Comparison of the in vitro effects of the anti-CD20 antibodies rituximab and GA101 on chronic lymphocytic leukaemia cells

The effects of two CD20 antibodies, namely rituximab, the current standard for treatment of chronic lymphocytic leukaemia (CLL) in combination with chemotherapy, and GA101, a glyco-engineered type II antibody were compared on CLL cells ex vivo. Antibody-induced phosphatidylserine exposure was examined in isolated CLL cells. For a more comprehensive assessment of antibody-mediated cell killing including Fc-mediated mechanisms, B cell depletion from whole blood samples was monitored. Treatment with rituximab or GA101 reduced the average viability of isolated CLL cells by 6% or 11%, and the ratio of B to T cells in whole blood samples by 12% or 33%, respectively. Combination with GA101 enhanced the cytotoxicity of the chemotherapeutic agent chlorambucil on isolated CLL cells. CD20 surface expression on CLL cells correlated with GA101-induced B cell depletion, but not with direct cell death induction. Treatment of whole blood samples from CLL patients with a CpG-containing oligonucleotide increased CD20 expression on CLL cells and GA101-dependent B cell depletion. Despite the variable responses of individual CLL samples, the CLL cell depletion from whole blood by GA101 was consistently much stronger than by rituximab, which argues for clinical investigation of GA101 in CLL patients.     

Enhanced killing of human B-cell lymphoma targets by combined use of cytokine-induced killer cell (CIK) cultures and anti-CD20 antibodies

We have investigated combining adoptive immunotherapy with cytokine-induced killer (CIK) cells and anti-CD20 monoclonal antibodies (mAb) GA101 or rituximab to optimize B-cell non-Hodgkin lymphoma (B-NHL) therapy. CIK cultures alone demonstrated significant cytotoxic activity against B-NHL cell lines or freshly isolated samples in either an autologous or allogeneic combination. This natural cytotoxicity (NC) was mainly due to the predominating CD3(+)CD56(+) CIK population (40%-75%) present in the cultures. The addition of anti-CD20 mAb GA101 or rituximab further increased cytotoxicity by 35% and 15%, respectively. This enhancement was mainly due to antibody-dependent cytotoxicity (ADCC) mediated by the 1%-10% NK cells contaminating CIK cultures. The addition of human serum (HS) inhibited NK-cell activation induced by rituximab, but not activation induced by GA101.Overall lysis in presence of serum, even of a resistant B-NHL cell line, was significantly increased by 100 μg/mL of rituximab, but even more so by GA101, with respect to CIK cultures alone. This was due to the combined action of complement-mediated cytotoxicity (CDC), ADCC, and CIK-mediated NC. These data suggest that rituximab, and even more so GA101, could be used in vivo to enhance CIK therapeutic activity in B-NHL.     

γδ T-cell killing of primary follicular lymphoma cells is dramatically potentiated by GA101, a type II glycoengineered anti-CD20 monoclonal antibody

Background

Anti-CD20 monoclonal antibodies are major therapeutic agents for patients with follicular lymphoma and work through complement-mediated cytotoxicity and antibody-dependent cellular cytotoxicity. Optimization of antibody-dependent cellular cytotoxicity, in particular by amplifying its effectors, could further increase the efficacy of anti-CD20 monoclonal antibodies.

Design and Methods

We investigated the cytotoxic activity of Vγ9Vδ2 T cells against follicular lymphoma cells and whether this killing could be increased by promoting antibody-dependent cellular cytotoxicity with anti-CD20 monoclonal antibodies, in particular a type-II glycoengineered anti-CD20. Vγ9Vδ2 T cells were expanded in vitro in the presence of bromohydrin pyrophosphate (Phosphostim) and interleukin-2 and their ability to kill follicular lymphoma primary cells or cell lines was evaluated by flow cytometry cytotoxic T-lymphocyte assays in the presence or absence of three anti-CD20 monoclonal antibodies: the afucosylated GA101, the chimeric rituximab or the humanized ofatumumab. The ability of these cells to release perforin/granzyme and secrete interferon-γ when co-cultured with follicular lymphoma primary cells or cell lines in the presence or not of the three anti-CD20 monoclonal antibodies was also evaluated by CD107a staining and Elispot assays.

Results

Phosphostim and interleukin-2 expanded Vγ9Vδ2 T cells were cytotoxic to primary follicular lymphoma cells and their cytotoxic potential was dramatically increased by GA101, a type II glycoengineered anti-CD20 monoclonal antibody, and to a lesser extent, by rituximab and ofatumumab. The increased cytotoxicity was associated with increased secretion of perforin/granzyme and interferon-γ.

Conclusions

In-vitro expanded Vγ9Vδ2 T cells efficiently kill primary follicular lymphoma cells and express CD16; anti-CD20 monoclonal antibodies, in particular GA101, dramatically increase the cytotoxic activity of expanded Vγ9Vδ2 T cells. These preclinical results prompt the development of clinical trials using this antibody dependent cellular cytotoxicity property of Vγ9Vδ2 T cells and anti-CD20 monoclonal antibodies.     

Geldanamycin and herbimycin A induce apoptotic killing of B chronic lymphocytic leukemia cells and augment the cells' sensitivity to cytotoxic drugs

We studied the actions of geldanamycin (GA) and herbimycin A (HMA), inhibitors of the chaperone proteins Hsp90 and GRP94, on B chronic lymphocytic leukemia (CLL) cells in vitro. Both drugs induced apoptosis of the majority of CLL isolates studied. Whereas exposure to 4-hour pulses of 30 to 100 nM GA killed normal B lymphocytes and CLL cells with similar dose responses, T lymphocytes from healthy donors as well as those present in the CLL isolates were relatively resistant. GA, but not HMA, showed a modest cytoprotective effect toward CD34+ hematopoietic progenitors from normal bone marrow. The ability of bone marrow progenitors to form hematopoietic colonies was unaffected by pulse exposures to GA. Both GA and HMA synergized with chlorambucil and fludarabine in killing a subset of CLL isolates. GA- and HMA-induced apoptosis was preceded by the up-regulation of the stress-responsive chaperones Hsp70 and BiP. Both ansamycins also resulted in down-regulation of Akt protein kinase, a modulator of cell survival. The relative resistance of T lymphocytes and of CD34+ bone marrow progenitors to GA coupled with its ability to induce apoptosis following brief exposures and to synergize with cytotoxic drugs warrant further investigation of ansamycins as potential therapeutic agents in CLL.     

Fas-ligand (CD178) and TRAIL synergistically induce apoptosis of CD40-activated chronic lymphocytic leukemia B cells

Chronic lymphocytic leukemia (CLL) B cells become sensitive to Fas (CD95)-mediated apoptosis 3 to 5 days after CD40 ligation. However, CD4+ cytotoxic T lymphocytes (CTLs) can kill CLL B cells via a Fas-ligand (CD178)-dependent process within 24 hours after CD40 cross-linking, when ligation of CD95 alone is insufficient to induce apoptosis. In addition to CD95, CD40-activated CLL cells also express DR5, a receptor for tumor-necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) that is expressed by CD4+ CTL. In addition, CD40 ligation in vitro and in vivo induces CLL cells to express the proapoptotic protein, BH3 interacting domain death agonist (Bid), which can facilitate crosstalk between mitochondrial-dependent, apoptosis-inducing pathways and death receptors, such as death receptor 5 (DR5). To evaluate whether ligation of CD95 and/or DR5 can induce apoptosis of CD40-activated CLL cells, we generated artificial cytotoxic effector cells that express both human TRAIL and CD178 (Chinese hamster ovary [CHO]-CD178/TRAIL) or only TRAIL (CHO-TRAIL) or CD178 (CHO-CD178). CHO-CD178/TRAIL cells were significantly more effective in killing CD40-activated CLL cells than either CHO-TRAIL or CHO-CD178 and, unlike the latter, could kill CLL cells 24 hours after CD40 ligation. We conclude that CD40 ligation induces CLL cells to express the proapoptotic molecule Bid and the death receptors CD95 and DR5, the latter of which can act synergistically to induce caspase-dependent apoptosis of CD40-activated CLL B cells.     

Antibody-induced nonapoptotic cell death in human lymphoma and leukemia cells is mediated through a novel reactive oxygen species-dependent pathway

Monoclonal antibodies (mAbs) have revolutionized the treatment of B-cell malignancies. Although Fc-dependent mechanisms of mAb-mediated tumor clearance have been extensively studied, the ability of mAbs to directly evoke programmed cell death (PCD) in the target cell and the underlying mechanisms involved remain under-investigated. We recently demonstrated that certain mAbs (type II anti-CD20 and anti-HLA DR mAbs) potently evoked PCD through an actin-dependent, lysosome-mediated process. Here, we reveal that the induction of PCD by these mAbs, including the type II anti-CD20 mAb GA101 (obinutuzumab), directly correlates with their ability to produce reactive oxygen species (ROS) in human B-lymphoma cell lines and primary B-cell chronic lymphocytic leukemia cells. ROS scavengers abrogated mAb-induced PCD indicating that ROS are required for the execution of cell death. ROS were generated downstream of mAb-induced actin cytoskeletal reorganization and lysosome membrane permeabilization. ROS production was independent of mitochondria and unaffected by BCL-2 overexpression. Instead, ROS generation was mediated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. These findings provide further insights into a previously unrecognized role for NADPH oxidase-derived ROS in mediating nonapoptotic PCD evoked by mAbs in B-cell malignancies. This newly characterized cell death pathway may potentially be exploited to eliminate malignant cells, which are refractory to conventional chemotherapy and immunotherapy.     

c-Abl kinase inhibitors overcome CD40-mediated drug resistance in CLL: implications for therapeutic targeting of chemoresistant niches

In lymph node (LN) proliferation centers in chronic lymphocytic leukemia (CLL), the environment protects from apoptotic and cytotoxic triggers. Here, we aimed to define the molecular basis for the increased drug resistance and searched for novel strategies to circumvent it. The situation in CLL LN could be mimicked by prolonged in vitro CD40 stimulation, which resulted in up-regulation of antiapoptotic Bcl-xL, A1/Bfl-1, and Mcl-1 proteins, and afforded resistance to various classes of drugs (fludarabine, bortezomib, roscovitine). CD40 stimulation also caused ERK-dependent reduction of Bim-EL protein, but ERK inhibition did not prevent drug resistance. Drugs combined with sublethal doses of the BH3-mimetic ABT-737 displayed partial and variable effects per individual CD40-stimulated CLL. The antiapoptotic profile of CD40-triggered CLL resembled BCR-Abl-dependent changes seen in chronic myeloid leukemia (CML), which prompted application of c-Abl inhibitors imatinib or dasatinib. Both compounds, but especially dasatinib, prevented the entire antiapoptotic CD40 program in CLL cells, and restored drug sensitivity. These effects also occurred in CLL samples with dysfunctional p53. Importantly, ex vivo CLL LN samples also displayed strong ERK activation together with high Bcl-xL and Mcl-1 but low Bim levels. These data indicate that CLL cells in chemoresistant niches may be sensitive to therapeutic strategies that include c-Abl inhibitors.     

Eradication of lymphoma by CD8 T cells following anti-CD40 monoclonal antibody therapy is critically dependent on CD27 costimulation

Growing evidence points to the potential of agonistic anti-CD40 mAbs as adjuvants for vaccination against cancer. These appear to act by maturing dendritic cells (DCs) and allowing them to prime CD8 cytotoxic T lymphocytes (CTLs). Although it is well established that optimal T-cell priming requires costimulation via B7:CD28, recent studies emphasize the contribution of TNF receptors to this process. To understand how anti-CD40 mAbs trigger effective antitumor immunity, we investigated the role of TNFR superfamily members CD27 and 4-1BB in the generation of this immunity and showed that, although partially dependent on 4-1BB:4-1BBL engagement, it is completely reliant on CD27:CD70 interactions. Importantly, blocking CD70, and to some extent 4-1BBL, during anti-CD40 treatment prevented accumulation of tumor-reactive T cells and subsequent tumor protection. However, it did not influence changes in DC number, phenotype, nor the activity of CTLs once immunity was established. We conclude that CD27:CD70 and 4-1BB:4-1BBL interactions are needed for DC-driven accumulation of antitumor CTLs following anti-CD40 mAb treatment. Finally, in support of the critical role for CD70:CD27, we show for the first time that agonistic anti-CD27 mAbs given without a DC maturation signal completely protect tumor-bearing mice and provide a highly potent reagent for boosting antitumor T-cell immunity.     

Resistance to CD95-mediated apoptosis of CD40-activated chronic lymphocytic leukemia B cells is not related to lack of DISC molecules expression

In B-cell chronic lymphocytic leukemia (CLL), accumulation of neoplastic B cells may be the result of dysregulated apoptosis. One of the major molecules triggering apoptosis, CD95 (FAS), is not expressed on CLL B cells at resting conditions. However, CD40 triggering of CLL B cells upregulates receptors belonging to the tumor necrosis factor (TNF) superfamily, like CD95. In the present study, we analyzed in B cells from 20 CLL patients the effect of CD40/CD40L interaction on: (i) CD95 modulation; (ii) CD95-mediated apoptosis and (iii) mRNA and protein expression of the death-inducing signaling complex (DISC) molecules.CD40 activation of CLL B cells was carried out by coculture with CD40L-transfected cells and cytofluorimetric analyses were performed to study CD95 modulation and apoptosis induction by an anti-CD95 moAb. Despite strong CD95 upregulation on the membrane of all the cases studied, only a minority of cases analyzed (3/20) proved weakly responsive to CD95-mediated apoptosis. Multiplex RT-PCR was used to analyze FLICE, FAS, FADD and TRADD mRNAs before and after CD40 triggering. In agreement with the cytofluorimetric data, FAS mRNA appeared significantly increased after CD40 triggering; the other molecules involved in DISC formation and in CD95-mediated apoptosis were also expressed without relevant differences between resting and activated conditions. Western blot analyses further confirmed FLICE and FADD protein expression by resting and activated CLL cells. Our findings demonstrate that, following CD40 triggering, CLL B cells are resistant to CD95-mediated apoptosis despite a strong CD95 upregulation on the membrane and a normal mRNA or protein expression of the DISC components.     

Analysis of TNF-receptor and ligand superfamily molecules in patients with lymphoproliferative disease of granular lymphocytes

In 21 patients with lymphoproliferative disease of granular lymphocytes (LDGL), we investigated the expression and the function of molecules belonging to TNF-receptor and TNF-ligand superfamilies (CD30/CD30L; CD40/CD40L; CD27/CD70; Fas [CD95]/FasL[CD95L]). Fourteen patients were characterized by a proliferation of granular lymphocytes (GLs) expressing the CD3(+)CD16(+) phenotype, whereas 7 cases showed the CD3(-)CD16(+) CD56 +/- phenotype. Our data show that both CD3(+) and CD3-GLs are preferentially equipped with CD30, CD40, CD40L, CD70, and CD95 antigens; this pattern is usually associated with the lack of CD27 and CD30L antigens expression. CD95L was demonstrated in the cytoplasm in 14 of 21 cases by flow cytometry, but a definite signal was demonstrated in all cases studied using polymerase chain reaction analysis. On functional grounds, a stimulatory activity on rIL-2 mediated redirected-cytotoxicity against Fcgamma+ P815 targets was demonstrated with anti-CD30, CD40, CD40L, CD70, CD95, and CD95L mAbs, although resting cells were unable to exhibit significant redirected-cell lysis. The addition of anti-CD30, CD30L, CD40, CD40L, CD95, and CD95L mAbs did not show any significant effect on cell proliferation at resting conditions or after rIL-2 stimulation, whereas anti-CD70 mAb mediated cell proliferation in 6 of 10 cases tested. This figure was not related to an increase in apoptotic cells, as investigated by Annexin-V expression. Our data indicate that both CD3(+) and CD3(-) GLs are equipped with different costimulatory antigens, supporting the concept that these cells are in vivo activated and suggesting that these molecules might play a role in the cytotoxic mechanisms of GLs. (Blood. 2000;96:647-654)     

Differential effect of agonistic anti-CD40 on human mature and immature dendritic cells: the Janus face of anti-CD40

Agonistic monoclonal antibodies to CD40 (CD40 mAbs) have a puzzling dual therapeutic effect in experimental animal models. CD40 mAbs induce tumor regression by potentiating antitumoral T-cell responses, yet they also have immunosuppressive activity in chronic autoimmune inflammatory processes. CD40 mAbs are thought to act on antigen presentation by dendritic cells (DCs) to T cells. DCs can be distinguished as either immature or mature by their phenotype and their ability to generate an effective T-cell response. Here we found that, on human cells, although anti-CD40 led immature DCs to mature and became immunogenic, it also reduced the capacity of lipopolysaccharide (LPS) and tumor necrosis factor alpha (TNF-alpha)-matured DCs to generate a specific CD4 T-cell response. This inhibitory effect was related to rapid and selective apoptosis of mature DCs. Anti-CD40-mediated apoptosis was due to an indirect mechanism involving cooperation with the death domain-associated receptor Fas, leading to activation of Fas-associated death domain protein (FADD) and caspase-8. On human cells, CD40 activation by such agonists could, therefore, trigger immune responses to antigens presented by immature DCs, which are otherwise nonimmunogenic, by inducing maturation. On the other hand, anti-CD40 mAbs, by rapidly inducing apoptosis, may reduce the capacity of inflammatory signal-matured immunogenic DCs to generate an effective T-cell response. These results call for caution in CD40 mAb-based immunotherapy strategies.     

Methyl-vitamin B12 blocks the CD28 co-stimulatory pathway in human T cells and its possible therapeutic application for T cell-mediated diseases, including rheumatoid arthritis

The effects of metyl-vitamin B12 have been examined on human T cell activation induced by stimulation of T cell receptor (TCR)-CD3 and an accessory molecule, CD28. When T cells in the presence of 10% mitomycin-treated non-T cells were stimulated with anti-CD3 mAb at the optimal concentration, methyl-B12 did not inhibit T cell proliferation. However, when T cells were stimulated with the suboptimal concentration of anti-CD3 and anti-CD28 mAbs, methyl-B12 exhibited potent inhibition of T cell proliferation. Methyl-B12 did not affect cell surface expression of the CD3 and CD28 molecules of T cells. Methyl-B12 inhibited a 29 kDa protein tyrosine phosphorylation that was specifically induced by anti-CD3 and anti-CD28 mAbs. Similarly, T cell proliferation of patients with rheumatoid arthritis (RA), which is representative of T cell-mediated disease was inhibited by methyl-B12, when T cells were stimulated by anti-CD3 and anti-CD28 mAbs. These results suggest that methyl-B12 modulates lymphocyte function through blockade of the CD28 signaling pathway and that methyl-B12 may have T cell inhibitory activity that is applicable for treating patients with RA.     

Latent sensitivity to Fas-mediated apoptosis after CD40 ligation may explain activity of CD154 gene therapy in chronic lymphocytic leukemia

Patients with chronic lymphocytic leukemia (CLL) treated with adenovirus (Ad)-CD154 (CD40L) gene therapy experience reductions in leukemia cell counts and lymph node size associated with induction of the death receptor Fas (CD95). CD4 T cell lines can induce apoptosis of CD40-activated CLL cells via a CD95 ligand (CD95-L)-dependent mechanism. To examine whether CD95-L was sufficient to induce cytolysis of CD40-activated CLL cells, we used Chinese hamster ovary cells transfected with CD95-L as cytotoxic effector cells. CD40-activated CLL cells were initially resistant to CD95-mediated apoptosis despite high-level expression of CD95. However, after 72 h, CLL cells from seven of seven patients became increasingly sensitive to CD95-mediated apoptosis. This sensitivity correlated with a progressive decline in Flice-inhibitory protein (FLIP), which was induced within 24 h of CD40 ligation. Down-regulation of FLIP with an antisense oligonucleotide or a pharmacologic agent, however, was not sufficient to render CLL cells sensitive to CD95-mediated apoptosis in the 24-72 h after CD40 activation. Although the levels of pro-Caspase-8 appeared sufficient, inadequate levels of Fas-associated death domain protein (FADD) and DAP3 may preclude assembly of the death-inducing signaling complex. Seventy-two hours after CD40 ligation, sensitivity to CD95 and a progressive increase in FADD and DAP3 were associated with the acquired ability of FADD and FLIP to coimmunoprecipitate with the death-inducing signaling complex after CD95 ligation. Collectively, these studies reveal that CD40 ligation on CLL B cells induces a programmed series of events in which the cells initially are protected and then sensitized to CD95-mediated apoptosis through shifts in the balance of the anti- and proapoptotic proteins FLIP and FADD.     

Combination of two anti‐CD5 monoclonal antibodies synergistically induces complement‐dependent cytotoxicity of chronic lymphocytic leukaemia cells

The treatment of chronic lymphocytic leukaemia (CLL) has been improved by introduction of monoclonal antibodies (mAbs) that exert their effect through secondary effector mechanisms. CLL cells are characterized by expression of CD5 and CD23 along with CD19 and CD20, hence anti‐CD5 Abs that engage secondary effector functions represent an attractive opportunity for CLL treatment. Here, a repertoire of mAbs against human CD5 was generated and tested for ability to induce complement‐dependent cytotoxicity (CDC) and antibody‐dependent cell‐mediated cytotoxicity (ADCC) both as single mAbs and combinations of two mAbs against non‐overlapping epitopes on human CD5. The results demonstrated that combinations of two mAbs significantly increased the level of CDC compared to the single mAbs, while no enhancement of ADCC was seen with anti‐CD5 mAb combinations. High levels of CDC and ADCC correlated with low levels of Ab‐induced CD5 internalization and degradation. Importantly, an anti‐CD5 mAb combination enhanced CDC of CLL cells when combined with the anti‐CD20 mAbs rituximab and ofatumumab as well as with the anti‐CD52 mAb alemtuzumab. These results suggest that an anti‐CD5 mAb combination inducing CDC and ADCC may be effective alone, in combination with mAbs against other targets or combined with chemotherapy for CLL and other CD5‐expressing haematological or lymphoid malignancies.     

CD40 stimulation of B-cell chronic lymphocytic leukaemia cells enhances the anti-apoptotic profile, but also Bid expression and cells remain susceptible to autologous cytotoxic T-lymphocyte attack

To enhance the poor antigen-presenting capacity of B-cell chronic lymphocytic leukaemia (B-CLL), CD40 triggering has been considered as an active immunotherapy. However, CD40 stimulation also has an anti-apoptotic effect and may further impair the dysregulated response of B-CLL to apoptotic stimuli. Therefore, we measured the expression of virtually all regulators of apoptosis before and after CD40 stimulation. These findings were correlated with sensitivity for chemotherapy- and death-receptor-induced apoptosis and T-cell-mediated killing. CD40 stimulation enhanced the constitutive anti-apoptotic profile of B-CLL cells by upregulation of Bcl-xL and Bfl-1 and downregulation of the BH3-only protein Harakiri. Unexpectedly, the BH3-only protein Bid was strongly induced. Functionally, CD40-stimulated B-CLL cells became resistant to drug-induced apoptosis and, despite upregulation of CD95 and Bid, were not sensitive to CD95L. In contrast, autologous T cell killing, triggered by loading CLL cells with viral (CMV) peptides, was very efficient both before and after CD40 stimulation. Upon CTL interaction, CLL targets underwent mitochondrial depolarization and caspase-3 activation. Thus, despite an increased anti-apoptotic profile, CD40 triggered B-CLL cells remain excellent targets for resident cytotoxic T cells. These data support therapeutic exploitation of CD40 stimulation in B-CLL, provided that a strong CTL component is induced.     

Methyl-vitamin B12 blocks the CD28 co-stimulatory pathway in human T cells and its possible therapeutic application for T cell-mediated diseases,including rheumatoid arthritis

Abstract

The effects of metyl-vitamin B12 have been examined on human T cell activation induced by stimulation of T cell receptor (TCR)-CD3 and an accessory molecule, CD28. When T cells in the presence of 10% mitomycin-treated non-T cells were stimulated with anti-CD3 mAb at the optimal concentration, methyl-B12 did not inhibit T cell proliferation. However, when T cells were stimulated with the suboptimal concentration of anti-CD3 and anti-CD28 mAbs, methyl-B12 exhibited potent inhibition of T cell proliferation. Methyl-B12 did not affect cell surface expression of the CD3 and CD28 molecules of T cells. Methyl-B12 inhibited a 29 kDa protein tyrosine phosphorylation that was specifically induced by anti-CD3 and anti-CD28 mAbs. Similarly, T cell proliferation of patients with rheumatoid arthritis (RA), which is representative of T cell-mediated disease was inhibited by methyl-B12, when T cells were stimulated by anti-CD3 and anti-CD28 mAbs. These results suggest that methyl-B12 modulates lymphocyte function through blockade of the CD28 signaling pathway and that methyl-B12 may have T cell inhibitory activity that is applicable for treating patients with RA.     

CD3-mediated activation of tumor-reactive lymphocytes from patients with advanced cancer

Lymphocytes from blood or tumors of patients with advanced cancer did not proliferate and produced very low levels of tumor necrosis factor and IFN-gamma when cultured with autologous tumor cells. Proliferation and lymphokine production dramatically increased in the presence of beads conjugated with mAbs to CD3 plus mAbs to CD28 and/or CD40, and the lymphocytes destroyed the tumor cells. Expression density of CD3 concomitantly increased from low to normal levels. Furthermore, beads providing a CD3 signal (in combination with CD28 or CD28 plus CD40) gave partial protection against the inhibitory effect of transforming growth factor type beta1 on lymphocyte proliferation and production of tumor necrosis factor and IFN-gamma. MHC class I-restricted cytolytic T cells lysing autologous tumor cells in a 4-h Cr(51) release assay were generated when peripheral blood leukocytes were activated in the presence of autologous tumor cells and anti-CD3/CD28 or anti-CD3/CD28/CD40 beads. Experiments performed in a model system using anti-V-beta1 or anti-V-beta2 mAbs to activate subsets of T cells expressing restricted T cell receptor showed that lymphocytes previously activated by anti-V-beta can respond to CD3 stimulation with vigorous proliferation and lymphokine production while retaining their specificity, also in the presence of transforming growth factor type beta1. Our results suggest that T lymphocytes from cancer patients can proliferate and form Th1 type lymphokines in the presence of autologous tumor cell when properly activated, and that antigen released from killed tumor cells and presented by antigen-presenting cells in the cultures facilitates the selective expansion of tumor-directed, CD8(+) cytolytic T cells.     

Bispecific anti-CD20/22 antibodies inhibit B-cell lymphoma proliferation by a unique mechanism of action

Combination immunotherapy with anti-CD20 and anti-CD22 mAbs shows promising activity in non-Hodgkin lymphoma. Therefore, bispecific mAbs (bsAbs) were recombinantly constructed from veltuzumab (humanized anti-CD20) and epratuzumab (humanized anti-CD22) and evaluated in vitro and in vivo. While none of the parental mAbs alone or mixed had notable antiproliferative activity against Burkitt lymphoma cells when not cross-linked, the bsAbs [eg, anti-CD20 IgG-anti-CD22 (scFv)(2)] were inhibitory without cross-linking and synergistic with B-cell antigen (BCR)-mediated inhibition. The bsAbs demonstrated higher antibody-dependent cellulary cytoxicity (ADCC) activity than the parental mAbs, but not complement-dependent cytoxicity (CDC) of the parental CD20 mAb. Cross-linking both CD20 and CD22 with the bsAbs resulted in the prominent redistribution of not only CD20 but also CD22 and BCR into lipid rafts. Surprisingly, appreciable translocation of CD22 into lipid rafts was also observed after treatment with epratuzumab. Finally, the bsAbs inhibited Daudi lymphoma transplant growth, but showed a significant advantage over the parental anti-CD20 mAb only at the highest dose tested. These results suggest that recombinantly fused, complementary, bispecific, anti-CD20/22 antibodies exhibit functional features distinct from their parental antibodies, perhaps representing new candidate therapeutic molecules.     

Phase 1 study results of the type II glycoengineered humanized anti-CD20 monoclonal antibody obinutuzumab (GA101) in B-cell lymphoma patients

Whereas the chimeric type I anti-CD20 Ab rituximab has improved outcomes for patients with B-cell malignancies significantly, many patients with non-Hodgkin lymphoma (NHL) remain incurable. Obinutuzumab (GA101) is a glycoengineered, humanized anti-CD20 type II Ab that has demonstrated superior activity against type I Abs in vitro and in preclinical studies. In the present study, we evaluated the safety, efficacy, and pharmacokinetics of GA101 in a phase 1 study of 21 patients with heavily pretreated, relapsed, or refractory CD20(+) indolent NHL. Patients received GA101 in a dose-escalating fashion (3 per cohort, range 50/100-1200/2000 mg) for 8 × 21-day cycles. The majority of adverse events (AEs) were grades 1 and 2 (114 of 132 total AEs). Seven patients reported a total of 18 grade 3 or 4 AEs. Infusion-related reactions were the most common AE, with most occurring during the first infusion and resolving with appropriate management. Three patients experienced grade 3 or 4 drug-related infusion-related reactions. The best overall response was 43%, with 5 complete responses and 4 partial responses. Data from this study suggest that GA101 was well tolerated and demonstrated encouraging activity in patients with previously treated NHL up to doses of 2000 mg. This trial is registered at www.clinicaltrials.gov as NCT00517530.     

T-cell responses against chronic lymphocytic leukemia cells: implications for immunotherapy

Chronic lymphocytic leukemia (CLL) cells are ineffective antigen-presenting cells (APCs) although CD40-activated CLL cells can stimulate proliferation of autologous and allogeneic T cells. We examined the antigen-presenting capacity of CD40-activated CLL cells as well as dendritic cells pulsed with apoptotic bodies of CLL cells to generate autologous and allogeneic immune responses against CLL cells. Both APC types were capable of generating T-cell lines that proliferate specifically in response to unstimulated CLL cells. Whereas cytotoxic responses against stimulated and unstimulated CLL cells could be repeatedly generated by allogeneic healthy donors, autologous cytotoxic immune responses against CD40-activated and native CLL cells were rarely detected. However, T cells isolated from patients with CLL could recognize and lyse allogeneic stimulated and unstimulated CLL cells, demonstrating that cytotoxic T cells from these tumor-bearing patients are functionally intact.