Christian Kleist

Generation of a large complex antibody library from multiple donors

We have generated a large complex library of single chain antibodies based on four individual libraries from each of 50 donors. DNA coding for the heavy and light chain variable domains of the IgM and IgG repertoires was amplified by PCR using two different sets of primers. Each individual library was composed of approximately 1-5x10(7) independent clones giving a final combined library of 4x10(9) members. Screening this library by phage display of single chain antibodies with small haptens, peptides and proteins yielded specific antibodies for each class of antigen.

Active-specific immunotherapy of human cancers with the heat shock protein Gp96-revisited.

The passive administration of specific antibodies that selectively target tumors is a well‐known strategy in cancer treatment. Active immunotherapy using peptide vaccines, in contrast, is expected to induce specific, cytolytic T cells in the patient, which react against tumor antigens and destroy malignant cells. Although several concepts exist, the identification and low immunogenicity of tumor‐specific peptides remain a serious problem. Heat shock proteins (HSPs), notably glycoprotein (Gp) 96, are of special interest, because they are able to take molecular peptide‐fingerprints of the protein array characteristic for a particular cell. Association of Gp96 with peptides has been shown to be essential for crosspresentation and activation of T cells. Consequently, Gp96–peptide complexes extracted from cancer cells harbor the tumor‐specific peptides and are immunogenic, thus offering a tool for active immunization against the tumor. Already, several immunotherapy studies of human cancers have been carried out, showing no severe adverse effects but unfortunately only limited improvement in the clinical outcome. Vitespen, a commercial HSP–peptide complex vaccine based on tumor‐derived Gp96, seems to induce an improved overall survival for subsets of early stage melanoma and kidney cancer patients. The limited access to vaccine material derived from the autologous tumor requires the development of alternative protocols. Moreover, counteracting immunosuppressive mechanisms induced by the malignancy might further improve the efficacy of vaccinations. This review critically analyzes the current state of clinical immunotherapy with Gp96, with special attention to Vitespen.

Mitomycin C-treated dendritic cells inactivate autoreactive T cells: Toward the development of a tolerogenic vaccine in autoimmune diseases

Treatment of autoimmune diseases remains a challenge for immunological research. An ideal therapy should inhibit the immune reaction against the diseased organ and leave the rest of the immune response intact. Our previous studies showed that donor-derived dendritic cells (DCs) treated in vitro with mitomycin C (MMC) suppress rat heart allograft rejection if injected into recipients before transplantation. Here we analyze their efficacy in controlling autoimmunity. MMC-DCs loaded with myelin-basic-protein (MBP) inhibited specific T cells derived from multiple sclerosis patients in vitro. If coincubated with MMC-DCs, T cells were arrested in the G0/G1 cell cycle phase. Microarray gene scan showed that MMC influences the expression of 116 genes in DCs, one main cluster comprising apoptotic and the second cluster immunosuppressive genes. Apparently, the combination of apoptosis with expression of tolerogenic molecules renders MMC-DCs suppressive. MBP-loaded MMC-DCs also inhibited mouse T cells in vitro and, in contrast to MBP-loaded naïve DCs, did not induce experimental autoimmune encephalitis. Most importantly, mice vaccinated with inhibitory DCs became resistant to the disease. Whereas this is not the first report on generation of suppressive DCs, it delineates a method using a clinically approved drug at nontoxic concentrations, which yields irreversibly changed DCs, effective across species in vitro and in vivo.

Suppressive dendritic cells as a tool for controlling allograft rejection in organ transplantation: promises and difficulties.

The most important antigen-presenting cells are dendritic cells (DCs), which play a central role in the initiation of immunity and tolerance. Their immunoregulatory properties offer the potential of donor-specific control of graft rejection after organ transplantation. It has not been clarified which DC subpopulations mediate tolerance, and the use of natural DCs for therapeutic applications is therefore problematic. Suppressive DCs can be generated in vitro by treating the cells with biologic, pharmacologic, or genetic agents. Here we discuss approaches for generating inhibitory DCs and present DC-based animal models for control of allograft rejection. A prerequisite of suppressive DCs for therapeutic application in clinical transplantation is a reproducible method for their generation as well as the induction of irreversible suppressive function. Based on lessons learned from the use of DCs as tools in clinical vaccine trials in cancer, we discuss the unknown aspects and risks of DC therapy in transplantation.

Melan-A/MART1 Analog Peptide Triggers Anti-myeloma T-cells Through Crossreactivity With HM1.24

The Melan-Aaa26–35 (EAAGIGILTV) peptide is a human leukocyte antigen (HLA)-A2-restricted T-cell epitope within the Melan-A/MART-1 tumor antigen expressed on malignant melanoma cells. Melan-A and Melan-A analog (ELAGIGILTV, Melan-Aaa26–35*A27L) specific T-cells can be expanded reliably for immunotherapeutic approaches in vitro. We studied the ability of Melan-A analog (ELAGIGILTV, Melan-Aaa26–35*A27L) specific T-cells to recognize the HM1.24aa22−30 (LLLGIGILV) peptide within the HM1.24 antigen presented by normal and malignant plasma cells. Peripheral blood mononuclear cells from HLA-A2+ healthy donors and HLA-A2+ multiple myeloma (MM) patients were stimulated with Melan-A analog peptide-loaded autologous dendritic cells, and expanded in vitro. T-cell activation was assessed by interferon-γ specific enzyme-linked immunosorbent spot and cytotoxicity by 51Chromium-release-assays. The frequency of Melan-A analog specific CD8+ T-cells was detected by using tetramers. Melan-A analog specific T-cells from HLA-A2+ healthy donors and HLA-A2+ MM patients showed a interferon-γ secretion mediated by HM1.24aa22−30 peptide-pulsed T2 cells and lysed the HLA-A2+ HM1.24+ U266 and XG-1 human myeloma derived cell-lines as well as the B-lymphoblastoid cell-line IM-9. Melan-A analog specific T-cells from MM patients specifically lysed autologous MM cells. The current data demonstrate that Melan-A analog specific T-cells crossreact with HM1.24aa22−30. They might be a tool for the future use in immunotherapy against MM.

Cytokine-induced killer cells targeted by the novel bispecific antibody CD19xCD5 (HD37xT5.16) efficiently lyse B-lymphoma cells

Due to their dual binding capacity, bispecific antibodies (bsAb) can be used to cross-link cytotoxic effector cells with malignant targets and may thereby improve adoptive immunotherapy. In this study, the development and preclinical testing of the quadroma-derived bsAb HD37xT5.16 of the specificity CD19xCD5 is reported. Effector cells used were a population of ex vivo expanded and activated T cells called cytokine-induced killer (CIK) cells expressing CD5. When combined with CIK cells, the cytolytic potency of HD37xT5.16 against CD19 positive B cell lymphoma lines was comparable to that observed with a previously described CD19xCD3 bsAb. Further on, we could demonstrate that bsAb CD19xCD5, in contrast to its CD3-binding counterpart, does not induce proliferation of resting T cells and causes only little activation-induced cell death. Therefore, this novel bsAb binding effector T cells via CD5 may be particularly useful in combination with adoptive transfer of ex vivo activated T cells, e.g., in the setting of adoptive immunotherapy after allogeneic stem cell transplantation. The in vitro studies outlined here support the experimental use of bsAb HD37xT5.16 in preclinical in vivo models for evaluation of its safety and efficacy profile.

Reduction of Skeletal Muscle Injury in Composite Tissue Allotransplantation by Heat Stress Preconditioning

Ischemia-reperfusion injury is a dominant factor limiting tissue survival in any microsurgical tissue transplantation, a fact that also applies to allogeneic hand transplantation. The clinical experience of the 12 human hand transplantations indicates that shorter ischemia times result in reduced tissue damage and, ultimately, in better hand function. Heat stress preconditioning and the accompanying up-regulation of the heat shock protein 72 have been shown to reduce the ischemia-reperfusion injury following ischemia of various organs, including organ transplantation. The aim of this study was to reduce the ischemia-reperfusion injury in a model of composite tissue allotransplantation. Allogeneic hind limb transplantations were performed from Lewis (donor) to Brown-Norway rats. Donor rats in group A (n = 10) received a prior heat shock whereas rats in group B (n = 10) did not receive any prior heat shock. Group C served as a control group without transplantation. The transplantations were performed 24 hours after the heat shock, at which time the heat shock protein 72 was shown to be up-regulated. The outcome was evaluated 24 hours after transplantation by nitroblue tetrazolium staining and wet-to-dry weight ratio of muscle slices (anterior tibial muscle). The nitroblue tetrazolium staining showed a significant reduction of necrotic muscle in group A (prior heat shock) (p = 0.005). The wet-to-dry ratio was significantly reduced in group A (prior heat shock), indicating less muscle edema and less tissue damage (p = 0.05). Heat shock preconditioning 24 hours before an ischemic event leads to an up-regulation of heat shock protein 72 in muscle and to a tissue protection reducing ischemia-reperfusion injury in composite tissue transplantation.

Ex vivo perfusion with mitomycin C containing solution prolongs heart graft survival in rats.

Mitomycin C (MMC) is an alkylating agent which suppresses allogeneic T-cell responses. We analyzed the effect of graft perfusion with MMC on transplant survival. Hearts from Brown-Norway (BN) rats were perfused ex vivo with MMC-containing solution, stored and implanted into Lewis (LEW) rats. In order to analyze the in vivo effect of MMC, recipients received MMC posttransplantation or were pretreated with MMC-incubated donor-derived peripheral blood mononuclear cells (PBMCs). The results show that MMC-perfusion significantly prolongs graft survival. Treatment of recipients with MMC has no effect, whereas MMC-treated donor PBMCs injected into the recipient prolong graft survival. Our findings indicate that the targeted perfusion of donor hearts with MMC-containing solution protects the graft from rejection.

Identification of HLA‐A2 restricted T‐cell epitopes within the conserved region of the immunoglobulin G heavy‐chain in patients with multiple myeloma

Objective:  The aim of this study is the identification of HLA‐A2 restricted T‐cell epitopes in the conserved region of the immunoglobulin‐G‐heavy‐chain (IgGH) that can be used for immunotherapy in multiple myeloma (MM) patients.

Generation of suppressive blood cells for control of allograft rejection.

Our previous studies in rats showed that incubation of monocytic dendritic cells (DCs) with the chemotherapeutic drug mitomycin C (MMC) renders the cells immunosuppressive. Donor-derived MMC-DCs injected into the recipient prior to transplantation prolonged heart allograft survival. Although the generation of DCs is labour-intensive and time-consuming, peripheral blood mononuclear cells (PBMCs) can be easily harvested. In the present study, we analyse under which conditions DCs can be replaced by PBMCs and examine their mode of action. When injected into rats, MMC-incubated donor PBMCs (MICs) strongly prolonged heart allograft survival. Removal of monocytes from PBMCs completely abrogated their suppressive effect, indicating that monocytes are the active cell population. Suppression of rejection was donor-specific. The injected MICs migrated into peripheral lymphoid organs and led to an increased number of regulatory T-cells (Tregs) expressing cluster of differentiation (CD) markers CD4 and CD25 and forkhead box protein 3 (FoxP3). Tolerance could be transferred to syngeneic recipients with blood or spleen cells. Depletion of Tregs from tolerogenic cells abrogated their suppressive effect, arguing for mediation of immunosuppression by CD4⁺CD25⁺FoxP3⁺ Tregs. Donor-derived MICs also prolonged kidney allograft survival in pigs. MICs generated from donor monocytes were applied for the first time in humans in a patient suffering from therapy-resistant rejection of a haploidentical stem cell transplant. We describe, in the present paper, a simple method for in vitro generation of suppressor blood cells for potential use in clinical organ transplantation. Although the case report does not allow us to draw any conclusion about their therapeutic effectiveness, it shows that MICs can be easily generated and applied in humans.