Key role for neutrophils in radiation-induced antitumor immune responses: Potentiation with G-CSF.

Radiation therapy (RT), a major modality for treating localized tumors, can induce tumor regression outside the radiation field through an abscopal effect that is thought to involve the immune system. Our studies were designed to understand the early immunological effects of RT in the tumor microenvironment using several syngeneic mouse tumor models. We observed that RT induced sterile inflammation with a rapid and transient infiltration of CD11b+Gr-1high+ neutrophils into the tumors. RT-recruited tumor-associated neutrophils (RT-Ns) exhibited an increased production of reactive oxygen species and induced apoptosis of tumor cells. Tumor infiltration of RT-Ns resulted in sterile inflammation and, eventually, the activation of tumor-specific cytotoxic T cells, their recruitment into the tumor site, and tumor regression. Finally, the concurrent administration of granulocyte colony-stimulating factor (G-CSF) enhanced RT-mediated antitumor activity by activating RT-Ns. Our results suggest that the combination of RT and G-CSF should be further evaluated in preclinical and clinical settings.

Identification of Human Anti-HIV gp160 Monoclonal Antibodies That Make Effective Immunotoxins.

The envelope (Env) glycoprotein of HIV is the only intact viral protein expressed on the surface of both virions and infected cells. Env is the target of neutralizing antibodies (Abs) and has been the subject of intense study in efforts to produce HIV vaccines. Therapeutic anti-Env Abs can also exert antiviral effects via Fc-mediated effector mechanisms or as cytotoxic immunoconjugates, such as immunotoxins (ITs). In the course of screening monoclonal antibodies (MAbs) for their ability to deliver cytotoxic agents to infected or Env-transfected cells, we noted disparities in their functional activities. Different MAbs showed diverse functions that did not correlate with each other. For example, MAbs against the external loop region of gp41 made the most effective ITs against infected cells but did not neutralize virus and bound only moderately to the same cells that they killed so effectively when they were used in ITs. There were also differences in IT-mediated killing among transfected and infected cell lines that were unrelated to the binding of the MAb to the target cells. Our studies of a well-characterized antigen demonstrate that MAbs against different epitopes have different functional activities and that the binding of one MAb can influence the interaction of other MAbs that bind elsewhere on the antigen. These results have implications for the use of MAbs and ITs to kill HIV-infected cells and eradicate persistent reservoirs of HIV infection. IMPORTANCE: There is increased interest in using antibodies to treat and cure HIV infection. Antibodies can neutralize free virus and kill cells already carrying the virus. The virus envelope (Env) is the only HIV protein expressed on the surfaces of virions and infected cells. In this study, we examined a panel of human anti-Env antibodies for their ability to deliver cell-killing toxins to HIV-infected cells and to perform other antiviral functions. The ability of an antibody to make an effective immunotoxin could not be predicted from its other functional characteristics, such as its neutralizing activity. Anti-HIV immunotoxins could be used to eliminate virus reservoirs that persist despite effective antiretroviral therapy.

Design and In Vivo Characterization of Immunoconjugates Targeting HIV gp160.

The envelope (Env) glycoprotein of HIV is expressed on the surface of productively infected cells and can be used as a target for cytotoxic immunoconjugates (ICs), in which cell-killing moieties, including toxins, drugs, or radionuclides, are chemically or genetically linked to monoclonal antibodies (MAbs) or other targeting ligands. Such ICs could be used to eliminate persistent reservoirs of HIV infection. We have found that MAbs which bind to the external loop of gp41, e.g., MAb 7B2, make highly effective ICs, particularly when used in combination with soluble CD4. We evaluated the toxicity, immunogenicity, and efficacy of the ICs targeted with 7B2 in mice and in simian-human immunodeficiency virus-infected macaques. In the macaques, we tested immunotoxins (ITs), consisting of protein toxins bound to the targeting agent. ITs were well tolerated and initially efficacious but were ultimately limited by their immunogenicity. In an effort to decrease immunogenicity, we tested different toxic moieties, including recombinant toxins, cytotoxic drugs, and tubulin inhibitors. ICs containing deglycosylated ricin A chain prepared from ricin toxin extracted from castor beans were the most effective in killing HIV-infected cells. Having identified immunogenicity as a major concern, we show that conjugation of IT to polyethylene glycol limits immunogenicity. These studies demonstrate that cytotoxic ICs can target virus-infected cells in vivo but also highlight potential problems to be addressed. IMPORTANCE: It is not yet possible to cure HIV infection. Even after years of fully effective antiviral therapy, a persistent reservoir of virus-infected cells remains. Here we propose that a targeted conjugate consisting of an anti-HIV antibody bound to a toxic moiety could function to kill the HIV-infected cells that constitute this reservoir. We tested this approach in HIV-infected cells grown in the lab and in animal infections. Our studies demonstrated that these immunoconjugates are effective both in vitro and in test animals. In particular, ITs constructed with the deglycosylated A chain prepared from native ricin were the most effective in killing cells, but their utility was blunted because they provoked immune reactions that interfered with the therapeutic effects. We then demonstrated that coating of the ITs with polyethylene glycol minimized the immunogenicity, as has been demonstrated with other protein therapies.

Thermostable ricin vaccine protects rhesus macaques against aerosolized ricin: Epitope-specific neutralizing antibodies correlate with protection.

Ricin toxin (RT) is the second most lethal toxin known; it has been designated by the CDC as a select agent. RT is made by the castor bean plant; an estimated 50,000 tons of RT are produced annually as a by-product of castor oil. RT has two subunits, a ribotoxic A chain (RTA) and galactose-binding B chain (RTB). RT binds to all mammalian cells and once internalized, a single RTA catalytically inactivates all of the ribosomes in a cell. Administered as an aerosol, RT causes rapid lung damage and fibrosis followed by death. There are no Food and Drug Administration-approved vaccines and treatments are only effective in the first few hours after exposure. We have developed a recombinant RTA vaccine that has two mutations V76M/Y80A (RiVax). The protein is expressed in Escherichia coli and is nontoxic and immunogenic in mice, rabbits, and humans. When vaccinated mice are challenged with injected, aerosolized, or orally administered (gavaged) RT, they are completely protected. We have now developed a thermostable, aluminum-adjuvant-containing formulation of RiVax and tested it in rhesus macaques. After three injections, the animals developed antibodies that completely protected them from a lethal dose of aerosolized RT. These antibodies neutralized RT and competed to varying degrees with a panel of neutralizing and nonneutralizing mouse monoclonal antibodies known to recognize specific epitopes on native RTA. The resulting antibody competition profile could represent an immunologic signature of protection. Importantly, the same signature was observed using sera from RiVax-immunized humans.

TLR-independent neutrophil-derived IFN-γ is important for host resistance to intracellular pathogens.

IFN-γ is a major cytokine that is critical for host resistance to a broad range of intracellular pathogens. Production of IFN-γ by natural killer and T cells is initiated by the recognition of pathogens by Toll-like receptors (TLRs). In an experimental model of toxoplasmosis, we have identified the presence of a nonlymphoid source of IFN-γ that was particularly evident in the absence of TLR-mediated recognition of Toxoplasma gondii. Genetically altered mice lacking all lymphoid cells due to deficiencies in Recombination Activating Gene 2 and IL-2Rγc genes also produced IFN-γ in response to the protozoan parasite. Flow-cytometry and morphological examinations of non-NK/non-T IFN-γ(+) cells identified neutrophils as the cell type capable of producing IFN-γ. Selective elimination of neutrophils in TLR11(-/-) mice infected with the parasite resulted in acute susceptibility similar to that observed in IFN-γ-deficient mice. Similarly, Salmonella typhimurium infection of TLR-deficient mice induces the appearance of IFN-γ(+) neutrophils. Thus, neutrophils are a crucial source for IFN-γ that is required for TLR-independent host protection against intracellular pathogens.

Ricin vaccine development.

In this chapter we discuss vaccines to protect against the highly toxic plant-derived toxin, ricin. Due to its prevalence, ease of use, and stability it has been used in sporadic incidents of espionage. There is also concern that it will be used as an agent of bioterrorism. As a result there has been a great deal of interest in developing a safe vaccine or antidote to protect humans, and in particular soldiers and first responders. Although multiple types of vaccines have been tested, at this time two recombinant vaccines are the leading candidates for the national vaccine stockpile. In terms of passive post-exposure protection, monoclonal neutralizing antibodies that passively protect animals are also under development. These vaccines and antibodies are discussed in the context of the toxicity and structure of ricin.

Engineering therapeutic monoclonal antibodies.

During last two decades, the chimerization and humanization of monoclonal antibodies (mAbs) have led to the approval of several for the treatment of cancer, autoimmune diseases, and transplant rejection. Additional approaches have been used to further improve their in vivo activity. These include combining them with other modalities such as chemotherapy and redesigning them for improved pharmacokinetics, effector function, and signaling activity. The latter has taken advantage of new insights emerging from an increased understanding of the cellular and molecular mechanisms that are involved in the interaction of immunoglobulin G with Fc receptors and complement as well as the negative signaling resulting from the hypercrosslinking of their target antigens. Hence, mAbs have been redesigned to include mutations in their Fc portions, thereby endowing them with enhanced or decreased effector functions and more desirable pharmacokinetic properties. Their valency has been increased to decrease their dissociation rate from cells and enhance their ability to induce apoptosis and cell cycle arrest. In this review we discuss these redesigned mAbs and current data concerning their evaluation both in vitro and in vivo.

Serum antibody profiling identifies vaccine-induced correlates of protection against aerosolized ricin toxin in rhesus macaques.

Inhalation of the biothreat agent, ricin toxin (RT), provokes a localized inflammatory response associated with pulmonary congestion, edema, neutrophil infiltration, and severe acute respiratory distress. The extreme toxicity of RT is the result of the toxin's B chain (RTB) promoting rapid uptake into alveolar macrophages and lung epithelial cells, coupled with the A chain's (RTA) potent ribosome-inactivating properties. We previously reported that intramuscular vaccination of rhesus macaques with a lyophilized, alum-adsorbed recombinant RTA subunit vaccine (RiVax®) was sufficient to confer protection against a lethal dose of aerosolized RT. That study implicated RT-specific serum IgG, toxin-neutralizing activity (TNA), and epitope-specific responses as being associated with immunity. However, it was not possible to define actual correlates of protection (COP) because all vaccinated animals survived the RT challenge. We addressed the issue of COP in the current study, by vaccinating groups of rhesus macaques with RiVax® following the previously determined protective regimen (100 µg on study days 0, 30 and 60) or one of two anticipated suboptimal regimens (100 µg on study days 30 and 60; 35 µg on study days 0, 30, and 60). Two unvaccinated animals served as controls. The animals were challenged with ~5 × LD50s of aerosolized RT on study day 110. We report that all vaccinated animals seroconverted prior to RT challenge, with the majority also having measurable TNA, although neither antibody levels nor TNA reached statistical significance with regard to a correlation with protection. By contrast, survival correlated with pre-challenge, epitope-specific serum IgG levels, derived from a competitive sandwich ELISA using a panel of toxin-neutralizing monoclonal antibodies directed against distinct epitopes on RiVax®. The identification of a species-neutral, competitive ELISA that correlates with vaccine-induced protection against RT in nonhuman represents an important advance in the development of medical countermeasures (MCM) against a persistent biothreat.

Structure of RiVax: a recombinant ricin vaccine.

RiVax is a recombinant protein that is currently under clinical development as part of a human vaccine to protect against ricin poisoning. RiVax includes ricin A-chain (RTA) residues 1-267 with two intentional amino-acid substitutions, V76M and Y80A, aimed at reducing toxicity. Here, the crystal structure of RiVax was solved to 2.1 Šresolution and it was shown that it is superposable with that of the ricin toxin A-chain from Ricinus communis with a root-mean-square deviation of 0.6 Šover 258 C(α) atoms. The RiVax structure is also compared with the recently determined structure of another potential ricin-vaccine immunogen, RTA 1-33/44-198 R48C/T77C. Finally, the locations and solvent-exposure of two toxin-neutralizing B-cell epitopes were examined and it was found that these epitopes are within or near regions predicted to be involved in catalysis. The results demonstrate the composition of the RiVax clinical material and will guide ongoing protein-engineering strategies to develop improved immunogens.

Chimeric, divalent and tetravalent anti-CD19 monoclonal antibodies with potent in vitro and in vivo antitumor activity against human B-cell lymphoma and pre-B acute lymphoblastic leukemia cell lines.

CD19 is an attractive therapeutic target for treating human B-cell tumors. In our study, chimeric (c) divalent (cHD37) and tetravalent (cHD37-DcVV) anti-CD19 monoclonal antibodies (MAbs) were constructed, expressed and evaluated for their binding to human 19-positive (CD19(+)) tumor cell lines. They were also tested for proapoptotic activity and the ability to mediate effector functions. The antitumor activity of these MAbs was further tested in mice xenografted with the CD19(+) Burkitt's lymphoma cell line, Daudi or the pre-B acute lymphoblastic leukemia (ALL) cell line, NALM-6. The cHD37 and cHD37-DcVV MAbs exhibited specific binding and comparable proapoptotic activity on CD19(+) tumor cell lines in vitro. In addition, the cHD37 and cHD37-DcVV MAbs were similar in their ability to mediate antibody-dependent cell-mediated phagocytosis (ADCP). However, the tetravalent cHD37-DcVV MAb bound more avidly, had a slower dissociation rate, and did not internalize as well. It also had enhanced antibody-dependent cellular cytotoxicity (ADCC) with human but not murine effector cells. The cHD37 and cHD37-DcVV MAbs exhibited comparable affinity for the human neonatal Fc receptor (FcRn) and similar pharmacokinetics (PKs) in mice. Moreover, all the HD37 constructs were similar in extending the survival of mice xenografted with Daudi or NALM-6 tumor cells. Therefore, the cHD37 and cHD37-DcVV MAbs have potent antitumor activity and should be further developed for use in humans. Although not evident in mice, due to its increased ability to mediate ADCC with human but not mouse effector cells, the cHD37-DcVV MAb should have superior therapeutic efficacy in humans.

The importance of cellular internalization of antibody-targeted carbon nanotubes in the photothermal ablation of breast cancer cells.

Single-walled carbon nanotubes (CNTs) convert absorbed near infrared (NIR) light into heat. The use of CNTs in the NIR-mediated photothermal ablation of tumor cells is attractive because the penetration of NIR light through normal tissues is optimal and the side effects are minimal. Targeted thermal ablation with minimal collateral damage can be achieved by using CNTs attached to tumor-specific monoclonal antibodies (MAbs). However, the role that the cellular internalization of CNTs plays in the subsequent sensitivity of the target cells to NIR-mediated photothermal ablation remains undefined. To address this issue, we used CNTs covalently coupled to an anti-Her2 or a control MAb and tested their ability to bind, internalize, and photothermally ablate Her2(+) but not Her2(-) breast cancer cell lines. Using flow cytometry, immunofluorescence, and confocal Raman microscopy, we observed the gradual time-dependent receptor-mediated endocytosis of anti-Her2-CNTs whereas a control MAb-CNT conjugate did not bind to the cells. Most importantly, the Her2(+) cells that internalized the MAb-CNTs were more sensitive to NIR-mediated photothermal damage than cells that could bind to, but not internalize the MAb-CNTs. These results suggest that both the targeting and internalization of MAb-CNTs might result in the most effective thermal ablation of tumor cells following their exposure to NIR light.

Thermal ablation of tumor cells with antibody-functionalized single-walled carbon nanotubes.

Single-walled carbon nanotubes (CNTs) emit heat when they absorb energy from near-infrared (NIR) light. Tissue is relatively transparent to NIR, which suggests that targeting CNTs to tumor cells, followed by noninvasive exposure to NIR light, will ablate tumors within the range of NIR. In this study, we demonstrate the specific binding of antibody-coupled CNTs to tumor cells in vitro, followed by their highly specific ablation with NIR light. Biotinylated polar lipids were used to prepare stable, biocompatible, noncytotoxic CNT dispersions that were then attached to one of two different neutralite avidin-derivatized mAbs directed against either human CD22 or CD25. CD22(+)CD25(-) Daudi cells bound only CNTs coupled to the anti-CD22 mAb; CD22(-)CD25(+) activated peripheral blood mononuclear cells bound only to the CNTs coupled to the anti-CD25 mAb. Most importantly, only the specifically targeted cells were killed after exposure to NIR light.

Mucoricin is a ricin-like toxin that is critical for the pathogenesis of mucormycosis.

Fungi of the order Mucorales cause mucormycosis, a lethal infection with an incompletely understood pathogenesis. We demonstrate that Mucorales fungi produce a toxin, which plays a central role in virulence. Polyclonal antibodies against this toxin inhibit its ability to damage human cells in vitro and prevent hypovolemic shock, organ necrosis and death in mice with mucormycosis. Inhibition of the toxin in Rhizopus delemar through RNA interference compromises the ability of the fungus to damage host cells and attenuates virulence in mice. This 17 kDa toxin has structural and functional features of the plant toxin ricin, including the ability to inhibit protein synthesis through its N-glycosylase activity, the existence of a motif that mediates vascular leak and a lectin sequence. Antibodies against the toxin inhibit R. delemar- or toxin-mediated vascular permeability in vitro and cross react with ricin. A monoclonal anti-ricin B chain antibody binds to the toxin and also inhibits its ability to cause vascular permeability. Therefore, we propose the name 'mucoricin' for this toxin. Not only is mucoricin important in the pathogenesis of mucormycosis but our data suggest that a ricin-like toxin is produced by organisms beyond the plant and bacterial kingdoms. Importantly, mucoricin should be a promising therapeutic target.

Endpoint and epitope-specific antibody responses as correlates of vaccine-mediated protection of mice against ricin toxin.

The successful licensure of vaccines for biodefense is contingent upon the availability of well-established correlates of protection (CoP) in at least two animal species that can be applied to humans, without the need to assess efficacy in the clinic. In this report we describe a multivariate model that combines pre-challenge serum antibody endpoint titers (EPT) and values derived from an epitope profiling immune-competition capture (EPICC) assay as a predictor in mice of vaccine-mediated immunity against ricin toxin (RT), a Category B biothreat. EPICC is a modified competition ELISA in which serum samples from vaccinated mice were assessed for their ability to inhibit the capture of soluble, biotinylated (b)-RT by a panel of immobilized monoclonal antibodies (mAbs) directed against four immunodominant toxin-neutralizing regions on the enzymatic A chain (RTA) of RT. In a test cohort of mice (n = 40) vaccinated with suboptimal doses of the RTA subunit vaccine, RiVax®, we identified two mAbs, PB10 and SyH7, which had EPICC inhibition values in pre-challenge serum samples that correlated with survival following a challenge with 5 × LD50 of RT administered by intraperitoneal (IP) injection. Analysis of a larger cohort of mice (n = 645) revealed that a multivariate model combining endpoint titers and EPICC values for PB10 and SyH7 as predictive variables had significantly higher statistical power than any one of the independent variables alone. Establishing the correlates of vaccine-mediated protection in mice represents an important steppingstone in the development of RiVax® as a medical countermeasure under the United States Food and Drug Administration's "Animal Rule."

Specific thermal ablation of tumor cells using single-walled carbon nanotubes targeted by covalently-coupled monoclonal antibodies.

CD22 is broadly expressed on human B cell lymphomas. Monoclonal anti-CD22 antibodies alone, or coupled to toxins, have been used to selectively target these tumors both in SCID mice with xenografted human lymphoma cell lines and in patients with B cell lymphomas. Single-walled carbon nanotubes (CNTs) attached to antibodies or peptides represent another approach to targeting cancer cells. CNTs convert absorbed near-infrared (NIR) light to heat, which can thermally ablate cells that have bound the CNTs. We have previously demonstrated that monoclonal antibodies (MAbs) noncovalently coupled to CNTs can specifically target and kill cells in vitro. Here, we describe the preparation of conjugates in which the MAbs are covalently conjugated to the CNTs. The specificity of both the binding and NIR-mediated killing of the tumor cells by the MAb-CNTs is demonstrated by using CD22+CD25- Daudi cells, CD22-CD25+ phytohemagglutinin-activated normal human peripheral blood mononuclear cells, and CNTs covalently modified with either anti-CD22 or anti-CD25. We further demonstrate that the stability and specificity of the MAb-CNT conjugates are preserved following incubation in either sodium dodecyl sulfate or mouse serum, indicating that they should be stable for in vivo use.

A phase 1 study of Combotox in pediatric patients with refractory B-lineage acute lymphoblastic leukemia.

BACKGROUND: Acute lymphoblastic leukemia (ALL) is the most common cancer in children. Combotox is a 1:1 mixture of RFB4-dgA and HD37-dgA which are immunotoxins that target the CD22 and CD19 antigens, respectively. Combotox has different toxicities and targets than chemotherapy and is, thus, a new candidate for the treatment of patients with relapsed ALL. Preclinical data have demonstrated which Combotox is effective in killing pre-B-ALL cell lines and cells from patients with pre-B ALL. METHODS: We designed and conducted a Phase 1 dose-escalation study using Combotox in children with refractory or relapsed B-lineage-ALL. Seventeen patients aged 1 to 16 years were enrolled in this multi-institution study. They were treated at 4-dose levels: 2 mg/m2, 4 mg/m2, 5 mg/m2, and 6 mg/m2. RESULTS: The maximum tolerated dose was 5 mg/m2 and graft versus host disease defined the maximum tolerated dose. Three patients experienced complete remission. Six additional patients experienced a decrease of >95% in their peripheral blood blast counts, and 1 patient experienced a decrease of 75%. CONCLUSIONS: Combotox can be safely administered to children with refractory leukemia. It has clinically important anticancer activity as a single agent. The recommended dose for future studies is 5 mg/m2/dose.

Rescue of rhesus macaques from the lethality of aerosolized ricin toxin.

Ricin toxin (RT) ranks at the top of the list of bioweapons of concern to civilian and military personnel alike, due to its high potential for morbidity and mortality after inhalation. In nonhuman primates, aerosolized ricin triggers severe acute respiratory distress characterized by perivascular and alveolar edema, neutrophilic infiltration, and severe necrotizing bronchiolitis and alveolitis. There are currently no approved countermeasures for ricin intoxication. Here, we report the therapeutic potential of a humanized mAb against an immunodominant epitope on ricin's enzymatic A chain (RTA). Rhesus macaques that received i.v. huPB10 4 hours after a lethal dose of ricin aerosol exposure survived toxin challenge, whereas control animals succumbed to ricin intoxication within 30 hours. Antibody intervention at 12 hours resulted in the survival of 1 of 5 monkeys. Changes in proinflammatory cytokine, chemokine, and growth factor profiles in bronchial alveolar lavage fluids before and after toxin challenge successfully clustered animals by treatment group and survival, indicating a relationship between local tissue damage and experimental outcome. This study represents the first demonstration, to our knowledge, in nonhuman primates that the lethal effects of inhalational ricin exposure can be negated by a drug candidate, and it opens up a path forward for product development.

The antitumor activity of an anti-CD54 antibody in SCID mice xenografted with human breast, prostate, non-small cell lung, and pancreatic tumor cell lines.

We have previously described the development and testing of a monoclonal anti-human CD54 antibody (UV3) in SCID mice xenografted with human multiple myeloma, lymphoma, and melanoma cell lines. In all 3 cases, UV3 was highly effective at slowing the growth of tumors and/or prolonging survival. Since CD54 (ICAM-1) is up-regulated on many different types of cancer cells, we have now investigated the anti-tumor activity of UV3 in several other CD54(+) epithelial tumors. A panel of 16 human breast, prostate, non-small cell (NSC) lung, and pancreatic tumor cell lines was examined for reactivity with UV3, and 13 were positive. A representative CD54(+) cell line from each cancer was grown subcutaneously in SCID mice. Once the tumors were established, UV3 was administered using different dose regimens. UV3 slowed the growth of all 4 tumors, although it was not curative. When UV3 or gemcitabine were administered to SCID mice xenografted with a NSC lung tumor cell line or a pancreatic tumor cell line, UV3 was as effective as the chemotherapy alone. When gemcitabine and UV3 were administered together, the best anti-tumor responses were observed. UV3 has been chimerized (cUV3) and both toxicology studies and clinical trials are planned to assess the safety and activity of cUV3 in patients with one or more of these tumors.

Generation of multidrug resistant lymphoma cell lines stably expressing P-glycoprotein.

The objective of this study was to generate new P-glycoprotein (P-gp)-expressing multidrug resistant (MDR) cell lines by drug selection. Since our previous studies have been carried out with cells infected with a P-gp-containing vector, it was important to confirm our findings in cells generated by drug selection. In this report, we describe three B-lymphoma cell lines which became drug-resistant by stepwise exposure to vincristine (VCR): Raji cells resistant to 18 nM VCR (R18V), Namalwa cells resistant to 21 nM VCR (N21V) and DHL-4 cells resistant to 12 nM VCR (DHL-4/12V). Cells overexpressed P-gp and continued to express CD19, CD20 and CD22, all of which are targets for monoclonal antibody (MAb) therapy. The P-gp pump in these new cells was functional as determined by the efflux of Rhodamine 123 and DIOC2, and the three cell lines were resistant to several chemotherapeutic drugs. We further determined that their P-gp phenotype was stable in xenografted SCID mice and that the tumors were also resistant to chemotherapy. We will now use these new MDR cells to determine whether monoclonal antibodies against CD19 and -20 can reverse P-gp, as we previously demonstrated using Namalwa cells infected with a human mdr1 gene-containing retrovirus.