Both activating and inhibitory Fc gamma receptors mediate rituximab-induced trogocytosis of CD20 in mice

Antigenic modulation by trogocytosis during anti-CD20 mAb treatment with rituximab (RTX) leads to loss of CD20 and therefore can compromise therapy. During trogocytosis, effector cells, such as macrophages, remove CD20 from the surface of antibody-coated cells in an Fc receptor-dependent manner. Importantly, Fcγ receptors (FcγRs) are also crucial in the anti-tumor effects of RTX by inducing antibody dependent cell-mediated cytotoxicity (ADCC). Here we studied the role of FcγR during RTX-induced trogocytosis of CD20 in an intraperitoneal tumor model with EL4-CD20 cells. We found marked RTX-induced trogocytosis of CD20 in FcγRI- or FcγRIII-deficient mice, similar to wild type mice, demonstrating a redundancy for activating FcγR in trogocytosis. Interestingly, in FcRγ-chain-deficient mice, trogocytosis was still apparent, indicating that the inhibitory receptor FcγRIIB alone can also mediate trogocytosis. These data were confirmed by in vitro analysis with blocking antibodies. Decreasing the amount of RTX in vivo resulted in less trogocytosis of CD20, supporting clinical studies with lower RTX dose. Importantly, we show that cells which undergo in vivo trogocytosis can still be killed ex vivo by ADCC but not by complement-mediated cytotoxicity (CDC), underscoring the clinical relevance of trogocytosis. Taken together, our study provides more insights into the mechanism and consequences of RTX-induced trogocytosis of CD20.     

Interaction Between B.7 and CD28 Costimulatory Molecules is Essential for the Activation of Effector Function Mediating Spontaneous Tumour Regression

The spontaneous regression of a rat histiocytoma, AK-5, is mediated by activated natural killer cells through antibody-dependent cellular cytotoxicity. In addition to the Fc-FcR interaction between the target and the effector cells demonstrated previously, we show the participation of costimulatory molecules B7 and CD28 in the efficient killing of the tumour cell. Blockade of the costimulatory interaction in vivo using anti-CD28 led to increased tumour growth and a suppressed cytokine response. Anti-CD28 antibody administration in vivo also diminished the cytotoxic potential of NK cells against AK-5 cells in vitro. Our studies also demonstrate the expression of B7.1 and B7.2 antigen on AK-5 tumour cells. The cytotoxic activity of natural killer cells was significantly inhibited when the effector/target cells were cultured in the presence of antibodies raised against B7.1, B7.2 and CD28. Administration of anti-CD28 in vivo also affected the efficiency of the formation of effector/target conjugates in vitro. Similarly, anti-CD28 injections affected expression of the adhesion molecules LFA 1 and ICAM 1 by splenocytes. Administration of anti-B7.1 and B7. 2 antibodies in AK-5 tumour-bearing animals showed a differential response. The cytotoxicity of natural killer cells was significantly inhibited after anti-B7.2 administration, suggesting the preferential participation of B7.2 molecules in vivo. These observations suggest an important role for B7-CD28 interaction in AK-5 tumour regression.     

Phenotypic Convergence and Divergence of Surface Immunoglobulin and CD40 Signals

Both anti-CD40 antibodies and anti-immunoglobulin (Ig) coupled to Sepharose induced proliferation of resting B cells and suppressed lipopolysaccharide (LPS)-induced B-cell differentiation to immunoglobulin secretion at comparable levels determined with the plaque-forming assay and Ig RNA steady state levels. Anti-CD40 antibodies also increased the proliferation of B cells stimulated by T helper cells in vitro while suppressing their differentiation to Ig secretion. Further, B cells preactivated by anti-Ig, anti-CD40 or a combination of the two mitogens could be restimulated by anti-CD40 but not by anti-Ig antibodies. Phenotypic divergence of Ig and CD40 signals regarding surface expression of activation markers was observed. Restimulation of anti-Ig- or anti-CD40-prestimulated cells with anti-Ig induced apoptosis whereas apoptosis could be inhibited when cells were recultivated with anti-CD40.     

Immunotherapeutic mechanisms of anti-CD20 monoclonal antibodies

The anti-CD20, B-cell-specific mAb rituximab (RTX) has been approved for treatment of non-Hodgkin's B cell lymphoma and rheumatoid arthritis. Under conditions of high B cell burden, exhaustion of the body's effector mechanisms, for example, NK-cell-mediated killing, may lead to substantial decreases in the immunotherapeutic efficacy of this mAb. Moreover, RTX treatment of patients with chronic lymphocytic leukemia and high levels of circulating B cells can lead to removal of CD20 from the cells, thus allowing them to persist and resist clearance. RTX therapy for several autoimmune diseases has proven to be effective, but in numerous instances there has been little correlation between reductions in disease activity and changes in titers of pathogenic autoantibodies. This paradox may be explained by a separate mechanism: Binding of RTX to B cells generates immune complexes that act as decoys to attract monoycte/macrophages and thus reduce their inflammatory activity in certain autoantibody-mediated diseases. Several second-generation anti-CD20 mAbs with enhanced cytotoxic action have been developed and are being tested in the clinic for treatment of cancer and autoimmune diseases. The application of these mAbs, potentially in combination with immune effector modifying drugs, may successfully address the shortcomings of current anti-CD20 immunotherapy.     

Mechanisms of killing by anti-CD20 monoclonal antibodies

CD20 is a cell-surface marker expressed on mature B cells and most malignant B cells, but not stem or plasma cells. It is an ideal target for monoclonal antibodies (mAb), such as rituximab and ofatumumab, as it is expressed at high levels on most B-cell malignancies, but does not become internalized or shed from the plasma membrane following mAb treatment. This allows mAb to persist on the cell surface for extended periods and deliver sustained immunological attack from complement and FcR-expressing innate effectors, particularly macrophages. CD20 can also generate transmembrane signals when engaged by certain mAb which, although unproven, might provide an important element of the therapeutic success of anti-CD20 mAb. These favourable characteristics have led to anti-CD20 mAb being developed and exploited for use in immunotherapy, where they have proven remarkably efficacious in both the treatment of malignant disease and autoimmune disorders by deleting malignant or normal B cells, respectively. In this review, we discuss how these mAb have driven research in the immunotherapy field over the last decade, detail their likely modes of action and their limitations in terms of effector exhaustion, and explore ways in which they might be enhanced and further exploited in the future.     

Mechanisms of killing by anti-CD20 monoclonal antibodies

CD20 is a cell-surface marker expressed on mature B cells and most malignant B cells, but not stem or plasma cells. It is an ideal target for monoclonal antibodies (mAb), such as rituximab and ofatumumab, as it is expressed at high levels on most B-cell malignancies, but does not become internalized or shed from the plasma membrane following mAb treatment. This allows mAb to persist on the cell surface for extended periods and deliver sustained immunological attack from complement and FcR-expressing innate effectors, particularly macrophages. CD20 can also generate transmembrane signals when engaged by certain mAb which, although unproven, might provide an important element of the therapeutic success of anti-CD20 mAb. These favourable characteristics have led to anti-CD20 mAb being developed and exploited for use in immunotherapy, where they have proven remarkably efficacious in both the treatment of malignant disease and autoimmune disorders by deleting malignant or normal B cells, respectively. In this review, we discuss how these mAb have driven research in the immunotherapy field over the last decade, detail their likely modes of action and their limitations in terms of effector exhaustion, and explore ways in which they might be enhanced and further exploited in the future.     

CD2-CD48 interactions promote cytotoxic T lymphocyte induction and function: anti-CD2 and anti-CD48 antibodies impair cytokine synthesis, proliferation, target recognition/adhesion, and cytotoxicity.

The role of CD2 signaling in cytotoxic T lymphocyte (CTL) development was examined by stimulating mouse T cells with anti-CD3 monoclonal antibody (mAb) in the absence or presence of anti-CD2 mAb or anti-CD48 mAb or both. Induction of nonspecific CTL and interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) synthesis were impaired in the absence of CD2-CD48 interactions. Anti-CD2 mAb also inhibited activation-induced expression of the high-affinity IL-2 receptor (IL-2R). In contrast, IFN-gamma receptor (IFNGR) expression was increased in the presence of anti-CD2 mAb. Reduced cytotoxicity by CTL induced in the absence of CD2-CD48 interactions was associated with a diminished ability of CTL to conjugate with target cells and reduced expression of granzyme B and perforin. Anti-CD2 mAb did not affect expression of Fas ligand and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) by anti-CD3-activated T cells. Cytotoxic effector function and granzyme B and perforin expression were rescued when exogenous IL-2 and IFN-gamma were added in combination with anti-CD2 mAb to anti-CD3-activated T cells at initiation of culture. We conclude that CD2-CD48 interactions during T cell activation are critical for the synthesis of sufficient IL-2 and IFN-gamma to drive CD8(+) T cells to differentiate into functional cytotoxic effector cells.     

Activation of macaque T cells and B cells with agonistic monoclonal antibodies

A series of mouse monoclonal antibodies (mAb) to human differentiation antigens known to have agonistic activity for human T or B cells was found to bind specifically to macaque T or B cell subsets. Most of these mAb also stimulated macaque lymphocyte proliferation, implying that they recognize functional homologues in monkeys. Anti-CD3, anti-CD28 (9.3), and anti-Lp220 (CD45R) mAb stimulated proliferation of both human and macaque T cells; similarly, anti-IgM and anti-CDw40 mAb stimulated both human and macaque B cells. In contrast, anti-CD20 and anti-CD39 mAb, which are known to stimulate human B cells, did not stimulate macaque B cells. A human low-molecular weight B cell growth factor (BCGF) and anti-IgM were co-stimulatory for macaque splenic B cells but not for blood B cells, suggesting that B cell subpopulations may differ in their responsiveness to BCGF. The results show that functional epitopes on some lymphocyte surface molecules such as CD28 or CDw40 are conserved in primate evolution. Functional epitopes on other cell surface molecules such as CD3 and CD20 may have more complex evolutionary constraints.     

Importance of antibody isotypes in antitumor immunity by monocytes and complement using human-immune tumor models

Monoclonal antibodies (mAbs) have revolutionized clinical medicine, especially in the field of cancer immunotherapy. The challenge now is to improve the response rates, as immunotherapy still fails for many patients. Strategies to enhance tumor cell death is a fundamental aim, but relevant model systems for human tumor immunology are lacking. Herein, we have developed a preclinical human immune – three-dimensional (3D) tumor model (spheroids) to map the efficiency of tumor-specific isotypes for improved tumor cell killing. Different anti-CD20 Rituximab (RTX) isotypes alone or in combination, were evaluated for mediating complement-dependent cytotoxicity and antibody-dependent phagocytosis by human monocytic cells in 3D spheroids, in parallel with monolayer cultures, of human CD20⁺ B-cell lymphomas. We demonstrate that the IgG3 variant of RTX has the greatest tumoricidal effect over other isotypes, and when combined with apoptosis-inducing RTX-IgG2 isotype the therapeutic effect can be substantially enhanced. The results show further that the treatment outcome by RTX isotypes is influenced by tumor morphology and expression of the complement inhibitor CD59. Hence, the human immune-3D tumor model is a clinical relevant and attractive ex vivo system to predict mAbs for best efficacy in cancer immunotherapy.     

Generation in vitro of B-cell chronic lymphocytic leukaemia-proliferative and specific HLA class-II-restricted cytotoxic T-cell responses using autologous dendritic cells pulsed with tumour cell lysate.

Immunotherapy using dendritic cells has shown encouraging results in both haematological and non-haematological malignancies. In this study, monocyte-derived dendritic cells from patients with B-CLL were cultured for 6 days in the presence of IL-4 and GM-CSF. Autologous B-CLL T-cells were cultured alone or with B-CLL lysate-pulsed and unpulsed autologous dendritic cells. IFN-gamma secretion was assessed using ELISA. Cytotoxicity was assessed, after 21 days in culture and re-stimulation, using flow cytometry with and without blockade by anti-HLA class I, anti-HLA class II, anti-CD4, anti-CD8 and anti-TCRalphabeta monoclonal antibodies. B-CLL T cells stimulated with B-CLL lysate-pulsed autologous dendritic cells showed a significant (P = 0.0004) increase in IFN-gamma secretion and a significant (P = 0.0008) increase in specific cytotoxicity to autologous B-cell targets, but none to autologous T cell or B cell targets from healthy individuals. B-CLL T cells cultured with (non-B-CLL) B-cell lysate-pulsed B-CLL dendritic cells showed no significant response. Pulsing dendritic cells from healthy volunteers with an autologous (non-B-CLL) B-cell lysate did not stimulate proliferation, cytokine production or cytotoxicity by autologous T cells. Pulsing B-CLL dendritic cells with allogeneic B-CLL lysates and culturing with autologous T-cells elicited cytotoxicity against autologous B-CLL targets in some cases, but not in others. Cytotoxicity was significantly reduced by blocking with anti-HLA class II (P = 0.001), anti-TCRalphabeta (P = 0.03) and anti-CD4 (P = 0.046) antibodies. Phenotyping of the responding T-cell population demonstrated the majority to be CD4 positive. Our data demonstrate that HLA class II-restricted proliferative and cytotoxic T-cell responses to B-CLL can be generated using autologous dendritic cells pulsed with tumour cell lysate.     

CD20单抗临床应用局限与免疫效应器耗竭的研究进展

CD20单克隆抗体(Rituximab),已被批准应用于非霍奇金B淋巴瘤的治疗,并取得了显著的免疫疗效。但在大负荷循环B细胞存在的情况下,由于机体效应器如效应细胞,效应因子的耗竭,继发表面抗原抗体结合物的削片反应,导致了肿瘤的耐药与复发,降低了Rituximab的疗效。此综述通过对近20年来CD20单抗研究现状的阐述,讨论其作用机制及在效应器耗竭方面的局限,探索适宜的改进方法,以拓展其应用前景。     

Pathways of human B-lymphocyte activation blocked by B-cell specific monoclonal antibodies.

The data presented in this report demonstrate that antibodies directed against B-cell specific surface antigens can block different modes of B-cell activation. The anti-CD 19 antibody HD37 strongly blocks the stimulation of thymidine incorporation induced by anti-mu and MLR supernatant and also partially inhibits the growth of long-term EBV-transformed cell lines. The anti-CD20 antibody B1, on the other hand, has little effect on anti-mu activation but prevents the stimulation of thymidine incorporation and transformation by EBV. We conclude that different mechanisms operate during B-cell activation by anti-mu and EBV involving different B-cell surface molecules.     

Flow cytometric assay detecting cytotoxicity against human endogenous retrovirus antigens expressed on cultured multiple sclerosis cells

Damage of target cells by cytotoxicity, either mediated by specific lymphocytes or via antibody‐dependent reactions, may play a decisive role in causing the central nervous system (CNS) lesions seen in multiple sclerosis (MS). Relevant epitopes, antibodies towards these epitopes and a reliable assay are all mandatory parts in detection and evaluation of the pertinence of such cytotoxicity reactions. We have adapted a flow cytometry assay detecting CD107a expression on the surface of cytotoxic effector cells to be applicable for analyses of the effect on target cells from MS patients expressing increased amounts of human endogenous retrovirus antigens. MS patients also have increased antibody levels to these antigens. The target cells are spontaneously growing peripheral blood mononuclear cells (PBMCs) of B cell lineage, expressing human endogenous retrovirus HERV epitopes on their surface. Polyclonal antibodies against defined peptides in the Env‐ and Gag‐regions of the HERVs were raised in rabbits and used in antibody‐dependent cell‐mediated cytotoxicity (ADCC) ‐assays. Rituximab® (Roche), a chimeric monoclonal antibody against CD20 expressed primarily on B cells, was used as control antibody. Without antibodies this system is suitable for analyses of natural killer cell activity. In optimization of the assay we have used effector lymphocytes from healthy donors. The most effective effector cells are CD56⁺ cells. CD8⁺ T cells also express CD107a in ADCC. Using the adapted assay, we demonstrate significant ADCC activity to target cells expressing HERV epitopes, and additionally a low level of NK activity.     

Antibody L26 recognizes an intracellular epitope on the B-cell-associated CD20 antigen.

Monoclonal antibody L26 is a highly selective marker of B cells and B-cell neoplasms in paraffin-embedded tissues, but it suffers from the drawback that the target molecule has not been identified. In this paper we provide evidence by two independent techniques that antibody L26 recognizes an intracellular epitope on the CD20 antigen (a pan B-cell marker). When this antigen was redistributed on the surface of unfixed viable B cells by incubation with monoclonal anti-CD20 followed by anti-mouse Ig, the diffuse cytoplasmic staining of L26 was abolished and replaced by coincident dotlike labeling for antibody L26 and the CD20 antigen. None of the other antibodies tested (covering 10 different B-cell-associated antigens) had this effect on the L26 staining pattern. Furthermore, COS-1 cells transfected with cDNA encoding the CD20 molecule gave positive staining with antibody L26 and with two other CD20 reagents, but not with antibodies to other pan B-cell markers (eg, CD19 and CD22).     

Selection and characterization of single domain antibodies against human CD20

CD20 is a membrane protein with four integral membrane regions and a large extracellular loop between residues 142 and 187, which serves as a target binding region for rituximab (RTX) and most other anti-CD20 monoclonal antibodies. It is highly expressed in B-lymphoma cells and B lymphocytes and often serves as a target for immunotherapy. In this study, we developed single domain antibodies (sdAbs) for the sensitive detection of CD20. To achieve this, an immune sdAb library was prepared from llamas immunized with a fusion between the large loop from CD20 and Hoc, a highly antigenic protein from the T4 bacteriophage, (CD20-Hoc). By subtracting binders to recombinant Hoc during the biopanning, potential anti-CD20 sdAbs were selected, sequenced, and characterized for their binding affinity to CD20-Hoc fusion versus Hoc. Twenty five clones grouped into three different families based on CDR3 sequence were identified as potential CD20 binders. The binding kinetics of representative sdAbs from each class and RTX were evaluated by surface plasmon resonance (SPR). Most of the sdAbs that were evaluated show binding affinities to CD20-Hoc in the nM range, and class A sdAbs, exhibited ≥40-fold increase in affinity for CD20-Hoc versus Hoc. When the binding of the sdAbs to CD20 on SU-DHL-4 cells was evaluated by flow cytometry, only class A sdAbs displayed strong binding to CD20 and recognized DHL cells in a concentration dependent manner.     

Chromosomal localization of the gene for human B-cell antigen CD40

CD40 is a surface glycoprotein expressed on all human B lymphocytes and plays an important role in B-cell development, growth, and differentiation. Anti-CD40 monoclonal antibodies cause isotype switching in B cells treated with IL-4. CD40 is a member of a family of proteins that include low-affinity nerve growth factor receptor, TNF receptor, and the antigen Fas. The ligand for CD40 had been recently identified and has been assigned to the X chromosome. Using a panel of human-rodent somatic cell hybrids, we now show that CD40 maps to human chromosome 20.     

The CD44 variant isoforms CD44v6 and CD44v7 are expressed by distinct leukocyte subpopulations and exert non-overlapping functional activities

We have described recently that anti-CD44s, anti-CD44v6 and anti-CD44v7 interfere with delayed-type hypersensitivity (DTH) reactions. Yet, TNBS-induced colitis can be cured only by anti-CD44v7. To clarify the mechanisms underlying the divergent functional activities of CD44v6 and CD44v7 we explored their contribution to lymphocyte activation in vivo and in vitro. CD44v6 and CD44v7 are distinctly expressed on subpopulations of activated lymphocytes. Expression of CD44v6 is mainly restricted to T cell blasts. CD44v7 has been detected on CD4(+) cells, B cells and monocytes. Mitogenic and antigenic stimulation of lymphocytes in vitro was impaired in the presence of anti-CD44v6 and anti-CD44v7. Accordingly, anti-CD44v6 and anti-CD44v7 mitigated the DTH reaction in 2,4-dinitro-1-fluorobenzene-sensitized and challenged mice. However, the seemingly similar effects of CD44v6- and CD44v7-specific antibodies resulted from different activities. Anti-CD44v6 treatment led to a down-regulation of IL-2 and IFN-gamma production predominantly by CD8(+) cells. In anti-CD44v7-treated mice expression of IL-12 was decreased. Elevated levels of IL-10 accompanied this reduction. The latter resulted from an anti-CD44v7-mediated blockade of interactions between CD4(+) cells and monocytes as well as an active triggering of B cells. Thus, anti-CD44v6 and anti-CD44v7 interfere with lymphocyte activation at very specific points. CD44v6 functions predominantly at the T cell level. CD44v7 influences production of proinflammatory cytokines by B cells as well as an interaction between CD4(+) cells and antigen-presenting cells. As CD44 isoforms do not differ in their intracytoplasmatic tail, the distinct activities must result from expression on different leukocyte subsets and interactions with distinct ligands.     

Adhesion of CD16+ K cells to antibody-coated targets is mediated by CD2 and CD18 receptors.

In order to investigate the function of CD2 and CD18 receptors in antibody-dependent cellular cytotoxicity (ADCC), Fab' fragments of the monoclonal antibodies 9.6 (anti-CD2) and 60.3 (anti-CD18) were preincubated with the human natural killer (NK) clone EB4.19 and tested for conjugate formation and cytotoxic function against the human B-cell line KMS and the mouse thymoma line SL-2. We concluded that: (1) the FcR CD16 does not participate in conjugate formation; (2) adhesion between target and effector cells mediated by CD2 and CD18 is a prerequisite for subsequent activation of the lytic programme through the CD16 receptor.     

Immunolocalization and characterization of the rat analogue of human CD59 in kidney and glomerular cells.

A mutant subclone of the murine thymoma EL-4, known as EL4B5, can strongly activate human B cells to proliferate and differentiate in a cell-cell contact-dependent manner. We have investigated whether interaction via CD40 plays a role in this helper activity. For this purpose, three newly generated anti-CD40 monoclonal antibodies (mAb) were used. In contrast with other anti-CD40 mAb described in the literature, these mAb did not co-stimulate proliferation of human B cells. On the other hand, these novel mAb could inhibit the co-stimulatory effect of the previously described anti-CD40 mAb S2C6 on anti-IgM-induced human B-cell proliferation. It was found that addition of these non-stimulatory anti-CD40 mAb could completely inhibit EL4B5-induced human B-cell proliferation. Maximal inhibition occurred already at a mAb concentration of 10 ng/ml. Similarly, a fusion protein, consisting of the extracellular portion of CD40 and human IgM constant domains CH2, CH3 and CH4, could completely inhibit EL4B5-induced human B-cell proliferation. Induction of human B-cell proliferation by EL4B5 cells was also inhibited by anti-CD40 mAb S2C6 and G28.5, but less effectively. In contrast, mAb against B-cell surface antigens CD20 or B7 had no inhibitory effects. It is concluded that interaction via CD40 is essential for the induction of human B-cell proliferation by EL4B5 cells.     

Anti-CD3ε induces splenic B220lo B-cell expansion following anti-CD20 treatment in a mouse model of allosensitization

Antibodies targeting T cells and B cells are increasingly used for immunosuppression in clinical transplantation. However, the impact of T-cell depletion by antibodies on B-cell homeostasis is poorly understood. Using a mouse model of allosensitization with skin allograft, we investigated whether targeting T cells by anti-CD3ε alters peripheral B-cell homeostasis and alloantibody responses following B-cell depletion by anti-CD20. We found that anti-CD3ε induced a discrete B220(lo), but not a conventional B220(hi) subset, in the spleens of the allosensitized mice 14 days after anti-CD20 treatment. The splenic B220(lo) cells were refractory to anti-CD20 depletion. Flow cytometry revealed that the splenic B220(lo) cells were phenotypically similar to the B220(lo) AA4.1(+) CD23(-) sIgM(lo) sIgD(-) developing B cells (pre-B to immature B) normally presented in the bone marrow. Despite the presence of the splenic B220(lo) cells, mice treated with combined anti-CD3ε/CD20 produced limited alloantibodies in response to the primary skin allografts. Alloantibody production increased significantly in the mice following re-immunization by donor-specific splenocytes. We conclude that anti-CD3ε can induce an expansion of B220(lo) B cells in the spleens after B-cell depletion by anti-CD20. These B cells are not producing alloantibodies, but re-immunization of the mice with alloantigen leads to risk of alloantibody response.