[The prediction of immunogenicity of therapeutic proteins]. / La prédiction de l'immunogénicité des protéines thérapeutiques.

Immunogenicity of therapeutic proteins is a nightmare for industrials because induced antibodies can neutralize the therapeutic activity and provoke autoimmune symptoms. It was believed that sequence humanization would be sufficient to tackle these problems but multiple clinical examples now demonstrate that humanization does not suffice to abrogate immune responses. In order to predict immunogenicity of therapeutic proteins, different approaches have been developed, among which the most relevant ones are based on the evaluation of the response of naïve CD4 T lymphocytes specific for therapeutic proteins. Other approaches also exist or are in development. This review is the state of art in the different technologies that are proposed to predict immunogenicity of therapeutic proteins.

Quantitative analysis of the CD4 T-cell repertoire specific to therapeutic antibodies in healthy donors.

Therapeutic antibodies are generally partially to fully humanized, yet they can show unwanted immunogenicity and lead to antibody response and adverse effects when administered to humans. As immunogenicity relies on a T-cell-dependent mechanism, we have evaluated in vitro the size of the preexisting CD4 T-cell repertoire specific to therapeutic antibodies in healthy donors. Specific CD4 T cells of individuals with different HLA-DR allotypes were amplified by in vitro stimulation and quantified. Well-known immunogenic proteins, KLH and a murine antibody, exhibited a strong in vitro T-cell response characterized by a mean of preexisting T cells >1 cell/10(6) cells. In contrast, the preexisting CD4 T-cell repertoires specific to 2 chimeric, 2 humanized, and 2 fully human antibodies remained generally inferior to this value, confirming the role of species-specific sequences in their shaping. Mean values ranged from 0.01 to 0.3 cell/10(6) cells and varied not necessarily in relationship with the humanization level of the therapeutic antibodies. Relationship with their known immunogenicity is discussed. These results contribute to a better understanding and prediction of immunogenicity of therapeutic antibodies in humans.

Quantification of the preexisting CD4 T-cell repertoire specific for human erythropoietin reveals its immunogenicity potential.

Antibody-mediated pure red cell aplasia is a rare but serious event resulting from the induction of neutralizing erythropoietin (Epo)-specific antibodies provoked by treatment with recombinant Epo. Because of the crucial role of CD4 T cells in humoral response, we have quantified the number of Epo-specific CD4 T cells in the blood of normal donors by in vitro stimulation. An important repertoire of preexisting Epo-specific T cells was observed in almost half of the donors, comparable with that of non-self-proteins. This observation suggests that, at the steady state, endogenous Epo weakly contributes to tolerance induction and may be ignored by the immune system. As a result, circulating Epo-specific CD4 T cells could be prone to be activated by altered batches of Epo, providing them with costimulatory signals. Our data also highlight the relevance of T-cell assays performed with normal donors to evaluate the potential immunogenicity of therapeutic proteins.

Dramatic efficacy improvement of a DC-based vaccine against AML by CD25 T cell depletion allowing the induction of a long-lasting T cell response.

Dendritic cell (DC)-based vaccination is a promising approach to enhance anti-tumor immunity that could be considered for acute myeloid leukemia (AML) patients with high-risk of relapse. Our purpose was to study the efficiency and to optimize the immunogenicity of a DC-based vaccine in a preclinical AML murine model. In this report, C57BL6 mice were vaccinated with DC pulsed with peptides eluted (EP) from the syngeneic C1498 myelomonocytic leukemic cell line in a prophylactic setting. In this model, a natural antileukemic immunity mediated by NK cells was observed in the control unloaded DC-vaccinated group. On the other hand, we showed that the cytotoxic antileukemic immune response induced by vaccination with eluted peptides pulsed-DC (DC/EP), in vitro and in vivo, was mainly mediated by CD4(+) T cells. Treatment with anti-CD25 antibody to deplete CD4(+) CD25(+) regulatory T cells before DC-vaccination dramatically improved the antileukemic immune response induced by immunization, and allowed the development of long-lasting immune responses that were tumor protective after a re-challenge with leukemic cells. Our results suggest that this approach could be successful against weakly immunogenic tumors such as AML, and could be translated in human.

DC-based vaccine loaded with acid-eluted peptides in acute myeloid leukemia: the importance of choosing the best elution method.

Tumor-associated peptides isolated by acid elution are frequently used for therapeutic immunization against various tumors both in mice and in humans. In acute myeloid leukemia (AML), the frequent accessibility of a large tumor burden allows for extraction of peptides from leukemia cells by using either citrate-phosphate (CP) or trifluoroacetic acid (TFA) buffer. To develop an optimal immunotherapeutic protocol for AML patients, we evaluated both in mice and in humans, the immunogenicity of peptides eluted from leukemia cells with the two acids (TFA or CP). Although ex vivo studies in mice showed that both prophylactic immunizations with mature dendritic cells (DC) loaded with TFA-peptides (DC/TFA), or CP-peptides (DC/CP), were able to stimulate specific antileukemia immune responses, only vaccination with DC/TFA was able to prevent leukemia outgrowth. Moreover, in humans, only DC/TFA generated significant antileukemia CD4(+) and cytotoxic CD8(+) T cell responses in vitro. In summary, these data demonstrate that the choice of the acid elution procedure to isolate immunogenic peptides strongly influences the efficacy of the antileukemia immune responses. These finding raise essential considerations for the development of immunotherapeutic protocols for cancer patients. In our model, our results argue for the use of the TFA elution method to extract immunogenic AML-associated peptides.

Autologous peptides eluted from acute myeloid leukemia cells can be used to generate specific antileukemic CD4 helper and CD8 cytotoxic T lymphocyte responses in vitro.

BACKGROUND AND OBJECTIVES: The poor prognosis of acute myeloid leukemia (AML) treated with conventional chemotherapy justifies seeking additional immunotherapeutic approaches to eliminate minimal residual disease. Hence, we evaluated the feasibility of generating in vitro antileukemic immune responses, which would bypass the need for epitope identification and rely on antigen presentation by autologous dendritic cells. DESIGN AND METHODS: Naturally processed peptides were extracted by acid elution from circulating AML cells of six patients at diagnosis. Mature dendritic cells (mDC) were derived from autologous monocytes obtained when the patients were in complete remission, and were loaded with the pool of eluted peptides to stimulate autologous T lymphocytes in vitro. RESULTS: We were able to induce in vitro antileukemic Th1 responses characterized by CD4(+) T-cell proliferation, significant interferon-gamma secretion by both CD4+ and CD8(+) T lymphocytes by recognition of autologous AML cells and generation of cytotoxic CD8(+) T lymphocytes. INTERPRETATION AND CONCLUSIONS: These results demonstrate that naturally processed peptides eluted from AML cells and presented by differentiated autologous mDC could be immunogenic in vitro. Although more in vitro data will be needed to check the safety of such an approach, notably to rule out possible autoimmune adverse effects, these results lay the basis for a potentially effective antileukemic immunotherapy for high-risk AML patients with minimal residual disease.

Ex vivo characterization of multiepitopic tumor-specific CD8 T cells in patients with chronic myeloid leukemia: implications for vaccine development and adoptive cellular immunotherapy.

Identification of tumor-associated Ags is a prerequisite for vaccine-based and adoptive immune therapies. Some tumor-associated Ags elicit specific CD8 T cells in patients with chronic myeloid leukemia (CML). Here, we characterized ex vivo responses of CD8 T cells from CML patients to extrajunction bcr-abl peptides and telomerase 540-548 hTert, PR1, and WT1 peptides. CML-specific CD8 T cells were present in most treated patients and were usually multiepitopic: WT1, hTert, PR1, and bcr74 tetramer(+) cells were detected in 85, 82, 67, and 61% of patients, respectively. The breadth and magnitude of these responses did not differ significantly according to treatment or disease status. CML-specific tetramer(+) CD8 T cells had a predominantly memory phenotype, an intermediate perforin content, and low intracellular IFN-gamma accumulation in the presence of the relevant peptide. However, in short-term culture with HLA-matched leukemia cells, the patients' memory T cells were specifically reactivated to become IFN-gamma-producing effector cells, suggesting that CD8 T cell precursors with lytic potential are present in vivo and can be activated by appropriate stimulation. In conclusion, this study shows that multiepitopic tumor-specific CD8 T cell responses occur naturally in most CML patients, opening the way to new strategies for enhancing anti-CML immunity, in particular in patients with minimal residual disease.

Absence or low expression of fas-associated protein with death domain in acute myeloid leukemia cells predicts resistance to chemotherapy and poor outcome.

In acute myeloid leukemia (AML), coexpression of death receptors and ligands of the tumor necrosis factor (TNF) receptor/TNF-alpha superfamily on leukemic cells after chemotherapy is not always accompanied by apoptosis, suggesting that the apoptotic death receptor signaling pathway is disrupted. Because Fas-associated protein with death domain (FADD) is the main adaptor for transmitting the Fas, TNF-related apoptosis-inducing ligand receptors, and TNF receptor 1 death signal, expression of FADD was analyzed by Western blot and immunocytochemistry in leukemic cells of 70 de novo AML patients treated with the European Organization of Research and Treatment of Cancer AML-10 randomized trial before initiation of induction chemotherapy. Thirty seven percent of patients (17 of 46) with FADD negative/low (FADD(-/low)) leukemic cells had a primary refractory disease compared with 12% of FADD(+) patients (3 of 24; P = 0.05). FADD(-/low) expression was significantly associated with a worse event-free survival [EFS (P = 0.04)] and overall survival (P = 0.04). In multivariate analysis, FADD(-/low) protein expression was independently associated with a poor EFS and overall survival (P = 0.002 and P = 0.026, respectively). Importantly, FADD(-/low) protein expression predicted poor EFS even in patients with standard- or good-risk AML (P = 0.009). Thus, we identified low or absent expression of the FADD protein in leukemic cells at diagnosis as a poor independent prognostic factor that can predict worse clinical outcome even for patients with standard- or good-risk AML.