David J. Munster

Antibody to the dendritic cell surface activation antigen CD83 prevents acute graft-versus-host disease

Allogeneic (allo) hematopoietic stem cell transplantation is an effective therapy for hematological malignancies but it is limited by acute graft-versus-host disease (GVHD). Dendritic cells (DC) play a major role in the allo T cell stimulation causing GVHD. Current immunosuppressive measures to control GVHD target T cells but compromise posttransplant immunity in the patient, particularly to cytomegalovirus (CMV) and residual malignant cells. We showed that treatment of allo mixed lymphocyte cultures with activated human DC-depleting CD83 antibody suppressed alloproliferation but preserved T cell numbers, including those specific for CMV. We also tested CD83 antibody in the human T cell–dependent peripheral blood mononuclear cell transplanted SCID (hu-SCID) mouse model of GVHD. We showed that this model requires human DC and that CD83 antibody treatment prevented GVHD but, unlike conventional immunosuppressants, did not prevent engraftment of human T cells, including cytotoxic T lymphocytes (CTL) responsive to viruses and malignant cells. Immunization of CD83 antibody-treated hu-SCID mice with irradiated human leukemic cell lines induced allo antileukemic CTL effectors in vivo that lysed 51Cr-labeled leukemic target cells in vitro without further stimulation. Antibodies that target activated DC are a promising new therapeutic approach to the control of GVHD.

Single step enrichment of blood dendritic cells by positive immunoselection

Dendritic cells (DC) for cancer immunotherapy protocols are generated most commonly by in vitro differentiation of monocytes with exogenous cytokines (Mo-DC). However, Mo-DC differ in their molecular phenotype and function from blood DC (BDC). Clinical isolation of BDC has been limited to the use of density gradients, which result in low yields of variable purity. We have developed a DC enrichment platform, which uses the CMRF-44 (IgM) or CMRF-56 (IgG) monoclonal antibodies (mAb) to select BDC that express these antigens after a short overnight incubation. After culture of peripheral blood mononuclear cells (PBMC) in autologous/AB serum, biotinylated CMRF-44 was used to select DC in a single step immuno-magnetic bead procedure; this produced populations containing up to 99% CMRF-44(+) cells, including up to 67% CMRF-44(+) CD14(-) CD19(-) DC, from an initial starting population of approximately 0.5%. We observed consistent differences in the purities obtained from individual donors with a mean of 54% CMRF-44(+) cells (range 19-99%). Similar results were obtained using biotinylated CMRF-56 mAb, an antibody identifying a comparable population in cultured PBMC. We recovered an average of 54% and 66% of the available BDC in separations performed with the CMRF-44 and CMRF-56 mAb, respectively. The reproducibility of the procedure and the ability to perform it in a closed sterile system makes it suitable for clinical use. Larger scale preparations starting from apheresis derived PBMC will produce sufficient BDC for immunotherapy protocols. The purified BDC elicited strong allogeneic mixed leukocyte reactions and HLA classes II- and I-restricted antigen-specific primary immune responses.

Activated Circulating Dendritic Cells After Hematopoietic Stem Cell Transplantation Predict Acute Graft-Versus-Host Disease

Background. Dendritic cells (DC) are central to the development of acute graft-versus-host disease (GVHD) following allogeneic hematopoietic stem cell transplantation (alloHSCT). We hypothesized that DC activation status determines the severity of GVHD and that activated DC may be detected in the circulation prior to clinical presentation of GVHD. Methods. Following transplant, blood samples were obtained twice weekly from alloHSCT patients. Myeloid (CD11c+) and plasmacytoid (CD123hi) DC were enumerated by flow cytometry, and activated myeloid DC were identified using the CMRF-44 monoclonal antibody. Results. Of 40 alloHSCT patients, 26 developed acute GVHD. Severity of GVHD was associated with low total blood DC counts (P=0.007) and with low myeloid and plasmacytoid DC numbers (P=0.015 and 0.003). The CMRF-44 antigen was expressed on blood CD11c+ DC in all cases prior to GVHD onset, whereas of the 14 patients without GVHD, seven had no CMRF-44+ CD11c+ DC. Patients with CMRF-44+ CD11c+ DC in more than 20% of samples were more likely to subsequently develop acute GVHD (P=0.001, odds ratio=37.1), while patients who developed grade 2–4 GVHD had prior higher percentages of CMRF-44+ CD11c+ DC compared to grade 0–1 GVHD patients (P=0.001). CMRF-44 expression on >7.9% CD11c+ DC predicted for subsequent development of GVHD with a sensitivity of 87.5% and specificity of 79.2%. Conclusions. Activation status, as assessed by CMRF-44 antigen expression, of blood CD11c+ DC is highly associated with acute GVHD and these cells may be targets for therapeutic intervention.

A method for rapid, ligation-independent reformatting of recombinant monoclonal antibodies

Recombinant monoclonal antibodies currently dominate the protein biologics marketplace. The path from target antigen discovery and screening, to a recombinant therapeutic antibody can be time-consuming and laborious. We describe a set of expression vectors, termed mAbXpress, that enable rapid and sequence-independent insertion of antibody variable regions into human constant region backbones. This method takes advantage of the In Fusion cloning system from Clontech, which allows ligation-free, high-efficiency insertion of the variable region cassette without the addition of extraneous amino acids. These modular vectors simplify the antibody reformatting process during the preliminary evaluation of therapeutic or diagnostic candidates. The resulting constructs can be used directly for transient or amplifiable, stable expression in mammalian cells. The effectiveness of this method was demonstrated by the creation of a functional, fully human anti-human CD83 monoclonal antibody.

Reduced Intensity Conditioning for Allogeneic Hematopoietic Stem-Cell Transplant Determines the Kinetics of Acute Graft-Versus-Host Disease.

Background. Preparative myeloablative conditioning regimens for allogeneic hematopoietic stem-cell transplantation (HSCT) may control malignancy and facilitate engraftment but also contribute to transplant related mortality, cytokine release, and acute graft-versus-host disease (GVHD). Reduced intensity conditioning (RIC) regimens have decreased transplant related mortality but the incidence of acute GVHD, while delayed, remains unchanged. There are currently no in vivo allogeneic models of RIC HSCT, limiting studies into the mechanism behind RIC-associated GVHD. Methods. We developed two RIC HSCT models that result in delayed onset GVHD (major histocompatibility complex mismatched (UBI-GFP/BL6 [H-2b]→BALB/c [H-2d]) and major histocompatibility complex matched, minor histocompatibility mismatched (UBI-GFP/BL6 [H-2b]→BALB.B [H-2b])) enabling the effect of RIC on chimerism, dendritic cell (DC) chimerism, and GVHD to be investigated. Results. In contrast with myeloablative conditioning, we observed that RIC-associated delayed-onset GVHD is characterized by low production of tumor necrosis factor-α, maintenance of host DC, phenotypic DC activation, increased T-regulatory cell numbers, and a delayed emergence of activated donor DC. Furthermore, changes to the peritransplant milieu in the recipient after RIC lead to the altered activation of DC and the induction of T-regulatory responses. Reduced intensity conditioning recipients suffer less early damage to GVHD target organs. However, as donor cells engraft, activated donor DC and rising levels of tumor necrosis factor-α are associated with a later onset of severe GVHD. Conclusions. Delineating the mechanisms underlying delayed onset GVHD in RIC HSCT recipients is vital to improve the prediction of disease onset and allow more targeted interventions for acute GVHD.

Immunosuppressive properties of mesenchymal stromal cell cultures derived from the limbus of human and rabbit corneas

BACKGROUND AIMS Mesenchymal stromal cells (MSCs) cultivated from the corneal limbus (L-MSCs) provide a potential source of cells for corneal repair. In the present study, we investigated the immunosuppressive properties of human L-MSCs and putative rabbit L-MSCs to develop an allogeneic therapy and animal model of L-MSC transplantation. METHODS MSC-like cultures were established from the limbal stroma of human and rabbit (New Zealand white) corneas using either serum-supplemented medium or a commercial serum-free MSC medium (MesenCult-XF Culture Kit; Stem Cell Technologies, Melbourne, Australia). L-MSC phenotype was examined by flow cytometry. The immunosuppressive properties of L-MSC cultures were assessed using mixed leukocyte reactions. L-MSC cultures were also tested for their ability to support colony formation by primary limbal epithelial (LE) cells. RESULTS Human L-MSC cultures were typically CD34⁻, CD45⁻ and HLA-DR⁻ and CD73⁺, CD90⁺, CD105⁺ and HLA-ABC⁺. High levels (>80%) of CD146 expression were observed for L-MSC cultures grown in serum-supplemented medium but not cultures grown in MesenCult-XF (approximately 1%). Rabbit L-MSCs were approximately 95% positive for major histocompatibility complex class I and expressed lower levels of major histocompatibility complex class II (approximately 10%), CD45 (approximately 20%), CD105 (approximately 60%) and CD90 (<10%). Human L-MSCs and rabbit L-MSCs suppressed human T-cell proliferation by up to 75%. Conversely, L-MSCs from either species stimulated a 2-fold to 3-fold increase in LE cell colony formation. CONCLUSIONS L-MSCs display immunosuppressive qualities in addition to their established non-immunogenic profile and stimulate LE cell growth in vitro across species boundaries. These results support the potential use of allogeneic L-MSCs in the treatment of corneal disorders and suggest that the rabbit would provide a useful pre-clinical model.

CMRF-44 antibody-mediated depletion of activated human dendridic cells: a potential means for improving allograft survival.

Background. Activated dendritic cells (DC) initiate immune responses by presenting antigen, including alloantigen from tissue grafts, to T lymphocytes. The potential to deplete or inactivate differentiated-activated DC during allogeneic transplantation represents a new approach to immunosuppression. Methods. The authors investigated the potential of the monoclonal antibody CMRF-44, which has specificity for a DC-associated differentiation-activation antigen, to induce complement-mediated lysis of activated human DC. Peripheral blood mononuclear cells (PBMC), or purified DC preparations, were cultured overnight to activate endogenous DC, resulting in the expression of CMRF-44 antigen and CD83. These were then treated with CMRF-44 and complement. Depletion of activated DC was monitored by flow cytometry. Results. Eighty-nine percent of activated (CD83+) DC in cultured PBMC were depleted by treatment with CMRF-44 and autologous serum (AS) (complement source; mean percentage of CD83+-CD14−-CD19− cells=0.06%; cf 0.50% for heat-inactivated AS controls, P <0.0005, n=7). Ninety-five percent of cultured purified myeloid DC were depleted by this treatment, compared with only 43% of similarly treated lymphoid DC. Overnight culture also increases CMRF-44 antigen on a proportion of B cells and mononuclears, but only 24% of these cells were depleted. This treatment considerably reduced the ability of PBMC to stimulate allogeneic CD4+ CD45RA+ T lymphocytes. Similarly, the T-cell proliferative responses to recall and naive antigens were significantly reduced. Conclusions. CMRF-44 may be a suitable candidate for a new selective immunosuppressive strategy, targeting differentiated-activated but not resting DC. It may have applications in preventing GVHD in allogeneic bone marrow transplantation and facilitate immunoacceptance of solid organ allografts.

Immunosuppressive human anti-CD83 monoclonal antibody depletion of activated dendritic cells in transplantation

Current immunosuppressive/anti-inflammatory agents target the responding effector arm of the immune response and their nonspecific action increases the risk of infection and malignancy. These effects impact on their use in allogeneic haematopoietic cell transplantation and other forms of transplantation. Interventions that target activated dendritic cells (DCs) have the potential to suppress the induction of undesired immune responses (for example, graft versus host disease (GVHD) or transplant rejection) and to leave protective T-cell immune responses intact (for example, cytomegalovirus (CMV) immunity). We developed a human IgG1 monoclonal antibody (mAb), 3C12, specific for CD83, which is expressed on activated but not resting DC. The 3C12 mAb and an affinity improved version, 3C12C, depleted CD83+ cells by CD16+ NK cell-mediated antibody-dependent cellular cytotoxicity, and inhibited allogeneic T-cell proliferation in vitro. A single dose of 3C12C prevented human peripheral blood mononuclear cell-induced acute GVHD in SCID mouse recipients. The mAb 3C12C depleted CMRF-44+CD83bright activated DC but spared CD83dim/- DC in vivo. It reduced human T-cell activation in vivo and maintained the proportion of CD4+ FoxP3+ CD25+ Treg cells and also viral-specific CD8+ T cells. The anti-CD83 mAb, 3C12C, merits further evaluation as a new immunosuppressive agent in transplantation.

Practical blood dendritic cell vaccination for immunotherapy of multiple myeloma.

Therapeutic vaccination combined with new drugs may cure multiple myeloma (MM). We have developed a bio‐process to purify CMRF‐56 monoclonal antibody (mAb) and a standard operating procedure to immunoselect blood dendritic cells (BDC). Leucopheresed mononuclear cells were cultured overnight, labelled with CMRF‐56 mAb and BDC prepared using a clinical scale immunoselection system. The mean BDC yield from healthy donors was 48% (n = 6, purity 28%). Preparations from MM patients (n = 6, yield 47%, purity 35%) primed cytotoxic T lymphocytes (CTL) to clinically relevant MM antigens. This procedure can be performed readily by clinical cell manufacturing units to facilitate BDC vaccination studies.

CMRF-56+ blood dendritic cells loaded with mRNA induce effective antigen-specific cytotoxic T-lymphocyte responses

ABSTRACT There are numerous transcriptional, proteomic and functional differences between monocyte-derived dendritic cells (Mo-DC) and primary blood dendritic cells (BDC). The CMRF-56 monoclonal antibody (mAb) recognizes a cell surface marker, which is upregulated on BDC following overnight culture. Given its unique ability to select a heterogeneous population of BDC, we engineered a human chimeric (h)CMRF-56 IgG4 mAb to isolate primary BDC for potential therapeutic vaccination. The ability to select multiple primary BDC subsets from patients and load them with in vitro transcribed (IVT) mRNA encoding tumor antigen might circumvent the issues limiting the efficacy of Mo-DC. After optimizing and validating the purification of hCMRF-56+ BDC, we showed that transfection of hCMRF-56+ BDC with mRNA resulted in efficient mRNA translation and antigen presentation by myeloid BDC subsets, while preserving superior DC functions compared to Mo-DC. Immune selected and transfected hCMRF-56+ BDC migrated very efficiently in vitro and as effectively as cytokine matured Mo-DC in vivo. Compared to Mo-DC, hCMRF-56+ BDC transfected with influenza matrix protein M1 displayed superior MHC peptide presentation and generated potent antigen specific CD8+ T-cell recall responses, while Wilms tumor 1 (WT1) transfected CMRF-56+ BDC generated effective primary autologous cytotoxic T-cell responses. The ability of the combined DC subsets within hCMRF-56+ BDC to present mRNA delivered tumor antigens merits phase I evaluation as a reproducible generic platform for the next generation of active DC immune therapies.