Biomedical subjects

Margaret A Lindorfer

Publications and source records attributed to Margaret A Lindorfer.

11 recordsLinked to original sources

PubMed2006

Thrice-weekly low-dose rituximab decreases CD20 loss via shaving and promotes enhanced targeting in chronic lymphocytic leukemia.

Treatment of chronic lymphocytic leukemia (CLL) patients with standard dose infusion of rituximab (RTX), 375 mg/m2, induces clearance of malignant cells from peripheral blood after infusion of 30 mg of RTX. After completion of the full RTX infusion, substantial recrudescence of CLL cells occurs, and these cells have lost > 90% of CD20. To gain insight into mechanism(s) of CD20 loss, we investigated the hypothesis that thrice-weekly low-dose RTX (20 or 60 mg/m2) treatment for CLL over 4 wk would preserve CD20 and enhance leukemic cell clearance. During initial infusions in all 12 patients, the first 30 mg of RTX promoted clearance of > 75% leukemic cells. Four of six patients receiving 20 mg/m2 RTX retained > or = 50% CD20, and additional RTX infusions promoted further cell clearance. However, four of six patients receiving 60 mg/m2 had CD20 levels < 20% baseline 2 days after initial infusions, and additional RTX infusions were less effective, presumably due to epitope loss. Our results suggest that when a threshold RTX dose is exceeded, recrudesced RTX-opsonized cells are not cleared, due to saturation of the mononuclear phagocytic system, but instead are shaved of RTX-CD20 complexes by acceptor cells. Thrice-weekly low-dose RTX may promote enhanced clearance of circulating CLL cells by preserving CD20.

Administration, Oral↗

PubMed2006

Quantitative analysis of protein co-localization on B cells opsonized with rituximab and complement using the ImageStream multispectral imaging flow cytometer.

Binding of the chimeric, humanized anti-CD20 mAb Rituximab (RTX) to B lymphocytes activates complement and promotes covalent deposition of C3 fragments (C3b/iC3b) on cells. Previous fluorescence microscopy studies, based on examination of B cell lines and of blood samples from RTX-treated CLL patients, suggest that C3b/iC3b is closely associated with cell-bound RTX. We examined Raji cells opsonized with serum and RTX with the ImageStream imaging flow cytometer. Cells were stained with fluorescently-labeled RTX and mAbs specific for C3b/iC3b fragments or for human IgG, and then imaged using the ImageStream cytometer and analyzed with an algorithm (Similarity Bright Detail Score, SBDS) which tests for co-localization of fluorescent probes. SBDS, calculated on 10,000 cells, verified that the majority of deposited C3b/iC3b is co-localized with bound RTX. In contrast, when cells were first opsonized in serum alone, washed and then reacted with RTX, SBDS confirmed that RTX and C3b/iC3b are poorly co-localized, thus demonstrating that cell-bound RTX directs deposition of C3b. In addition, a sulfhydryl-specific probe, maleimide conjugated to AF488, exhibited substantial co-localization with an anti-C3b/iC3b mAb on Raji cells opsonized with RTX and serum, thus validating maleimide labeling as an alternative for detecting cell-bound C3b/iC3b. The digital imaging method described should have wide applicability for quantitative analysis of co-localization.

Algorithms↗

PubMed2006

Low doses of antigen coupled to anti-CR2 mAbs induce rapid and enduring IgG immune responses in mice and in cynomolgus monkeys.

The complement system and B cell complement receptor 2 (CR2), specific for C component C3dg, play important roles in both the innate and adaptive immune response. We used hapten and protein conjugates of anti-CR2 mAbs as models for C3dg-opsonized antigens and immune complexes to examine the handling of and immune response to these reagents in mice and in non-human primates (NHP). Mice immunized and boosted i.v. with only 100 ng of Alexa 488 rat anti-mouse CR1/2 mAb 7G6 had strong IgG immune responses to the Alexa 488 hapten and to rat IgG, compared to very weak immune responses in mice treated with a comparable isotype control; larger doses of Alexa 488 mAb 7G6 did not increase the immune response. A vaccine constructed by cross-linking anthrax protective antigen to mAb 7G6 proved to be effective at low doses in generating sufficiently high titer serum IgG antibodies to neutralize anthrax lethal toxin in vitro and to protect mice from i.v. challenge with anthrax lethal toxin. When biotinylated HB135, a mouse mAb specific for human CR2, was injected i.v. into NHP, the probe manifested the same initial marginal zone B cell binding and subsequent localization to follicular dendritic cells as we have previously reported for comparable experiments in mice. Moreover, i.v. immunization of NHP with 1 microg/kg of Alexa 488 mAb HB135 promoted an IgG immune response to the Alexa 488 hapten and to mouse IgG. Taken together, these results demonstrate the efficacy of using anti-CR2 mAbs as antigen carriers for i.v. immunization with small amounts of antigens without adjuvant.

Animals↗

PubMed2006

The shaving reaction: rituximab/CD20 complexes are removed from mantle cell lymphoma and chronic lymphocytic leukemia cells by THP-1 monocytes.

Clinical investigations have revealed that infusion of immunotherapeutic mAbs directed to normal or tumor cells can lead to loss of targeted epitopes, a phenomenon called antigenic modulation. Recently, we reported that rituximab treatment of chronic lymphocytic leukemia patients induced substantial loss of CD20 on B cells found in the circulation after rituximab infusion, when rituximab plasma concentrations were high. Such antigenic modulation can severely compromise therapeutic efficacy, and we postulated that B cells had been stripped (shaved) of the rituximab/CD20 complex by monocytes or macrophages in a reaction mediated by FcgammaR. We developed an in vitro model to replicate this in vivo shaving process, based on reacting rituximab-opsonized CD20(+) cells with acceptor THP-1 monocytes. After 45 min at 37 degrees C, rituximab and CD20 are removed from opsonized cells, and both are demonstrable on acceptor THP-1 cells. The reaction occurs equally well in the presence and absence of normal human serum, and monocytes isolated from peripheral blood also promote shaving of CD20 from rituximab-opsonized cells. Tests with inhibitors and use of F(ab')(2) of rituximab indicate transfer of rituximab/CD20 complexes to THP-1 cells is mediated by FcgammaR. Antigenic modulation described in previous reports may have been mediated by such shaving, and our findings may have profound implications for the use of mAbs in the immunotherapy of cancer.

Antibodies, Monoclonal↗

PubMed2005

Selective and efficient inhibition of the alternative pathway of complement by a mAb that recognizes C3b/iC3b.

The alternative pathway (AP) of the complement system plays an important role in tissue damage and inflammation associated with certain autoimmune diseases and with ischemia-reperfusion injury. Selective inhibition of the AP could prevent such pathologies while allowing the classical and lectin pathways of complement activation to continue to provide protection. Here we present data describing selective inhibition of the AP of complement by anti-C3b/iC3b monoclonal antibody (mAb) 3E7, and by a chimeric, "deimmunized" form of this mAb, H17, which contains the human IgG1 Fc region and was further modified by substitution of amino acids in order to remove T cell epitopes. Both mAbs block AP-mediated deposition of C3b onto zymosan or Sepharose 4B, and they also inhibit AP-promoted lysis of rabbit erythrocytes. MAbs 3E7 and H17 also successfully compete with both factors B and H for binding to C3b-opsonized substrates, and the ability of both mAbs to inhibit the AP is blocked by pre-incubation with two different sources of C3(H2O). Kinetic measurements demonstrate that mAb 3E7 effectively stops progression of C3b deposition after AP activation is initiated. Our results therefore suggest that these mAbs block activation of the AP by binding to both C3(H2O) and to C3b, and thus prevent binding and activation of factor B. Based on these and other observations, mAb H17 may find future use in therapeutic applications focused on selective inhibition of the AP.

Antibodies, Monoclonal↗

PubMed2004

Analyses of the in vivo trafficking of stoichiometric doses of an anti-complement receptor 1/2 monoclonal antibody infused intravenously in mice.

Complement plays a critical role in the immune response by opsonizing immune complexes (IC) and thymus-independent type 2 Ags with C3 breakdown product C3dg, a CR2-specific ligand. We used a C3dg-opsonized IC model, anti-CR1/2 mAb 7G6, to investigate how such substrates are processed. We used RIA, whole body imaging, flow cytometry, and fluorescence immunohistochemistry to examine the disposition of 0.1- to 2-microg quantities of mAb 7G6 infused i.v. into BALB/c mice. The mAb is rapidly taken up by the spleen and binds preferentially to marginal zone (MZ) B cells; within 24 h, the MZ B cells relocate and transfer mAb 7G6 to follicular dendritic cells (FDC). Transfer occurs coincident with loss of the extracellular portion of MZ B cell CR2, suggesting that the process may be mediated by proteolysis of CR2. Intravenous infusion of an FDC-specific mAb does not induce comparable splenic localization or cellular reorganization, emphasizing the importance of MZ B cells in intrasplenic trafficking of bound substrates. We propose the following mechanism: binding of C3dg-opsonized IC to noncognate MZ B cells promotes migration of these cells to the white pulp, followed by CR2 proteolysis, which allows transfer of the opsonized IC to FDC, thus facilitating presentation of intact Ags to cognate B cells.

Animals↗

PubMed2004

Rituximab infusion promotes rapid complement depletion and acute CD20 loss in chronic lymphocytic leukemia.

Complement plays an important role in the immunotherapeutic action of the anti-CD20 mAb rituximab, and therefore we investigated whether complement might be the limiting factor in rituximab therapy. Our in vitro studies indicate that at high cell densities, binding of rituximab to human CD20(+) cells leads to loss of complement activity and consumption of component C2. Infusion of rituximab in chronic lymphocytic leukemia patients also depletes complement; sera of treated patients have reduced capacity to C3b opsonize and kill CD20(+) cells unless supplemented with normal serum or component C2. Initiation of rituximab infusion in chronic lymphocytic leukemia patients leads to rapid clearance of CD20(+) cells. However, substantial numbers of B cells, with significantly reduced levels of CD20, return to the bloodstream immediately after rituximab infusion. In addition, a mAb specific for the Fc region of rituximab does not bind to these recirculating cells, suggesting that the rituximab-opsonized cells were temporarily sequestered by the mononuclear phagocytic system, and then released back into the circulation after the rituximab-CD20 complexes were removed by phagocytic cells. Western blots provide additional evidence for this escape mechanism that appears to occur as a consequence of CD20 loss. Treatment paradigms to prevent this escape, such as use of engineered or alternative anti-CD20 mAbs, may allow for more effective immunotherapy of chronic lymphocytic leukemia.

Antibodies, Monoclonal↗

PubMed2004

Staphylococcus aureus bound to complement receptor 1 on human erythrocytes by bispecific monoclonal antibodies is phagocytosed by acceptor macrophages.

Antibiotic resistant strains of Staphylococcus aureus pose a growing threat to public health, and immunotherapy offers potential modalities to combat antibiotic resistance. We prepared bispecific monoclonal antibody complexes (heteropolymers, HP), specific for the primate erythrocyte complement C3b receptor (CR1) and type 5 capsular polysaccharide of the T5 isolate of S. aureus. Fluorescence microscopy and flow cytometry revealed that HP promote binding of S. aureus to human erythrocytes. Incubation of erythrocyte-bound, HP-opsonized S. aureus with human monocyte/macrophages or mouse macrophage cell lines led to transfer, internalization and killing of bacteria by macrophages with little erythrocyte loss. This reaction is similar to the process in which C3b-opsonized substrates, bound to erythrocyte CR1 by immune adherence, are transferred to acceptor phagocytes. Our results provide the basis for development of an in vivo paradigm focused on immunotherapeutic approaches for treatment of infections due to antibiotic resistant bacteria.

Animals↗

PubMed2003

B cell complement receptor 2 transfer reaction.

The B cell C receptor specific for C3dg (CR2) shares a number of features with the primate E C receptor (CR1). Previously, we have demonstrated, both in vitro and in animal models, that immune complexes (IC) bound to primate E CR1, either via C opsonization or by means of bispecific mAb complexes, can be transferred to acceptor macrophages in a process that also removes CR1 from the E. We have now extended this paradigm, the transfer reaction, to include B cell CR2. We used both flow cytometry and fluorescence microscopy to demonstrate that IC bound to Raji cell CR2, either via C opsonization or through the use of an anti-CR2 mAb, are transferred to acceptor THP-1 cells. This reaction, which appears to require Fc recognition of IgG bound to Raji cell CR2, also leads to transfer of CR2. Additional support for the B cell transfer reaction is provided in a prototype study in a monkey model in which IC bound to B cell CR2 are localized to the spleen. These findings may have important implications with respect to defining the role of C in IC handling during the normal immune response.

Animals↗

PubMed2002

Concerted clearance of immune complexes bound to the human erythrocyte complement receptor: development of a heterologous mouse model.

Experiments in primates have demonstrated that immune complexes (IC) bound to erythrocytes (E) via complement receptor 1 (CR1) are cleared to the liver in a process which removes CR1, but otherwise spares the E. Human E are stabilized for >1 h in the circulation of the mouse if the terminal complement pathway is blocked, and we used this paradigm to examine clearance in a mouse model. Human E were opsonized with an anti-CR1 mAb cross-linked to dsDNA (antigen-based heteropolymer, AHP), and then incubated with systemic lupus erythematosus (SLE) plasmas containing IgG anti-dsDNA to form IC in situ. These IC stably bind to E CR1 in the complete absence of complement, thus allowing analysis in a model which does not require human C3b to facilitate E binding. Dual label experiments, based on RIA, flow cytometry and fluorescence microscopy, were employed to monitor separately E and IC. When opsonized E-IC were injected into A/J mice, >90% of the IC were rapidly removed from the E coincident with loss of CR1. The E remained in the circulation while IC were localized to the liver, mainly to Kupffer cells. Preliminary experiments in NZB/W mice, which spontaneously develop IgG anti-dsDNA, indicated that infusion of E-AHP led to rapid binding of murine IgG to the E-AHP, followed by removal of the nascent IC from E, and loss of CR1 in a concerted reaction. These studies provide additional evidence that E CR1 functions as a privileged site for IC clearance, and that the key step in clearance requires removal of CR1 from E to release bound IC for uptake by acceptor macrophages. This model can be extended to genetically altered mice to investigate the role of specific Fc gamma receptors as well as complement receptors in IC clearance.

Animals↗

PubMed2002

An anti-C3b(i) mAb enhances complement activation, C3b(i) deposition, and killing of CD20+ cells by rituximab.

We investigated deposition of the complement protein fragment C3b and its breakdown products (collectively designated as C3b(i)) on CD20-positive cells treated with rituximab (RTX) in the presence of normal human serum (NHS). Radioimmunoassay (RIA) demonstrates that about 500 000 C3b(i) molecules deposit per cell, and fluorescence microscopy reveals that C3b(i) colocalizes with bound RTX. Use of mAb 3E7, specific for C3b(i) bound to substrates, enhances C3b(i) deposition; > 1 million C3b(i) deposit when cells are incubated with NHS, RTX and mAb 3E7. Treatment of Raji cells in NHS plus RTX leads to robust cell killing (95%) after 24 to 48 hours, and mAb 3E7 significantly enhances RTX-mediated killing of Raji and DB cells. A cynomolgus monkey model based on intravenous infusion of RTX followed by mAb 3E7 demonstrated that RTX rapidly binds to B cells and promotes complement activation and C3b(i) deposition; fluorescence microscopy analyses revealed the same pattern of colocalization of C3b(i) on cell-bound RTX in vivo as observed in vitro. Preliminary in vitro studies with blood samples from patients with chronic lymphocytic leukemia lead to similar findings. These experiments suggest that complement plays a key role in the mechanism of action of RTX; moreover, the in vivo molecular form of RTX (and possibly other antitumor mAbs) in the circulation or in tissues may include C3b(i) molecules covalently bound to the therapeutic mAb, thus allowing it to interact with cells containing both Fc and complement receptors.

Animals↗