Internalizing antibodies are necessary for improved therapeutic efficacy of antibody-targeted liposomal drugs

Direct experimental proof has been sought for the hypothesis that liposomal drugs targeted against internalizing epitopes (e.g., CD19) will have higher therapeutic efficacies than those targeted against noninternalizing epitopes (e.g., CD20). Anti-CD19-targeted liposomes were rapidly internalized into human B-lymphoma (Namalwa) cells, whereas those targeted with anti-CD20 were not internalized. Similar in vitro binding and cytotoxicity were observed for anti-CD19-targeted and anti-CD20-targeted liposomal formulations of doxorubicin (DXR). Therapeutic experiments were performed in severe combined immunodeficient mice inoculated i.v. with Namalwa cells. Administration of single i.v. doses of DXR-loaded anti-CD19-targeted liposomes resulted in significantly greater survival times than either DXR-loaded anti-CD20-targeted liposomes or nontargeted liposomes. The therapeutic advantage of targeting internalizing versus noninternalizing epitopes has been directly demonstrated.     

Improved therapeutic responses in a xenograft model of human B lymphoma (Namalwa) for liposomal vincristine versus liposomal doxorubicin targeted via anti-CD19 IgG2a or Fab' fragments.

PURPOSE: Monoclonal antibody-mediated targeting of liposomal anticancer drugs to surface antigens expressed on malignant B cells can be an effective strategy for treating B-cell malignancies. In a murine model of human B-cell lymphoma, we have made in vitro and in vivo comparisons of long-circulating sterically stabilized (Stealth) immunoliposome (SIL) formulations of two anticancer drugs, vincristine (VCR) and doxorubicin (DXR), with different mechanisms of action and drug release rates. EXPERIMENTAL DESIGN: SIL formulations of VCR or DXR were conjugated to the monoclonal antibody anti-CD19 (SIL[alphaCD19]) or its Fab' fragments (SIL[Fab']). Specific binding of SILs to Namalwa cells was studied using radiolabeled liposomes, and cytotoxicities of DXR- or VCR-loaded SILs were quantitated by a tetrazolium assay. Pharmacokinetic and drug leakage experiments were performed in mice using dual-labeled liposomes, and the therapeutic responses of SILs were evaluated in a Namalwa (human B lymphoma) cell xenograft model. RESULTS: SIL[alphaCD19] or SIL[Fab'] had higher association with and cytotoxicity against Namalwa cells than nontargeted liposomes. SIL[Fab'] had longer circulation times than SIL[alphaCD19], and VCR had faster release rates from the liposomes than DXR. SIL formulations of either VCR or DXR had significantly better therapeutic outcomes than nontargeted liposomes or free drugs. SILs loaded with VCR were superior to those loaded with DXR. SIL[Fab'] had better therapeutic outcomes than SIL[alphaCD19] for the drug DXR but were equally efficacious for the drug VCR. CONCLUSIONS: Treatment of a B lymphoma model with single injections of anti-CD19-targeted liposomal formulations of VCR resulted in high levels of response and long-term survivors. Responses to anti-CD19-targeted liposomal DXR were more modest, although the longer circulation times of SIL[Fab'] versus SIL[alphaCD19] led to superior therapeutics for DXR-loaded immunoliposomes.     

Tumor targeting of vincristine by mBAFF-modified PEG liposomes in B lymphoma cells

Malignant hematologic diseases are highly malignant and refractory to conventional therapies. Ligand-mediated targeting of liposomal anticancer drugs to surface receptors expressed on malignant B cells can be an effective strategy for treating B-cell malignancies. BAFF plays an important role in the maintenance of normal B-cell development and homeostasis and the expression of its receptors is significantly increased in numerous B-cell malignancies. mBAFF (a soluble BAFF mutant with amino acid 217–224 being replaced by two glycine residues) may be used as a competitive inhibitor for BAFF to treat relevant malignant hematologic diseases. It may also hold promise as a novel ligand for targeted anticancer therapy. In this study, we show that liposomes that are sterically stabilized by PEG and surface decorated with mBAFF exhibited strong affinity and specificity to cultured human Raji B lymphoma cells. Vincristine formulated in the targeted liposomes showed significantly higher levels of cytotoxicity towards Raji cells than the nontargeted liposomal drug. Therapeutic experiments in SCID mice implanted with Raji cells showed significantly prolonged survival time with targeted liposomal vincristine compared to either free VCR or vincristine formulated in nontargeted liposomes. These studies suggest the potential of the mBAFF-modified liposomal drugs in targeted therapy of B-cell malignancies.     

Epidermal growth factor receptor-targeted immunoliposomes significantly enhance the efficacy of multiple anticancer drugs in vivo

We previously reported the development of epidermal growth factor receptor (EGFR)-targeted immunoliposomes that bind and internalize in tumor cells which overexpress EGFR and/or mutant EGFR variant III (EGFRvIII), enabling intracellular delivery of potent anticancer agents in vitro. We now describe in vivo proof-of-concept for this approach for the delivery of multiple anticancer drugs in EGFR-overexpressing tumor models. Anti-EGFR immunoliposomes were constructed modularly with Fab' fragments of cetuximab (IMC-C225), covalently linked to liposomes containing probes and/or anticancer drugs. Pharmacokinetic and biodistribution studies confirmed long circulation times (t(1/2) = 21 hours) and efficient accumulation in tumors (up to 15% ID/g) irrespective of the presence of the targeting ligand. Although total accumulations of anti-EGFR immunoliposomes and nontargeted liposomes in EGFR-overexpressing tumors were comparable, only immunoliposomes internalized extensively within tumor cells (92% of analyzed cells versus <5% for nontargeted liposomes), indicating different mechanisms of delivery at the cellular level. In vivo therapy studies in a series of xenograft models featuring overexpression of EGFR and/or EGFRvIII showed the superiority of immunoliposomal delivery of encapsulated drugs, which included doxorubicin, epirubicin, and vinorelbine. For each of these drugs, anti-EGFR immunoliposome delivery showed significant antitumor effects and was significantly superior to all other treatments, including the corresponding free or liposomal drug (P < 0.001-0.003). We conclude that anti-EGFR immunoliposomes provide efficient and targeted drug delivery of anticancer compounds and may represent a useful new treatment approach for tumors that overexpress the EGFR.     

Doxorubicin-loaded Fab' fragments of anti-disialoganglioside immunoliposomes selectively inhibit the growth and dissemination of human neuroblastoma in nude mice

Neuroblastoma (NB) is the most common extracranial solid tumor in children. Intensive therapeutic intervention does not prolong the overall disease-free survival rate for this tumor. NB tumor, but not normal tissues, overexpress the disialoganglioside (GD(2)) at the cell surface. Anti-GD(2) whole antibodies (aGD(2)) or their corresponding Fab' fragments were covalently coupled to Stealth immunoliposomes (aGD(2)-SIL or Fab'-SIL), and their binding to GD(2)-positive NB cells was measured. Cytotoxic effects of immunoliposomes loaded with doxorubicin (DXR) were determined. Radiolabelled immunoliposomes were used to evaluate pharmacokinetics (PK). The effectiveness of different liposomal formulations of DXR was tested against a metastatic model of human NB in nude mice. aGD(2)-SIL and Fab'-SIL showed concentration-dependent specific binding and uptake by GD(2)-positive NB cells. DXR entrapped in aGD(2)-SIL or Fab'-SIL (aGD(2)-SIL[DXR], Fab'-SIL[DXR]) showed higher cytotoxicities than nontargeted liposomes (SL[DXR]). DXR-loaded Fab'-SIL (Fab'-SIL[DXR]) also showed specific binding, uptake, and cytotoxic effects on several GD(2)-positive NB cells in vitro. PK studies showed that Fab'-SIL had long-circulating profiles in blood compared with aGD(2)-SIL, with the PK profile for Fab'-SIL being almost identical to that obtained with nontargeted Stealth liposomes. In vivo, long-term survivors were obtained in mice treated with Fab'-SIL[DXR] but not in untreated animals, or those treated with free aGD(2) Fab', Fab'-SIL (no drug), free-DXR, or nontargeted Stealth liposomes[DXR] (no antibody; P < 0.0001). Immunoliposomes containing DXR prevented the establishment and growth of the tumor in all of the organs examined. In conclusion, Fab'-SIL[DXR] formulations led to the total inhibition of metastatic growth of human NB in a nude mouse metastatic model. This formulation should receive clinical evaluation as adjuvant therapy of NB.     

A novel strategy inducing autophagic cell death in Burkitt's lymphoma cells with anti-CD19-targeted liposomal rapamycin

Relapsed or refractory Burkitt''s lymphoma often has a poor prognosis in spite of intensive chemotherapy that induces apoptotic and/or necrotic death of lymphoma cells. Rapamycin (Rap) brings about autophagy, and could be another treatment. Further, anti-CD19-targeted liposomal delivery may enable Rap to kill lymphoma cells specifically. Rap was encapsulated by anionic liposome and conjugated with anti-CD19 antibody (CD19-GL-Rap) or anti-CD2 antibody (CD2-GL-Rap) as a control. A fluorescent probe Cy5.5 was also liposomized in the same way (CD19 or CD2-GL-Cy5.5) to examine the efficacy of anti-CD19-targeted liposomal delivery into CD19-positive Burkitt''s lymphoma cell line, SKW6.4. CD19-GL-Cy5.5 was more effectively uptaken into SKW6.4 cells than CD2-GL-Cy5.5 in vitro. When the cells were inoculated subcutaneously into nonobese diabetic/severe combined immunodeficiency mice, intravenously administered CD19-GL-Cy5.5 made the subcutaneous tumor fluorescent, while CD2-GL-Cy5.5 did not. Further, CD19-GL-Rap had a greater cytocidal effect on not only SKW6.4 cells but also Burkitt''s lymphoma cells derived from patients than CD2-GL-Rap in vitro. The specific toxicity of CD19-GL-Rap was cancelled by neutralizing anti-CD19 antibody. The survival period of mice treated with intravenous CD19-GL-Rap was significantly longer than that of mice treated with CD2-GL-Rap after intraperitoneal inoculation of SKW6.4 cells. Anti-CD19-targeted liposomal Rap could be a promising lymphoma cell-specific treatment inducing autophagic cell death.     

Development of Fab' fragments of anti-GD(2) immunoliposomes entrapping doxorubicin for experimental therapy of human neuroblastoma

Neuroblastoma (NB) is the most common extra-cranial solid tumor in children. Since intensive therapeutic intervention does not prolong the overall disease-free survival rate for this tumor, novel therapeutic strategies are required. NB tumor, but not normal tissues, over-express the disialoganglioside (GD(2)) at the cell surface. In this study we developed a novel immunoliposomal formulation by covalently coupled Fab' fragments of the monoclonal antibody anti-GD(2) to Stealth liposomes (Fab'-SIL). In vitro experiments showed specific, competitive binding to, and uptake by various NB cell lines. Moreover, doxorubicin-loaded immunoliposomes (Fab'-SIL[DXR]) presented increased selectivity and efficacy in inhibiting NB cell proliferation compared to free drug and non-targeted liposomes (SL[DXR]). The in vivo cytotoxic effectiveness of different liposomal formulations encapsulating DXR was tested against an experimental metastatic model of human NB in nude mice. Long term survivors were obtained in mice treated with Fab'-SIL[DXR], but not in untreated animals or those treated with free anti-GD(2) Fab' fragments, Fab'-SIL (no drug), free-DXR or SL[DXR] (P<0.0001). Fab'-SIL[DXR] prevented the establishment and the metastatic growth of the tumor cells in all organs examined. In conclusion, Fab'-SIL[DXR] formulations should receive clinical evaluation as adjuvant therapy of neuroblastoma.     

A novel Raji-Burkitt's lymphoma model for preclinical and mechanistic evaluation of CD52-targeted immunotherapeutic agents.

PURPOSE: To date, efforts to study CD52-targeted therapies, such as alemtuzumab, have been limited due to the lack of stable CD52 expressing transformed B-cell lines and animal models. We describe generation and utilization of cell lines that stably express CD52 both in vitro and in vivo. EXPERIMENTAL DESIGN: By limiting dilution, we have established several clones of Raji-Burkitt's lymphoma cell line that express surface CD52. Immunophenotype and cytogenetic characterization of these clones was done. In vivo usefulness of the CD52(high) cell line to evaluate the therapeutic efficacy of CD52-directed antibody was investigated using a SCID mouse xenograft model. RESULTS: Stable expression of CD52 was confirmed in cells cultured in vitro up to 52 weeks of continuous growth. The functional integrity of the expressed CD52 molecule was shown using alemtuzumab, which induced cytotoxic effects in vitro in the CD52(high) but not the CD52(low) clone. Compared with control antibody, alemtuzumab treatment in CD52(high) inoculated mice resulted in significantly increased median survival. Comparable levels of CD52-targeted direct cytotoxicity, complement-dependent cytotoxicity, and antibody-dependent cytotoxicity and anti-CD52 immunoliposome-mediated delivery of synthetic oligodeoxyribo nucleotides in CD52(high) clone and primary B-chronic lymphocytic leukemia cells implicated potential in vivo application of this model for evaluation of CD52-targeted antibody and immunoliposomes encapsulating therapeutic agents. CONCLUSIONS: These results show the in vitro utility of the cloned Raji cell lines that stably express high levels CD52. The disseminated leukemia-lymphoma mouse model described herein using these stable cell lines can serve as an excellent system for in vivo therapeutic and mechanistic evaluation of existing and novel antibodies directed against CD52 molecule.     

A bispecific recombinant immunotoxin, DT2219, targeting human CD19 and CD22 receptors in a mouse xenograft model of B-cell leukemia/lymphoma.

A novel bispecific single-chain fusion protein, DT2219, was assembled consisting of the catalytic and translocation domains of diphtheria toxin (DT(390)) fused to two repeating sFv subunits recognizing CD19 and CD22 and expressed in Escherichia coli. Problems with yield, purity, and aggregation in the refolding step were solved by incorporating a segment of human muscle aldolase and by using a sodium N-lauroyl-sarcosine detergent-based refolding procedure. Problems with reduced efficacy were addressed by combining the anti-CD19 and anti-CD22 on the same single-chain molecule. DT2219 had greater anticancer activity than monomeric or bivalent immunotoxins made with anti-CD19 and anti-CD22 sFv alone and it showed a higher level of binding to patient leukemia cells and to CD19(+)CD22(+) Daudi or Raji cells than did anti-CD19 and anti-CD22 parental monoclonal antibodies. The resulting DT2219, mutated to enhance its avidity, was cytotoxic to Daudi cells in vitro (IC(50) = 0.3 nmol/L). In vivo, DT2219 was effective in a flank tumor therapy model in which it significantly inhibited tumor growth (P < 0.05) and in a systemic model in which it significantly prolonged survival of severe combined immunodeficient mice with established Daudi (P < 0.008) compared with controls. DT2219 has broader reactivity in recognizing B-cell malignancies, has more killing power, and requires less toxin than using individual immunotoxin, which warrants further investigation as a new drug for treating B leukemia/lymphoma.     

Efficacy of immunoliposomes on cancer models in a cell-surface-antigen-density-dependent manner

We have recently established a cancer-reactive human monoclonal antibody, GAH, with a positive ratio of over 90% against stomach cancer. GAH was formulated as polyethyleneglycol (PEG)-modified immunoliposomal doxorubicin (DXR) (ILD) and its efficacy was examined against gastrointestinal human cancers. In in vitro studies, a comparison of ILD with PEG-modified liposomal DXR (LD) demonstrated that ILD had dose-dependent cytotoxicity for GAH-reactive B37 cancer cells, but not LD. In concordance with this result, microscopic observations showed that ILD was bound to and GAH-dependently internalised by B37 cells. In in vivo studies, ILD exhibited significantly greater antitumour activity on cancer xenograft models than LD or free DXR. The relation between efficacy and antigen density was examined on 10 xenograft models bearing cancer cells with varying GAH reactivity. Immunoliposomal doxorubicin therapeutic activity correlated with the antigen density, with a minimum number being required. Also, ILD revealed strong antitumour activity on cancers with low sensitivity to DXR or LD, suggesting that ILD overcame the DXR resistance of antigen-positive cancer cells. Thus, these results show that GAH endows liposomes with targeting activity, resulting in strong efficacy against gastrointestinal cancers.     

Potent and specific antitumor efficacy of CMC-544, a CD22-targeted immunoconjugate of calicheamicin, against systemically disseminated B-cell lymphoma.

PURPOSE: CMC-544 is a CD22-targeted immunoconjugate of calicheamicin and exerts a potent cytotoxic effect against CD22+ B-cell lymphoma. This study evaluated antitumor efficacy of CMC-544 against systemically disseminated B-cell lymphoma. EXPERIMENTAL DESIGN: Scid mice received i.v. injections of CD22+ Ramos B-cell lymphoma cells for their systemic dissemination. CMC-544, G5/44, CD33-targeted CMA-676 (control conjugate) or rituximab were given i.p. 3, 9, 15, or 21 days after B-cell lymphoma dissemination. Diseased mice were monitored daily for hind-limb paralysis and death. Histopathological examination of CMC-544-treated and vehicle-treated diseased mice was also performed. RESULTS: Mice with disseminated B-cell lymphoma developed hind-limb paralysis within 35 days. When given up to 15 days after B-cell lymphoma dissemination, CMC-544 extended survival of the diseased mice to >100 days, and these mice were considered cured. CMC-544 was efficacious when given during both the early initiation phase and the late established phase of the disease. A single dose of CMC-544 was effective in delaying the occurrence of hind-limb paralysis. In contrast, neither CMA-676 nor unconjugated G5/44 was effective. Rituximab was effective when given early in the disease process but not when the disease was established. Histopathological analysis revealed B-cell lymphoma infiltration in brain, spinal cord, bone marrow, and kidney in vehicle-treated but not in CMC-544-treated diseased mice. Consistent with its efficacy against the disseminated B-cell lymphoma, CMC-544 also caused regression of established Ramos B-cell lymphoma xenografts in scid mice. CONCLUSIONS: CMC-544 confers strong therapeutic activity against systemic disseminated B-cell lymphoma and protects mice from hind-limb paralysis and death. These results support clinical evaluation of CMC-544 in the treatment of CD22+ lymphoid malignancies.     

Sterically stabilized anti-idiotype immunoliposomes improve the therapeutic efficacy of doxorubicin in a murine B-cell lymphoma model

A liposome containing diverse synthetic lipid derivatives of polyethylene glycol (PEG) results in smaller distribution volume and longer circulation time in blood and, thus, may improve drug targeting. The characteristics and therapeutic efficacy of immunoliposomes with similar liposomal formulation have never been studied in lymphoma models. We have developed immunoliposomes conjugated with S5A8 monoclonal antibody, an anti-idiotype antibody to 38C13 murine B-cell lymphoma, and loaded them with doxorubicin using an ammonium sulfate gradient. Purified antibodies were covalently coupled to the termini of PEG on the surface of small unilamellar liposomes. Cell binding and internalization ability of these immunoliposomes were estimated by a fluorescence assay using a pH-sensitive fluorescent dye (HPTS). The in vitro cytotoxicity of doxorubicin encapsulated in immunoliposomes was greater for idiotype-positive 38C13 cells than for the idiotype-negative variant of this cell line. In syngeneic C3H/HeN mice, doxorubicin encapsulated in immunoliposomes exhibited a long circulation time and was more effective at prolonging survival of mice bearing 38C13 tumor than non-targeted liposomal doxorubicin or free doxorubicin plus empty immunoliposomes. Our results demonstrate the superiority of targeted therapy with these immunoliposomes and its potential in lymphoma treatment.     

Cure of Burkitt's lymphoma in severe combined immunodeficiency mice by T cells, tetravalent CD3 x CD19 tandem diabody, and CD28 costimulation

To increase the valency, stability, and therapeutic potential of bispecific antibodies, we have constructed a tetravalent tandem diabody (Tandab) that is specific to both human CD3 (T-cell antigen) and CD19 (B-cell marker; S. M. Kipriyanov et al., J. Mol. Biol., 293: 41-56, 1999). It was generated by the functional dimerization of a single chain molecule that contained four antibody variable domains (V(H) and V(L)) in an orientation that prevented intramolecular pairing. Compared with a previously constructed heterodimeric CD3 x CD19 diabody, the Tandab exhibited a higher apparent affinity to both CD3+ and CD19+ cells and longer blood retention when injected into mice. Biodistribution studies in mice bearing Burkitt's lymphoma xenografts demonstrated specific accumulation of the radioiodinated Tandab in a tumor site with tumor-to-blood ratios of 1.5, 8.1, and 13.3 at 3, 18, and 24 h, respectively. Treatment of severe combined immunodeficiency mice bearing established Burkitt's lymphoma (5 mm in diameter) with human peripheral blood lymphocytes, Tandab, and anti-CD28 MAbs resulted in the complete elimination of tumors in all of the animals within 10 days. In contrast, mice receiving human peripheral blood lymphocytes in combination with either the diabody alone or the diabody plus anti-CD28 MAbs showed only partial tumor regression. These data demonstrate that the CD3 x CD19 Tandab may be a promising tool for the immunotherapy of human B-cell leukemias and lymphomas.     

Anti-CD19-targeted liposomal doxorubicin improves the therapeutic efficacy in murine B-cell lymphoma and ameliorates the toxicity of liposomes with varying drug release rates.

Some formulations of liposomal doxorubicin with intermediate rates of drug release have shown increased levels of toxicity in mice. Because antibody-mediated targeting of liposomal drugs influences the pharmacokinetics, mechanism of uptake, and selectivity of the associated drugs, we hypothesized that anti-CD19-mediated targeting of liposomal doxorubicin might moderate the toxicity of the problem formulations. Phosphatidylcholine/cholesterol liposomal formulations of doxorubicin having faster, intermediate, and slower drug release rates were prepared by altering the fatty acyl chain length or degree of saturation of the phosphatidylcholine component. Pharmacokinetic and biodistribution studies and in vivo drug release rates were determined in mice using liposomes dual labeled with [3H]cholesteryl hexadecylether and [14C]doxorubicin. Therapeutic studies were done in xenograft models of human B lymphoma (Namalwa cells). The rate of clearance of the liposomal lipid was similar for all formulations (average t1/2, 18 hours), but the rate of clearance of doxorubicin was dependent on the release rate of the formulation (t1/2, 2-315 hours). Liposomes with the slowest drug release rates showed no toxicity and exhibited therapeutic activity that was superior to the other formulations when targeted with anti-CD19; liposomes with the most rapid drug release rates also showed no toxicity but showed little therapeutic effect even when targeted. Liposomes with intermediate drug release rates exhibited varying degrees of toxicity. The toxicities could be reduced and even overcome by targeting with anti-CD19 antibodies. For these formulations, therapeutic effects were intermediate between those found for liposomes with the fastest and slowest drug release rates.     

Antitumor efficacy of a combination of CMC-544 (inotuzumab ozogamicin), a CD22-targeted cytotoxic immunoconjugate of calicheamicin, and rituximab against non-Hodgkin's B-cell lymphoma.

PURPOSE: CMC-544 is a CD22-targeted cytotoxic immunoconjugate, currently being evaluated in B-cell non-Hodgkin's lymphoma (B-NHL) patients. Rituximab is a CD20-targeted antibody commonly used in B-NHL therapy. Here, we describe antitumor efficacy of a combination of CMC-544 and rituximab against B-cell lymphoma (BCL) in preclinical models. EXPERIMENTAL DESIGN: BCLs were cultured in vitro with CMC-544, rituximab, or their combination. BCLs were injected either s.c. or i.v. to establish localized s.c. BCL in nude mice or disseminated BCL in severe combined immunodeficient mice, respectively. I.p. treatment with CMC-544 or rituximab was initiated at various times either alone or in combination and its effect on s.c. BCL growth or survival of mice with disseminated BCL was monitored. RESULTS: In vitro growth-inhibitory activity of CMC-544 combined with rituximab was additive. Rituximab but not CMC-544 exhibited effector functions, such as antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. Rituximab was less effective in inhibiting growth of established BCL xenografts than developing xenografts. In contrast, CMC-544 was equally effective against both developing and established BCL xenografts. Although CMC-544 and rituximab individually caused partial inhibition of the growth of BCL xenografts at suboptimal doses examined, their combination suppressed xenograft growth by >90%. In a disseminated BCL model, 60% of CMC-544-treated mice and 20% of rituximab-treated mice survived for 125 days. In contrast, 90% of mice treated with the combination of CMC-544 and rituximab survived for longer than 125 days. CONCLUSION: The demonstration of superior antitumor activity of a combination of CMC-544 and rituximab described here provides the preclinical basis for its clinical evaluation as a treatment option for B-NHL.     

Targeted liposomal c-myc antisense oligodeoxynucleotides induce apoptosis and inhibit tumor growth and metastases in human melanoma models.

PURPOSE: Melanoma is a highly malignant and increasingly common tumor. Because the cure rate of metastatic melanoma by conventional treatment is very low, new therapeutic approaches are needed. We previously reported that coated cationic liposomes (CCL) targeted with a monoclonal antibody against the disialoganglioside (GD(2)) and containing c-myb antisense oligodeoxynucleotides (asODNs) resulted in a selective inhibition of the proliferation of GD(2)-positive neuroblastoma cells in vitro. EXPERIMENTAL DESIGN: Here, we tested the in vivo antitumor effects of this novel antisense liposomal formulation by targeting the c-myc oncogene on melanoma, a neuroectodermal tumor sharing with neuroblastoma the expression of GD(2). RESULTS: Our methods produced GD(2)-targeted liposomes that stably entrapped 90% of added c-myc asODNs. These liposomes showed a selective binding for GD(2)-positive melanoma cells in vitro. Melanoma cell proliferation was inhibited to a greater extent by GD(2)-targeted liposomes containing c-myc asODNs (aGD(2)-CCL-myc-as) than by nontargeted liposomes or free asODNs. The pharmacokinetic results obtained after i.v. injection of [(3)H]-myc-asODNs, free or encapsulated in nontargeted CCLs or GD(2)-targeted CCLs, showed that free c-myc-asODNs were rapidly cleared, with less than 10% of the injected dose remaining in blood at 30 min after injection. c-myc-asODNs encapsulated within either CCL or aGD(2)-CCL demonstrated a more favorable profile in blood, with about 20% of the injected dose of each preparation remaining in vivo at 24 h after injection. In an in vivo melanoma experimental metastatic model, aGD(2)-CCL-myc-as, at a total dose of only 10 mg of asODN per kilogram, significantly inhibited the development of microscopic metastases in the lung compared with animals treated with myc-asODNs, free or entrapped in nontargeted liposomes, or aGD(2)-CCL encapsulating scrambled asODNs (P < 0.01). Moreover, mice bearing established s.c. human melanoma xenografts treated with aGD(2)-CCL-myc-as exhibited significantly reduced tumor growth and increased survival (P < 0.01 versus control mice). The mechanism for the antitumor effects appears to be down-regulation of the expression of the c-myc protein and interruption of c-myc-mediated signaling: induction of p53 and inhibition of Bcl-2 proteins, leading to extensive tumor cell apoptosis. CONCLUSION: These results suggest that inhibition of c-myc proto-oncogene by GD(2)-targeted antisense therapy could provide an effective approach for the treatment of melanoma in an adjuvant setting.     

In vivo inhibition of human CD19-targeted effector T cells by natural T regulatory cells in a xenotransplant murine model of B cell malignancy

Human T cells genetically modified to express chimeric antigen receptors (CAR) specific to the B cell tumor antigen CD19 can successfully eradicate systemic human CD19(+) tumors in immunocompromised SCID (severe combined immunodeficient)-Beige mice. However, in the clinical setting, CD4(+) CD25(hi) T regulatory cells (Treg) present within the tumor microenvironment may be potent suppressors of tumor-targeted effector T cells. In order to assess the impact of Tregs on CAR-modified T cells in the SCID-Beige xenotransplant model, we isolated, genetically targeted and expanded natural T regulatory cells (nTreg). In vitro nTregs modified to express CD19-targeted CARs efficiently inhibited the proliferation of activated human T cells, as well as the capacity of CD19-targeted 19-28z(+) effector T cells to lyse CD19(+) Raji tumor cells. Intravenous infusion of CD19-targeted nTregs into SCID-Beige mice with systemic Raji tumors traffic to sites of tumor and recapitulate a clinically relevant hostile tumor microenvironment. Antitumor efficacy of subsequently infused 19-28z(+) effector T cells was fully abrogated as assessed by long-term survival of treated mice. Optimal suppression by genetically targeted nTregs was dependent on nTreg to effector T-cell ratios and in vivo nTreg activation. Prior infusion of cyclophosphamide in the setting of this nTreg-mediated hostile microenvironment was able to restore the antitumor activity of subsequently infused 19-28z(+) effector T cells through the eradication of tumor-targeted nTregs. These findings have significant implications for the design of future clinical trials utilizing CAR-based adoptive T-cell therapies of cancer.     

Therapy of B-cell lymphoma with anti-CD20 antibodies can result in the loss of CD20 antigen expression.

Rituximab is a chimeric antibody with human gamma-1 and kappa constant regions and murine variable regions. It recognizes the CD20 antigen, a pan B-cell marker. Therapeutic trials in patients with B-cell non-Hodgkin's lymphoma (NHL) have shown significant efficacy with a primary response rate of 50%, and a secondary response rate of 44% after repeat treatments in prior responders. The selection for proliferating tumor cells that no longer express CD20 may compromise repeated treatment. We have identified a patient who developed a transformed NHL that lost CD20 protein expression after two courses of therapy with rituximab. In a pretreatment lymph node biopsy, 83% of B cells (as defined by CD19 and surface immunoglobulin) expressed surface CD20. A biopsy from the recurrent tumor after two courses of rituximab revealed a diffuse large cell NHL where 0% of B cells expressed CD20 with no evidence of bound rituximab. Cytoplasmic staining showed no CD20 protein. Sequencing of immunoglobulin heavy chain cDNA identified identical variable sequences in the initial and recurrent lymphomas, confirming the association between the two tumors. Literature and database review suggests that approximately 98% of diffuse large cell lymphomas express CD20, which suggests that these tumors rarely survive without CD20. This is the first identified case of loss of CD20 expression in a lymphoma that has relapsed after rituximab therapy, although several other cases have since been identified. Considering the significant number of patients treated with anti-CD20 antibodies, this may occur only rarely and is unlikely to preclude recurrent therapy with anti-CD20 antibodies in the majority of patients. However, because many patients have relapsed after anti-CD20 antibody therapy and have not been biopsied to identify clones with down-regulated CD20 antigen, we do not currently know the true frequency of this phenomenon. When possible, patients should undergo evaluation for CD20 expression before repeated courses of anti-CD20 therapy.     

Trastuzmab F(ab′)2修饰紫杉醇免疫脂质体对人大肠癌HT-29细胞的杀伤作用

 目的 构建Trastuzmab F(ab′)2修饰的紫杉醇免疫脂质体并测定其对体外生长的人大肠癌HT-29细胞的杀伤作用。方法 采用逆相蒸发法合成紫杉醇脂质体,使用胃蛋白酶在Tastuzumab抗体J链Fc段侧切断抗体以获得抗体F(ab′)2段,抗体交联法制备Trastuzmab F(ab′)2段修饰的紫杉醇免疫脂质体;透射电子显微镜观察免疫脂质体的形态及粒径分布;采用反相高效液相色谱（RP-HPLC）及荧光标记法测定免疫脂质体包封率和活性;MTT检测细胞的杀伤率。结果 成功构建了Trastuzmab F(ab′)2修饰的紫杉醇免疫脂质体;所得免疫脂质体平均粒径为210nm,粒径小于200nm者占91.37%;且其具有较高的包封率和稳定性;相同时间内,HT-29细胞对紫杉醇免疫脂质体的摄取要明显高于对照组;Trastuzmab F(ab′)2修饰的紫杉醇免疫脂质体对HT-29细胞的杀伤作用强于对照组(P<0.01),且该作用具有时间依赖性(P<0.01)。结论 成功构建Trastuzmab F(ab′)2修饰的紫杉醇免疫脂质体,其对体外生长的大肠癌细胞具有较强的杀伤作用。     

Radiotherapy of CD19 expressing Daudi tumors in nude mice with Yttrium-90-labeled anti-CD19 antibody

Studies were performed to determine the suitability of using two different anti-CD19 monoclonal antibodies to deliver the high energy beta-particle emitting isotope 90Y to B-cell lymphoma grown as flank tumors in athymic nude mice. The antibodies BU12 and HD37, both of the IgG1 subclass, recognize CD19, an internalizing B-lineage-specific membrane glycoprotein and member of the Ig supergene family. The antibodies were readily labeled with 90Y using the highly stable chelate, 1B4M-MX-DTPA. The radioimmunoconjugates selectively bound to the CD19 expressing B cell line Daudi, but not to CD19 negative control cells. Significantly more 90Y anti-CD19 bound to Daudi tumors growing in nude mice than did a control non-binding antibody (p = 0.001). The biodistribution data correlated with an anti-tumor effect. Anti-tumor activity was dose dependent and the best results were observed in mice receiving a single dose of approximately 300 uCi. The anti-CD19 antibody had significantly better anti-tumor activity as compared to a control 90Y-labeled antibody and most mice survived over 119 days with no evidence of tumor (p < 0.003). Histology studies showed no significant injury to the kidney, liver, or small intestine. Because radiolabeled anti-CD19 antibody can be used to deliver radiation selectively to lymphohematopoietic tissue, these data support the use of 90Y anti-CD19 antibodies in treating B-cell malignancies.