Reduction of Minimal Residual Disease in Pediatric B-lineage Acute Lymphoblastic Leukemia by an Fc-optimized CD19 Antibody.

Prognosis of primary refractory and relapsed pediatric B-lineage acute lymphoblastic leukemia (ALL) is very poor. Relapse rates significantly correlate with persistent minimal residual disease (MRD). In MRD, favorable effector-target ratios prevail and thus this situation might be optimally suited for immunotherapy with antibodies recruiting immunological effector cells. We here report on the generation, preclinical characterization and first clinical application in B-lineage ALL of an Fc-optimized CD19 antibody. This third-generation antibody (4G7SDIE) mediated enhanced antibody-dependent cellular cytotoxicity (ADCC) against leukemic blasts with effector cells from healthy volunteers and B-lineage ALL patients. The antibody was produced in a university-owned production unit and was applied on a compassionate use basis to 14 pediatric patients with refractory and relapsed B-lineage ALL at the stage of MRD. In 10/14 patients, MRD was reduced by ≥ 1 log or below the patient-individual detection limit, and 5/14 patients have achieved ongoing complete molecular remission with a median leukemia-free survival of 428 days. Two additional patients died in complete molecular remission due to complications not related to antibody therapy. Besides profound in vivo B-cell depletion, side effects were negligible. A clinical phase 1/2 study to further assess the therapeutic activity of 4G7SDIE is in preparation.

A novel category of antigens enabling CTL immunity to tumor escape variants: Cinderella antigens.

Deficiencies in MHC class I antigen presentation are a common feature of tumors and allows escape from cytotoxic T lymphocyte (CTL)-mediated killing. It is crucial to take this capacity of tumors into account for the development of T-cell-based immunotherapy, as it may strongly impair their effectiveness. A variety of escape mechanisms has been described thus far, but progress in counteracting them is poor. Here we review a novel strategy to target malignancies with defects in the antigenic processing machinery (APM). The concept is based on a unique category of CD8+ T-cell epitopes that is associated with impaired peptide processing, which we named TEIPP. We characterized this alternative peptide repertoire emerging in MHC-I on tumors lacking classical antigen processing due to defects in the peptide transporter TAP (transporter associated with peptide processing). These TEIPPs exemplify interesting parallels with the folktale figure Cinderella: they are oppressed and neglected by a stepmother (like functional TAP prevents TEIPP presentation), until the suppression is released and Cinderella/TEIPP achieves unexpected recognition. TEIPP-specific CTLs and their cognate peptide-epitopes provide a new strategy to counteract immune evasion by APM defects and bear potential to targeting escape variants observed in a wide range of cancers.

T Cells Engaging the Conserved MHC Class Ib Molecule Qa-1b with TAP-Independent Peptides Are Semi-Invariant Lymphocytes.

The HLA-E homolog in the mouse (Qa-1b) is a conserved MHC class Ib molecule presenting monomorphic peptides to germline-encoded natural killer receptor CD94/NKG2A. Previously, we demonstrated the replacement of this canonical peptide by a diverse peptidome upon deficiency of the TAP peptide transporter. Analysis of this Qa-1b-restricted T cell repertoire against these non-mutated neoantigens revealed characteristics of conventional hypervariable CD8+ T cells, but also of invariant T cell receptor (TCR)αß T cells. A shared TCR Vα chain was used by this subset in combination with a variety of Vß chains. The TCRs target peptide ligands that are conserved between mouse and man, like the identified peptide derived from the transcriptional cofactor Med15. The thymus selection was studied in a TCR-transgenic mouse and emerging naïve CD8+ T cells displayed a slightly activated phenotype, as witnessed by higher CD122 and Ly6C expression. Moreover, the Qa-1b protein was dispensable for thymus selection. Importantly, no self-reactivity was observed as reported for other MHC class Ib-restricted subsets. Naïve Qa-1b restricted T cells expanded, contracted, and formed memory cells in vivo upon peptide vaccination in a similar manner as conventional CD8+ T cells. Based on these data, the Qa-1b restricted T cell subset might be positioned closest to conventional CD8+ T cells of all MHC class Ib populations.

Nutritional control of IL-23/Th17-mediated autoimmune disease through HO-1/STAT3 activation.

The nutritional curcumin (CUR) is beneficial in cell-mediated autoimmune diseases. The molecular mechanisms underlying this food-mediated silencing of inflammatory immune responses are poorly understood. By investigating antigen-specific immune responses we found that dietary CUR impairs the differentiation of Th1/Th17 cells in vivo during encephalomyelitis and instead promoted Th2 cells. In contrast, feeding CUR had no inhibitory effect on ovalbumin-induced airway inflammation. Mechanistically, we found that CUR induces an anti-inflammatory phenotype in dendritic cells (DC) with enhanced STAT3 phosphorylation and suppressed expression of Il12b and Il23a. On the molecular level CUR readily induced NRF2-sensitive heme oxygenase 1 (HO-1) mRNA and protein in LPS-activated DC. HO-1 enhanced STAT3 phosphorylation, which enriched to Il12b and Il23a loci and negatively regulated their transcription. These findings demonstrate the underlying mechanism through which a nutritional can interfere with the immune response. CUR silences IL-23/Th17-mediated pathology by enhancing HO-1/STAT3 interaction in DC.

CD19-specific triplebody SPM-1 engages NK and γδ T cells for rapid and efficient lysis of malignant B-lymphoid cells.

Triplebodies are antibody-derived recombinant proteins carrying 3 antigen-binding domains in a single polypeptide chain. Triplebody SPM-1 was designed for lysis of CD19-bearing malignant B-lymphoid cells through the engagement of CD16-expressing cytolytic effectors, including NK and γδ T cells.SPM-1 is an optimized version of triplebody ds(19-16-19) and includes humanization, disulfide stabilization and the removal of potentially immunogenic sequences. A three-step chromatographic procedure yielded 1.7 - 5.5 mg of purified, monomeric protein per liter of culture medium. In cytolysis assays with NK cell effectors, SPM-1 mediated potent lysis of cancer-derived B cell lines and primary cells from patients with various B-lymphoid malignancies, which surpassed the ADCC activity of the therapeutic antibody Rituximab. EC50-values ranged from 3 to 86 pM. Finally, in an impedance-based assay, SPM-1 mediated a particularly rapid lysis of CD19-bearing target cells by engaging and activating both primary and expanded human γδ T cells from healthy donors as effectors.These data establish SPM-1 as a useful tool for a kinetic analysis of the cytolytic reactions mediated by γδ T and NK cells and as an agent deserving further development towards clinical use for the treatment of B-lymphoid malignancies.

γδ T Cell-Mediated Antibody-Dependent Cellular Cytotoxicity with CD19 Antibodies Assessed by an Impedance-Based Label-Free Real-Time Cytotoxicity Assay.

γδ T cells are not MHC restricted, elicit cytotoxicity against various malignancies, are present in early post-transplant phases in novel stem cell transplantation strategies and have been shown to mediate antibody-dependent cellular cytotoxicity (ADCC) with monoclonal antibodies (mAbs). These features make γδ T cells promising effector cells for antibody-based immunotherapy in pediatric patients with B-lineage acute lymphoblastic leukemia (ALL). To evaluate combination of human γδ T cells with CD19 antibodies for immunotherapy of B-lineage ALL, γδ T cells were expanded after a GMP-compliant protocol and ADCC of both primary and expanded γδ T cells with an Fc-optimized CD19 antibody (4G7SDIE) and a bi-specific antibody with the specificities CD19 and CD16 (N19-C16) was evaluated in CD107a-degranulation assays and intracellular cytokine staining. CD107a, TNFα, and IFNγ expression of primary γδ T cells were significantly increased and correlated with CD16-expression of γδ T cells. γδ T cells highly expressed CD107a after expansion and no further increased expression by 4G7SDIE and N19-C16 was measured. Cytotoxicity of purified expanded γδ T cells targeting CD19-expressing cells was assessed in both europium-TDA release and in an impedance-based label-free method (using the xCELLigence system) measuring γδ T cell lysis in real-time. Albeit in the 2 h end-point europium-TDA release assay no increased lysis was observed, in real-time xCELLigence assays both significant antibody-independent cytotoxicity and ADCC of γδ T cells were observed. The xCELLigence system outperformed the end-point europium-TDA release assay in sensitivity and allows drawing of conclusions to lysis kinetics of γδ T cells over prolonged periods of time periods. Combination of CD19 antibodies with primary as well as expanded γδ T cells exhibits a promising approach, which may enhance clinical outcome of patients with pediatric B-lineage ALL and requires clinical evaluation.

Natural killer cell mediated antibody-dependent cellular cytotoxicity in tumor immunotherapy with therapeutic antibodies.

In the last decade several therapeutic antibodies have been Federal Drug Administration (FDA) and European Medicines Agency (EMEA) approved. Although their mechanisms of action in vivo is not fully elucidated, antibody-dependent cellular cytotoxicity (ADCC) mediated by natural killer (NK) cells is presumed to be a key effector function. A substantial role of ADCC has been demonstrated in vitro and in mouse tumor models. However, a direct in vivo effect of ADCC in tumor reactivity in humans remains to be shown. Several studies revealed a predictive value of FcγRIIIa-V158F polymorphism in monoclonal antibody treatment, indicating a potential effect of ADCC on outcome for certain indications. Furthermore, the use of therapeutic antibodies after allogeneic hematopoietic stem cell transplantation is an interesting option. Studying the role of the FcγRIIIa-V158F polymorphism and the influence of Killer-cell Immunoglobuline-like Receptor (KIR) receptor ligand incompatibility on ADCC in this approach may contribute to future transplantation strategies. Despite the success of approved second-generation antibodies in the treatment of several malignancies, efforts are made to further augment ADCC in vivo by antibody engineering. Here, we review currently used therapeutic antibodies for which ADCC has been suggested as effector function.