Fc Receptor Blocking Antibodies Research Articles

Antigenic Modulation of CD6 By Itolizumab Is a New Mechanism for Effector T Cell Inhibition

IntroductionAcute Graft-Versus-Host Disease (aGVHD) is a serious complication of hematopoietic stem cell transplantation (HSCT) that is primarily driven by alloreactive T cells. CD6 is a costimulatory receptor primarily expressed on T cells that promotes synapse formation, T cell activation, and migration into tissues by engaging with its ligand, activated leukocyte cell adhesion molecule (ALCAM). CD6 is expressed on reconstituting T cells soon after HSCT (Rambaldi et al., 2019) and early studies have shown that ex-vivo depletion of CD6 + donor cells prior to HSCT decreases the incidence of aGVHD (Soiffer et al., 1992; Soiffer et al., 1998). The reduced levels of aGVHD were attributed to an increased prevalence of CD6 - T cells that were less alloreactive (Rasmussen et al., 1994). Therefore, modulating activity of the CD6-ALCAM pathway may ameliorate aGVHD. Itolizumab is a humanized anti-CD6 monoclonal antibody that was previously described to block the engagement of ALCAM, thereby inhibiting T cell activity and trafficking. Here, we elucidate a second and highly novel mechanism in which antibody-mediated loss of CD6 from the surface of T cells results in CD6 low T cells that are hyporesponsive to T cell stimulation.MethodsTo assess the molecular mechanisms for itolizumab-induced loss of cell surface CD6, peripheral blood mononuclear cells (PBMCs) were exposed to different conditions including treatment with selected protease inhibitors, a membrane inhibitor, or Fc receptor blocking antibodies in the presence of itolizumab. Furthermore, B cells, NK cells, and monocytes were isolated from PBMCs and mixed with T cells in combination with itolizumab to evaluate cell contact requirements for loss. Following treatment, cell surface levels of CD6 protein were assessed by flow cytometry using a non-competing anti-CD6 detection antibody while analysis of full length CD6 was detected by western blot from total protein lysate. The soluble form of CD6 (sCD6) was analyzed by electrochemiluminescence and immunoprecipitation from supernatant of treated cells.ResultsFollowing treatment with itolizumab, the levels of cell surface CD6 was reduced as assessed by flow cytometry and western blot. Concurrent with a loss of cell surface CD6, levels of sCD6 in the cell supernatant increased, suggesting CD6 is primarily cleaved from the cell surface rather than internalized. Characterization of the cleaved sCD6 product by western blot revealed a 30KDa product. Itolizumab-induced changes in both levels of cell surface CD6 and sCD6 was abrogated by both 4-benzenesulfonyl fluoride hydrochloride (AEBSF), a serine protease inhibitor and cytochalasin D, an inhibitor of actin polymerization that prevents movement within the cell membrane and blocks endocytic trafficking. This suggests that a membrane-bound serine protease is responsible for cleavage.Itolizumab-induced CD6 cleavage did not occur with T cells alone and required the presence of other immune cells including monocytes and NK cells, but not B cells (Figure 1). In addition, when monocytes and T cells were separated by a membrane, cleavage of CD6 was not observed in the presence of itolizumab, indicating that cell-to-cell contact is required. Furthermore, blocking antibodies indicated that functional Fc receptors, especially FcγRIA, were required, suggesting that binding of itolizumab to both an Fc receptor on monocytes and CD6 on T cells predicated the cleavage event. The resulting CD6 low T cells were hyporesponsive to T cell stimulation, even in the absence of itolizumab, as indicated by reduced expression of PD-1, CD71 and CD25 as well as inflammatory cytokines (Figure 2). Inhibition was observed for both naïve and effector/memory T cell subsets.ConclusionsOur results reveal a novel mechanism of antigenic modulation by itolizumab in which CD6 is cleaved from the T cell surface and released in a soluble form. Cleavage of CD6 occurs via a membrane-bound serine protease and appears dependent upon the engagement of itolizumab with Fc receptor(s) present on monocytes and NK cells. The loss of cell surface CD6 results in T cells with reduced responses to TCR-mediated stimulation. As such, treatment of aGVHD patients with itolizumab may reduce the alloreactivity of donor T cells, ameliorating disease symptoms and improving the clinical outcomes in these patients. [Display omitted] DisclosuresChu: Equillium: Current Employment. Marrocco: Equillium, Inc.: Current Employment. Tiet: Equillium, Inc.: Current Employment. Ampudia: Equillium, Inc.: Current Employment. Connelly: Equillium: Current Employment, Divested equity in a private or publicly-traded company in the past 24 months, Membership on an entity's Board of Directors or advisory committees. Ng: Equillium: Current Employment, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months.

Selective 5-Lipoxygenase Expression in Langerhans Cells and Impaired Dendritic Cell Migration in 5-LO-Deficient Mice Reveal Leukotriene Action in Skin

5-Lipoxygenase (5-LO) catalyzes the initial steps in the formation of leukotrienes (LTs), which are implicated in immune reactions. Recently, it was shown that FITC-triggered epidermal Langerhans cell (LC) emigration to draining lymph nodes (LNs) is impaired in LTC4 export pump (multidrug resistance-associated protein 1)-deficient mice. Here, we sought genetic evidence for a role of endogenous LTs in dendritic cell function through the study of 5-LO-deficient mice. Though DC numbers in skin, spleen, and peripheral LNs were similar in both 5-LO-deficient and wild-type (WT) mice, DC homing from skin to draining LNs induced by FITC was reduced by 75% in 5-LO-deficient mice. Moreover, in WT mice, all epidermal LCs, dermal langerin+ LCs, and subsets of dermal macrophages and langerin+ LCs in T-cell areas of skin-draining LNs markedly expressed 5-LO. However, the enzyme was noticeably absent in all DC subsets of the dermis, thymus, spleen, Peyer's patches, mesenteric LNs, and mucosal surfaces of lung and intestine. As all epidermal cells other than LCs lacked 5-LO and because differentiation and activation of DCs generated from 5-LO-deficient mice in vitro were normal, these data support a selective role of endogenous LTs in DC homing following skin sensitization.

Prophylaxis and Therapy of Inhalational Anthrax by a Novel Monoclonal Antibody to Protective Antigen That Mimics Vaccine-Induced Immunity

The neutralizing antibody response to the protective antigen (PA) component of anthrax toxin elicited by approved anthrax vaccines is an accepted correlate for vaccine-mediated protection against anthrax. We reasoned that a human anti-PA monoclonal antibody (MAb) selected on the basis of superior toxin neutralization activity might provide potent protection against anthrax. The fully human MAb (also referred to as MDX-1303 or Valortim) was chosen from a large panel of anti-PA human MAbs generated using transgenic mice immunized with recombinant PA solely on the basis of in vitro anthrax toxin neutralization. This MAb was effective in prophylactic and postsymptomatic treatment of rabbits exposed to aerosolized anthrax spores, and a single intramuscular injection of 1 mg/kg of body weight fully protected cynomolgus monkeys challenged with aerosolized anthrax spores. Importantly, MAb 1303 defines a novel neutralizing epitope that requires Fc receptor engagement for maximal activity. F(ab')2 fragments of MAb 1303, which retain equivalent affinity for PA, are 10- to 100-fold less potent in neutralizing anthrax toxin in vitro. Addition of Fc receptor-blocking antibodies also greatly reduced the activity of MAb 1303. Moreover, we found that the neutralizing activity of mouse, rabbit, and human antisera elicited by PA vaccines was effectively abrogated by blocking Fc receptors. Selection of an anti-PA MAb by using a functional assay that is a surrogate for protection has resulted in the identification of a fully human MAb with potent activity in vivo and uncovered a previously unrecognized mechanism of antibody-mediated toxin neutralization that is important for currently used anthrax vaccines.

A Unique CD72 Epitope Suggests a Potential Interaction with FcγRII/CD32 on B Lineage Lymphocytes

It has long been known that ligation of the transmembrane CD72 glycoprotein delivers signals to B lymphocytes, with the outcome depending on context. Of particular interest is its ability to function as a counter-receptor/ ligand for the CD100 semaphorin protein. We have now obtained evidence that CD72 physically interacts on the lymphocyte membrane with Fcgamma receptor II (CD32). The association was first revealed with a new monoclonal antibody that recognizes polymorphic determinants on murine CD72. Although the specificity for CD72 was clear from immunoblotting, transfection and other experiments, staining with this reagent was inhibited when cells were pretreated with an Fc receptor-blocking antibody (CD16/CD32 specific). Furthermore, confocal microscopy revealed that the two molecules co-distributed on viable B cells. We also used the antibody to determine when CD72 becomes available to maturing lymphocytes. The marker is first acquired as large pre-B cells and enter the IL-7 independent phase of maturation within bone marrow. Subsequent interactions between CD72 and CD32 may cooperatively deliver negative signals that modulate humoral immune responses.

Antistreptokinase platelet-activating antibodies are common and heterogeneous

Platelet activation by antistreptokinase (SK) antibodies could impair the clinical effect of SK administration. To better describe anti-SK antibodies with particular emphasis on procoagulant activities as a result of platelet activation. Sera were collected from 146 patients with coronary artery disease: non-SK-treated, 95 from mainland France, 31 from French Polynesia; 20 patients from mainland in year 2 after SK treatment. Serum-induced SK-dependent platelet activation resulting in procoagulant activities was assessed with washed platelets from five donors representative of the known patterns of reactivities to IgG. Concentrations (2-5252 microg mL(-1)) and fibrinolytic neutralization titres (< 10 to > 1280) were found in the expected wide range and correlated (rho = 0.66, P < 0.0001). Platelet activation was detected with 145 samples, but varied in intensity and pattern (depending on the donors), although there was no systematic hierarchy; it was presumably due to IgG (inhibited by an IgG Fc receptor-blocking antibody and recovered in the IgG fraction) and only partially affected by aspirin. Marked platelet activation could be detected in samples with concentration as low as 2 microg mL(-1), and/or no detectable neutralizing titers. The way of immunization to SK was not found to influence the functional profile of antibodies. Anti-SK platelet-activating antibodies are widespread, heterogeneous, poorly predictable on the basis of their antifibrinolytic effect and strong enough to trigger procoagulant activities. Their clinical relevance should be formally assessed, using patients' own platelets for detection owing to the variation of platelet reactivity.

FcαRI (CD89) as a Novel Trigger Molecule for Bispecific Antibody Therapy

AbstractPromising results from clinical trials with unconjugated antibodies stimulated renewed interest in immune effector mechanisms of monoclonal antibodies (MoAbs). We investigated the potential of IgA as antibody isotype for cell- or complement-mediated tumor cell lysis and assessed the potential of its myeloid Fc receptor, FcαRI (CD89), as trigger molecule for bispecific antibody (BsAb)-mediated immunotherapy. Comparing hapten-directed antibodies of human IgA2 with IgG1 or IgG3 isotypes, we found all three to mediate effective killing of sensitized tumor target cells in whole blood assays. Analysis of effector mechanisms showed IgG-mediated lysis to be predominantly complement-dependent, whereas IgA-dependent killing was primarily effector cell-mediated. A comparison of effector cell populations in antibody-dependent cell-mediated cytotoxicity (ADCC) showed neutrophils to be most important for IgA-dependent tumor cell killing, involving FcαRI as shown with Fc receptor blocking antibodies. Reverse ADCC experiments against target cells sensitized with Fc receptor antibodies, or assays with FcαRI-directed bispecific antibodies confirmed FcαRI as effective trigger molecule in polymorphonuclear neutrophil (PMN)-mediated lysis. During granulocyte colony-stimulating factor (G-CSF ) therapy, (FcαRI × HER-2/neu) bispecific antibodies induced enhanced killing of HER-2/neu positive SK-BR–3 breast cancer cells in whole blood assays. This enhanced cytotoxicity was paralleled by increased PMN counts, which lead to higher effector to target cell ratios in G-CSF–primed blood. Furthermore, bispecific antibodies, directed to FcαRI and Candida albicans, enhanced neutrophils' phagocytosis of fungi. In summary, these results identify IgA as an effective antibody isotype for immunotherapy, working primarily via FcαRI on neutrophils. They suggest FcαRI-directed bispecific antibodies and G-CSF to be an attractive combination for malignant or infectious diseases.

FcαRI (CD89) as a Novel Trigger Molecule for Bispecific Antibody Therapy

Promising results from clinical trials with unconjugated antibodies stimulated renewed interest in immune effector mechanisms of monoclonal antibodies (MoAbs). We investigated the potential of IgA as antibody isotype for cell- or complement-mediated tumor cell lysis and assessed the potential of its myeloid Fc receptor, FcαRI (CD89), as trigger molecule for bispecific antibody (BsAb)-mediated immunotherapy. Comparing hapten-directed antibodies of human IgA2 with IgG1 or IgG3 isotypes, we found all three to mediate effective killing of sensitized tumor target cells in whole blood assays. Analysis of effector mechanisms showed IgG-mediated lysis to be predominantly complement-dependent, whereas IgA-dependent killing was primarily effector cell-mediated. A comparison of effector cell populations in antibody-dependent cell-mediated cytotoxicity (ADCC) showed neutrophils to be most important for IgA-dependent tumor cell killing, involving FcαRI as shown with Fc receptor blocking antibodies. Reverse ADCC experiments against target cells sensitized with Fc receptor antibodies, or assays with FcαRI-directed bispecific antibodies confirmed FcαRI as effective trigger molecule in polymorphonuclear neutrophil (PMN)-mediated lysis. During granulocyte colony-stimulating factor (G-CSF ) therapy, (FcαRI × HER-2/neu) bispecific antibodies induced enhanced killing of HER-2/neu positive SK-BR–3 breast cancer cells in whole blood assays. This enhanced cytotoxicity was paralleled by increased PMN counts, which lead to higher effector to target cell ratios in G-CSF–primed blood. Furthermore, bispecific antibodies, directed to FcαRI and Candida albicans, enhanced neutrophils' phagocytosis of fungi. In summary, these results identify IgA as an effective antibody isotype for immunotherapy, working primarily via FcαRI on neutrophils. They suggest FcαRI-directed bispecific antibodies and G-CSF to be an attractive combination for malignant or infectious diseases.

Fc receptor blocking antibodies after active immunization for the treatment of recurrent spontaneous abortion

In a prospective study 140 couples who had at least three spontaneous abortions (RSA) were studied for the presence of Fc receptor blocking antibodies detected by the erythrocyte antibody rosette inhibition (EAI) assay, for anti-paternal cytotoxic antibodies (APCA), and for mixed lymphocyte culture inhibiting (MLCI) antibodies before and after active immunization with paternal lymphocytes. The comparative analysis revealed the EAI assay to possess a higher sensitivity than the APCA and/or MLCI tests in monitoring the specific immune response after active immunization. The success of pregnancy in EAI positive post-immunization patients was not influenced by the presence or absence of APCA or MLCI. In the light of a successful pregnancy outcome of 85.7% ( n = 37) in this study we conclude that the monitoring of Fc receptor blocking antibodies is useful in active immunization protocols for RSA patients.

Non-cytotoxic antibodies in chronic lymphocytic leukaemia.

Non-cytotoxic Fc receptor blocking antibodies against autologous B lymphocytes were sought in sera from patients with chronic lymphocytic leukaemia (CLL), using a rosette inhibition assay. They were found in 11 of 52 (21%) of patients with CLL, but were not associated with previous blood transfusion or pregnancy, suggesting that they were unlikely to have resulted from allogeneic stimulation. Fc receptor blockade was more commonly detected in sera from patients with stage B rather than stage A CLL (Binet classification), though this did not achieve significance beyond the 90% level, and it was noted in 62.5% of those with lymphocyte doubling times of less than one year, compared with 36.3% of those whose lymphocyte doubling time was more than one year. The results indicate that autologous Fc receptor blocking antibody activity occurs in sera from patients with CLL, is more likely to be generated by the tumour itself than by allogeneic stimulation, and is associated with increased tumour load. Such antibodies may permit tolerance of tumour by the host.

Blocking antibodies and T cell subsets in long-term survivors of renal allografts.

Fc-receptor blocking antibodies as well as T cell subsets were studied in a group of 12 long-term survivors of renal allografts. The absolute number of T suppressor cells was similar to that found in a control group, but there was a significant decrease in the total number of T helper lymphocytes and a decreased helper/suppressor ratio. Sera from all patients tested showed inhibitory activity in the erythrocyte antibody model against leukemic B lymphocytes, demonstrating the presence of Fc-receptor blocking antibodies. Also inhibited was the ability of normal human lymphocytes to form E rosettes and to induce a local xenogeneic graft-versus-host reaction. These findings indicate that the Fc blocking antibodies are beneficial to the patient, possibly by abrogating the cellular and humoral immune mechanism that is detrimental to the kidney allograft.

Further investigation of nonspecific biological substance in anti-Rh(D) preparations.

Fc receptor-blocking activity, based on the EA rosetting inhibition of anti-Rh(D) gamma-globulin preparations, was determined during biological analyses. After sample purification by means of precipitation with a low concentration of ammonium sulphate, fractions were obtained containing anti-D activity and Fc receptor-blocking activity separately. Fc receptor-blocking antibodies inhibited both the formation of EA rosettes and the antibody-dependent cellular cytotoxicity activity. It is suggested that Fc receptor-blocking antibodies obtained during hyperimmunization probably result in the immunosuppressive effect of Rh immune globulin (via the blocking of Fc receptors of activated lymphocytes) that decreases in turn the progress of Rh immunization.

Differential expression of trophoblast lymphocyte cross-reactive (TLX) antigens on T and B lymphocytes.

Heterologous sera raised to human trophoblast (TLX antisera) have been shown to recognize peripheral blood lymphocytes (PBL); in unrelated studies noncytotoxic Fc receptors blocking B lymphocyte antibodies have been found in the sera of women during normal pregnancies. This study aimed to determine whether (a) there was any relation between Fc receptor blocking and cytotoxic anti-TLX activity in ten TLX sera and (b) different reactivity patterns arose when ten TLX antisera were tested in the cytotoxicity assay against separated T and B lymphocytes. Independent factor analysis showed four antisera with T lymphocytotoxicity patterns giving a loading high on one mathematical factor and low on a second. Three sera shared the opposite pattern and three were intermediate. The groups were similar but more discrete than those obtained when PBL were used as targets. Patterns of B lymphocytoxicity were dissimilar from T and both differed from the erythrocyte antibody rosette inhibition activity. Activity did not correlate with the HLA-A, -B, or -DR types carried by the panel cells. These data indicate that TLX antisera contain antibodies directly cytotoxic to antigens differentially expressed on T and B lymphocytes and that noncytotoxic Fc receptor blocking antibodies are not associated with any TLX groupings.

Further Investigation of Nonspecific Biological Substance in Anti-Rh(D) Preparations

Fc receptor-blocking activity, based on the EA resetting inhibition of anti-Rh(D) gamma-globulin preparations, was determined during biological analyses. After sample purification by means of precipitation with a low concentration of ammonium sulphate, fractions were obtained containing anti-D activity and Fc receptorblocking activity separately. Fc receptor-blocking antibodies inhibited both the formation of EA rosettes and the antibody-dependent cellular cytotoxicity activity. It is suggested that Fc receptor-blocking antibodies obtained during hyperimmunization probably result in the immunosuppressive effect of Rh immune globulin (via the blocking of Fc receptors of activated lymphocytes) that decreases in turn the progress of Rh immunization.

Evidence that protective Fc-receptor-blocking antibodies in renal transplantation are alloantibodies not autoantibodies.

We have previously shown that the presence in pretransplant recipient sera of Fc receptor blocking antibodies detected by the EA inhibition assay is correlated with improved allograft survival. Twenty-four such sera were assessed for the presence of autoantibodies by the EA inhibition and lymphocytotoxicity assays. No autolymphocytotoxic antibodies were found, and autologous EA inhibition was noted in only one case. EA-inhibiting alloantibodies did occur, and their presence was correlated with improved allograft survival. Sera from 37 dialysis patients were also studied, and neither autologous EA inhibiting nor autologous lymphocytotoxic antibodies were present. Thus Fc receptor blocking alloantibodies that were correlated with improved renal transplant survival were not autoantibodies.

POSSIBLE MECHANISM OF ACTION OF TRANSFUSION EFFECT IN RENAL TRANSPLANTATION

The mechanism by which blood transfusions given before renal transplantation improves allograft survival was studied in 31 transplant recipients. The presence of non-cytotoxic, Fc receptor blocking antibodies to donor and leukaemic B lymphocytes in pre-transplant sera correlated with both improved graft survival (p<0 ·03 and <0·1, respectively) and the number of blood transfusions given (p<0 ·05 and <0 ·03, respectively). Moreover, 6 out of 10 previously untransfused prospective transplant recipients developed these potentially protective antibodies during a course of elective blood transfusions. These results indicate that such non-cytotoxic, Fc receptor blocking antibodies in pretransplant recipient sera (a) are associated with improved allograft survival, (b) correlate with the number of blood transfusions given, and (c) can develop in response to blood transfusion.