of both binding and release of the peptide are very fast in living cells; using glutaraldehyde-fixed cells, the kinetics of the two phenomena are slow, closely resembling those observed with the same peptide and purified, immobilized DR molecules. As confirmed by the study of a specific T-cell clone activation, the Class II-MA 17-29-Y complexes are short-living ones, with an average half-life of 55 min, and the DR molecules that bind exogenous peptides continuously undergo peptidic exchange. These data, taken together, suggest that the APC are endowed with cellular mechanisms that increase the efficiency of both the loading and the unloading of Class II HLA with exogenous peptides. These mechanisms do not appear to require ATP or to involve newly synthesized Class II molecules, intracellular acidic compartments, or the microtubule-microfilament system. On the other hand, an undamaged cell membrane appears to be crucial for an efficient binding.

Expression of the IL2 receptor alpha, beta and gamma chains in hairy cell leukemia.

The expression of the interleukin 2 (IL2) receptor chains was investigated by immunofluorescence and mRNA analysis in typical and variant hairy cell leukemia (HCL) cases. While typical hairy cells express both the alpha and the beta chains, variant HCL express the IL2 receptor beta chain on the surface membrane but not the alpha chain. Both typical and variant HCL have mRNA transcript of the p64 molecule (gamma chain). In B-cell chronic lymphocytic leukemia (B-CLL), EBV-transformed lymphoblastoid cell lines and normal B lymphocytes the expression of the IL2 receptor alpha and beta chains is very weak or negative. Furthermore, in B-CLL the IL2 receptor gamma chain shows a weaker expression compared to HCL. Both classic and variant HCL cases appear capable of proliferating in the presence of exogenous IL2, though the proliferative response of variant HCL appears lower than that of typical cases. Since in variant HCL the IL2 receptor alpha chain could not be induced by different stimuli, it appears that the CD25 antigen may not be essential in the process of IL2-promoted growth, while p75 and p64 play a primary role in IL2 activation. Taken together, it is suggested that the expression of IL2 receptors is up- or down-regulated during the process of B-cell lineage activation and differentiation.

Immunotoxins containing saporin linked to different CD2 monoclonal antibodies: in vitro evaluation.

In this study we describe immunotoxins prepared with different CD2 monoclonal antibodies (mAbs) and a ribosome-inactivating protein, saporin. The CD2 immunotoxins were tested on different models. Anti-CD2-saporin conjugates inhibited protein synthesis by a neoplastic CD2+ cell line (SKW-3) and by an interleukin 2 dependent polyclonal CD2+ lymphoid cell culture (T lymphoblasts), with IC50s ranging from 10(-13) M to 10(-11) M (as saporin). Similar results were obtained with proliferation inhibition tests (3H-thymidine incorporation) on phytohaemagglutinin (PHA) driven lymphoid cultures and on mixed lymphocyte culture activated lymphocytes. Moreover a CD2-ricin A chain conjugate was less effective than an analogous immunotoxin containing the same CD2 mAb and saporin in inhibiting lymphocyte proliferation induced by PHA (IC50 approximately 10(-9) M as ricin A chain versus 10(-12) M as saporin). The conjugates were not toxic on bone marrow stem cells. These results suggest that CD2-saporin immunotoxins could represent an effective tool for CD2+ lymphomas or leukaemias, and for T-dependent immune disorders, such as transplanted organ rejection and graft-versus-host disease.

Cultured human NK cells express the Ki-1/CD30 antigen.

In this study we show that in vitro cultured human polyclonal NK cell lines and clones express the Ki-1/CD30 Hodgkin-associated antigen, identified by the BER-H2 monoclonal antibody. The percentage of BER-H2+ cells ranged from 19% to 67% in five polyclonal NK cell lines and was 31% and 20% in two NK clones. The intensity of BER-H2 mAb staining on cultured NK cells was remarkably lower than that found on the L540 Hodgkin's lymphoma cell line. Resting PBL populations that had been enriched for NK cells failed to react with the BER-H2 mAb. Western blot analysis performed on cell lysates from a polyclonal NK cell line and from the NK3.3 NK-like cell line revealed that BER-H2-reactive molecules consisted of two major bands of approximately 110 kD and 100 kD. Two bands displaying an identical electrophoretic mobility were also found in lysates of the L540 cell line. The BER-H2 mAb failed to inhibit the nonspecific activated killer activity of cultured NK cells against both K562 and MeWo tumour target cells. In addition, the BER-H2 mAb was unable to trigger the cytolytic activity of NK cells in a redirected killing assay. The observation that cultured human NK cells express the Ki-1/CD30 antigen may be of relevance for the possible lineage assignment of K11/CD30+ lymphoid cell neoplasia with unrearranged TCR genes.

Phenotypic analysis of hairy cell leukemia: "variant" cases express the interleukin-2 receptor beta chain, but not the alpha chain (CD25).

Hairy cell leukemia (HCL) is a B-cell chronic lymphoproliferative disorder in which the pathologic cells show a strong expression of CD25 (interleukin-2 [IL-2] receptor alpha chain or p55). "Variant" cases of HCL, characterized by hyperleukocytosis, neoplastic elements with a prominent nucleolus and a higher nucleo/cytoplasmic ratio, and an easily obtained bone marrow aspirate, lack surface CD25 determinants. Limited information is available on the expression of the IL-2 receptor beta chain (p75) on normal and neoplastic B cells. In this study, we have assessed by immunofluorescence and mRNA analysis the presence of the IL-2 receptor alpha and beta chains on 12 cases of classic HCL, as well as on 3 variant cases. The results obtained show that, while the alpha chain of the IL-2 receptor is present only on classic HCL, the IL-2 receptor beta chain (p75) is expressed on both the classic and variant form. Unlike hairy cells, only 8 of the 15 B-cell chronic lymphocytic leukemia cases tested showed a weak expression of the p75 antigen on a small proportion of cells. Purified B lymphocytes from normal healthy controls, as well as Epstein-Barr virus-transformed lymphoblastoid cell lines, showed a weak staining for the p75 determinant, while being CD25-. The results of this study suggest that the expression of the alpha and beta chains of the IL-2 receptor appears to be upregulated or downregulated during the process of B-cell-lineage activation and differentiation.

Heterogeneous immunophenotype of granular lymphocyte expansions: differential expression of the CD8 alpha and CD8 beta chains.

In this study, we have evaluated 14 large granular lymphocyte (LGL) expansions, 11 of which were CD8+. Analysis of the membrane expression of the alpha and beta chains of the CD8 antigen, using specific monoclonal antibodies (MoAbs), has shown that LGL expansions with the CD3+, CD4+, CD8+, CD57+ T-cell receptor (TcR) alpha beta phenotype bear the CD8 alpha/alpha isoform, while the CD3+, CD4-, CD8+, CD57+ TcR alpha beta samples were positive for both the CD8 alpha and CD8 beta chains. These data were confirmed also by messenger RNA analysis. One additional case, with a peculiar phenotype (CD3-, CD2-, CD4-, CD8+, CD57-) and a germline configuration of the TcR beta and gamma chain genes, expressed only the CD8 alpha chain. After additional phenotypic analysis with a wider panel of MoAbs, it was found that the beta chain of the interleukin-2 receptor was constitutively expressed on the majority of the samples tested, and that most of the monoclonal samples coexpressed CD45RA/R0 antigens. Using MoAbs directed against the variable regions of the TcR beta chain, we could show a preferential V beta region restriction in the CD8+ monoclonal cases. This more extensive characterization of CD8+ LGL expansions has further documented the marked heterogeneity within this rare condition and allowed a better phenotypic dissection between the monoclonal and polyclonal cases.

B-B10 (anti-CD25)-saporin immunotoxin--a possible tool in graft-versus-host disease treatment.

An immunotoxin containing the B-B10 MoAb, directed against the CD25 determinant, and the ribosome-inactivating protein saporin, inhibits 3H-TdR incorporation in phytohemagglutin, allogeneic-stimulated lymphocytes (primary and secondary mixed-lymphocyte reaction), and in an alloreactive T cell clone. A lower degree of inhibition was obtained with the B-B10 MoAb, which is known to inhibit IL-2 activity, as well as with the unconjugated compounds. These results suggest that the in vivo administration of the conjugate might be a more effective tool in the treatment of patients affected by graft-versus-host disease than B-B10 alone, by inducing an efficient killing of allogeneic-reacting T lymphocytes.

Ber-H2 (anti-CD30)-saporin immunotoxin: a new tool for the treatment of Hodgkin's disease and CD30+ lymphoma: in vitro evaluation.

An immunotoxin containing an anti-CD30 monoclonal antibody (Ber-H2) and saporin, a ribosome-inactivating protein type 1, is described. It specifically inhibits protein synthesis by Hodgkin derived target cell lines with a very high efficiency (IC50 ranging from 5 x 10(-12) M to 5 x 10(-14) M, as saporin), while irrelevant immunotoxins do not. Present results suggest that this immunotoxin could be used for in vivo therapy as well as for ex vivo bone marrow purging in Hodgkin's disease and CD30+ lymphomas.

Polymorphism of the HLA-DP beta region detected by Southern blot hybridization.

A panel of homozygous cell lines, previously typed by primed lymphocyte test for their DPw specificity, have been studied by restriction fragment length polymorphism analysis, using a DP beta-specific probe. Highly stringent hybridization and washing conditions were used to prevent cross-hybridization with DR- and DQ-specific fragments. Three out of six enzymes employed allowed us to distinguish clustered or single DPw specificities, and by MspI digestion it was possible to detect different patterns within a single specificity such as DPw4. Some of the cell lines have been further studied with synthetic oligonucleotides derived from the polymorphic regions of the second exon of DP beta 1 gene, and, in general, a correlation with the primed lymphocyte test--defined specificities and restriction fragment length polymorphism was found. These data suggest a more extended complexity of the DP region, in addition to that defined as the DPw1-DPw6 segregant series.