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1.
Proc Natl Acad Sci U S A ; 121(25): e2312499121, 2024 Jun 18.
Article in English | MEDLINE | ID: mdl-38857395

ABSTRACT

Ex vivo expansion of human CD34+ hematopoietic stem and progenitor cells remains a challenge due to rapid differentiation after detachment from the bone marrow niche. In this study, we assessed the capacity of an inducible fusion protein to enable sustained ex vivo proliferation of hematopoietic precursors and their capacity to differentiate into functional phagocytes. We fused the coding sequences of an FK506-Binding Protein 12 (FKBP12)-derived destabilization domain (DD) to the myeloid/lymphoid lineage leukemia/eleven nineteen leukemia (MLL-ENL) fusion gene to generate the fusion protein DD-MLL-ENL and retrovirally expressed the protein switch in human CD34+ progenitors. Using Shield1, a chemical inhibitor of DD fusion protein degradation, we established large-scale and long-term expansion of late monocytic precursors. Upon Shield1 removal, the cells lost self-renewal capacity and spontaneously differentiated, even after 2.5 y of continuous ex vivo expansion. In the absence of Shield1, stimulation with IFN-γ, LPS, and GM-CSF triggered terminal differentiation. Gene expression analysis of the obtained phagocytes revealed marked similarity with naïve monocytes. In functional assays, the novel phagocytes migrated toward CCL2, attached to VCAM-1 under shear stress, produced reactive oxygen species, and engulfed bacterial particles, cellular particles, and apoptotic cells. Finally, we demonstrated Fcγ receptor recognition and phagocytosis of opsonized lymphoma cells in an antibody-dependent manner. Overall, we have established an engineered protein that, as a single factor, is useful for large-scale ex vivo production of human phagocytes. Such adjustable proteins have the potential to be applied as molecular tools to produce functional immune cells for experimental cell-based approaches.


Subject(s)
Cell Differentiation , Phagocytes , Humans , Phagocytes/metabolism , Hematopoietic Stem Cells/metabolism , Oncogene Proteins, Fusion/genetics , Oncogene Proteins, Fusion/metabolism , Recombinant Fusion Proteins/metabolism , Recombinant Fusion Proteins/genetics , Myeloid-Lymphoid Leukemia Protein/metabolism , Myeloid-Lymphoid Leukemia Protein/genetics , Leukemia/genetics , Leukemia/pathology , Leukemia/metabolism , Protein Engineering/methods , Phagocytosis
2.
Blood ; 143(26): 2735-2748, 2024 Jun 27.
Article in English | MEDLINE | ID: mdl-38518105

ABSTRACT

ABSTRACT: Acute lymphoblastic leukemia (ALL) arises from the uncontrolled proliferation of B-cell precursors (BCP-ALL) or T cells (T-ALL). Current treatment protocols obtain high cure rates in children but are based on toxic polychemotherapy. Novel therapies are urgently needed, especially in relapsed/refractory (R/R) disease, high-risk (HR) leukemias and T-ALL, in which immunotherapy approaches remain scarce. Although the interleukin-7 receptor (IL-7R) plays a pivotal role in ALL development, no IL-7R-targeting immunotherapy has yet reached clinical application in ALL. The IL-7Rα chain (CD127)-targeting IgG4 antibody lusvertikimab (LUSV; formerly OSE-127) is a full antagonist of the IL-7R pathway, showing a good safety profile in healthy volunteers. Here, we show that ∼85% of ALL cases express surface CD127. We demonstrate significant in vivo efficacy of LUSV immunotherapy in a heterogeneous cohort of BCP- and T-ALL patient-derived xenografts (PDX) in minimal residual disease (MRD) and overt leukemia models, including R/R and HR leukemias. Importantly, LUSV was particularly effective when combined with polychemotherapy in a phase 2-like PDX study with CD127high samples leading to MRD-negativity in >50% of mice treated with combination therapy. Mechanistically, LUSV targeted ALL cells via a dual mode of action comprising direct IL-7R antagonistic activity and induction of macrophage-mediated antibody-dependent cellular phagocytosis (ADCP). LUSV-mediated in vitro ADCP levels significantly correlated with CD127 expression levels and the reduction of leukemia burden upon treatment of PDX animals in vivo. Altogether, through its dual mode of action and good safety profile, LUSV may represent a novel immunotherapy option for any CD127+ ALL, particularly in combination with standard-of-care polychemotherapy.


Subject(s)
Xenograft Model Antitumor Assays , Animals , Humans , Mice , Receptors, Interleukin-7/antagonists & inhibitors , Mice, SCID , Phagocytosis/drug effects , Interleukin-7 Receptor alpha Subunit , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology , Precursor Cell Lymphoblastic Leukemia-Lymphoma/immunology , Female , Mice, Inbred NOD , Antibodies, Monoclonal, Humanized/therapeutic use , Antibodies, Monoclonal, Humanized/pharmacology , Cell Line, Tumor , Antineoplastic Agents, Immunological/pharmacology , Antineoplastic Agents, Immunological/therapeutic use
3.
Blood ; 140(1): 45-57, 2022 07 07.
Article in English | MEDLINE | ID: mdl-35452517

ABSTRACT

Acute lymphoblastic leukemia (ALL) is the most common malignant disease affecting children. Although therapeutic strategies have improved, T-cell acute lymphoblastic leukemia (T-ALL) relapse is associated with chemoresistance and a poor prognosis. One strategy to overcome this obstacle is the application of monoclonal antibodies. Here, we show that leukemic cells from patients with T-ALL express surface CD38 and CD47, both attractive targets for antibody therapy. We therefore investigated the commercially available CD38 antibody daratumumab (Dara) in combination with a proprietary modified CD47 antibody (Hu5F9-IgG2σ) in vitro and in vivo. Compared with single treatments, this combination significantly increased in vitro antibody-dependent cellular phagocytosis in T-ALL cell lines as well as in random de novo and relapsed/refractory T-ALL patient-derived xenograft (PDX) samples. Similarly, enhanced antibody-dependent cellular phagocytosis was observed when combining Dara with pharmacologic inhibition of CD47 interactions using a glutaminyl cyclase inhibitor. Phase 2-like preclinical in vivo trials using T-ALL PDX samples in experimental minimal residual disease-like (MRD-like) and overt leukemia models revealed a high antileukemic efficacy of CD47 blockade alone. However, T-ALL xenograft mice subjected to chemotherapy first (postchemotherapy MRD) and subsequently cotreated with Dara and Hu5F9-IgG2σ displayed significantly reduced bone marrow infiltration compared with single treatments. In relapsed and highly refractory T-ALL PDX combined treatment with Dara and Hu5F9-IgG2σ was required to substantially prolong survival compared with single treatments. These findings suggest that combining CD47 blockade with Dara is a promising therapy for T-ALL, especially for relapsed/refractory disease harboring a dismal prognosis in patients.


Subject(s)
Precursor Cell Lymphoblastic Leukemia-Lymphoma , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma , Animals , Antibodies, Monoclonal/pharmacology , Antibodies, Monoclonal/therapeutic use , CD47 Antigen , Humans , Mice , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/drug therapy
4.
J Immunol ; 209(9): 1724-1735, 2022 11 01.
Article in English | MEDLINE | ID: mdl-36104113

ABSTRACT

In this work, we have generated novel Fc-comprising NK cell engagers (NKCEs) that bridge human NKp30 on NK cells to human epidermal growth factor receptor (EGFR) on tumor cells. Camelid-derived VHH single-domain Abs specific for human NKp30 and a humanized Fab derived from the EGFR-specific therapeutic Ab cetuximab were used as binding arms. By combining camelid immunization with yeast surface display, we were able to isolate a diverse panel of NKp30-specific VHHs against different epitopes on NKp30. Intriguingly, NKCEs built with VHHs that compete for binding to NKp30 with B7-H6, the natural ligand of NKp30, were significantly more potent in eliciting tumor cell lysis of EGFR-positive tumor cells than NKCEs harboring VHHs that target different epitopes on NKp30 from B7-H6. We demonstrate that the NKCEs can be further improved with respect to killing capabilities by concomitant engagement of FcγRIIIa and that soluble B7-H6 does not impede cytolytic capacities of all scrutinized NKCEs at significantly higher B7-H6 concentrations than observed in cancer patients. Moreover, we show that physiological processes requiring interactions between membrane-bound B7-H6 and NKp30 on NK cells are unaffected by noncompeting NKCEs still eliciting tumor cell killing at low picomolar concentrations. Ultimately, the NKCEs generated in this study were significantly more potent in eliciting NK cell-mediated tumor cell lysis than cetuximab and elicited a robust release of proinflammatory cytokines, both features which might be beneficial for antitumor therapy.


Subject(s)
Cytokines , Natural Cytotoxicity Triggering Receptor 3 , Humans , B7 Antigens/metabolism , Cell Death , Cetuximab/pharmacology , Epitopes , ErbB Receptors , Killer Cells, Natural , Ligands , Natural Cytotoxicity Triggering Receptor 3/metabolism
5.
J Immunol ; 206(1): 225-236, 2021 01 01.
Article in English | MEDLINE | ID: mdl-33268483

ABSTRACT

Activating NK cell receptors represent promising target structures to elicit potent antitumor immune responses. In this study, novel immunoligands were generated that bridge the activating NK cell receptor NKp30 on NK cells with epidermal growth factor receptor (EGFR) on tumor cells in a bispecific IgG-like format based on affinity-optimized versions of B7-H6 and the Fab arm derived from cetuximab. To enhance NKp30 binding, the solitary N-terminal IgV domain of B7-H6 (ΔB7-H6) was affinity matured by an evolutionary library approach combined with yeast surface display. Biochemical and functional characterization of 36 of these novel ΔB7-H6-derived NK cell engagers revealed an up to 45-fold-enhanced affinity for NKp30 and significantly improved NK cell-mediated, EGFR-dependent killing of tumor cells compared with the NK cell engager based on the wild-type ΔB7-H6 domain. In this regard, potencies (EC50 killing) of the best immunoligands were substantially improved by up to 87-fold. Moreover, release of IFN-γ and TNF-α was significantly increased. Importantly, equipment of the ΔB7-H6-based NK cell engagers with a human IgG1 Fc part competent in Fc receptor binding resulted in an almost 10-fold superior killing of EGFR-overexpressing tumor cells compared with molecules either triggering FcγRIIIa or NKp30. Additionally, INF-γ and TNF-α release was increased compared with molecules solely triggering FcγRIIIa, including the clinically approved Ab cetuximab. Thus, incorporating affinity-matured ligands for NK cell-activating receptors might represent an effective strategy for the generation of potent novel therapeutic agents with unique effector functions in cancer immunotherapy.


Subject(s)
B7 Antigens/metabolism , Immunotherapy/methods , Killer Cells, Natural/immunology , Natural Cytotoxicity Triggering Receptor 3/metabolism , Neoplasms/immunology , Antibodies, Bispecific/genetics , Antibodies, Bispecific/metabolism , B7 Antigens/genetics , Cell Line, Tumor , Cetuximab/genetics , Cytokines/metabolism , Cytotoxicity, Immunologic , ErbB Receptors/immunology , ErbB Receptors/metabolism , Genetic Engineering , Humans , Immunoglobulin Fab Fragments/genetics , Inflammation Mediators/metabolism , Killer Cells, Natural/transplantation , Lymphocyte Activation , Natural Cytotoxicity Triggering Receptor 3/immunology , Neoplasms/therapy , Protein Binding , Signal Transduction
6.
Cell ; 134(4): 577-86, 2008 Aug 22.
Article in English | MEDLINE | ID: mdl-18691745

ABSTRACT

Evaluation of the therapeutic potential of RNAi for HIV infection has been hampered by the challenges of siRNA delivery and lack of suitable animal models. Using a delivery method for T cells, we show that siRNA treatment can dramatically suppress HIV infection. A CD7-specific single-chain antibody was conjugated to oligo-9-arginine peptide (scFvCD7-9R) for T cell-specific siRNA delivery in NOD/SCIDIL2rgamma-/- mice reconstituted with human lymphocytes (Hu-PBL) or CD34+ hematopoietic stem cells (Hu-HSC). In HIV-infected Hu-PBL mice, treatment with anti-CCR5 (viral coreceptor) and antiviral siRNAs complexed to scFvCD7-9R controlled viral replication and prevented the disease-associated CD4 T cell loss. This treatment also suppressed endogenous virus and restored CD4 T cell counts in mice reconstituted with HIV+ peripheral blood mononuclear cells. Moreover, scFvCD7-9R could deliver antiviral siRNAs to naive T cells in Hu-HSC mice and effectively suppress viremia in infected mice. Thus, siRNA therapy for HIV infection appears to be feasible in a preclinical animal model.


Subject(s)
HIV Infections/genetics , HIV Infections/therapy , RNA Interference , RNA, Small Interfering/metabolism , T-Lymphocytes/metabolism , Animals , Antigens, CD7/metabolism , Disease Models, Animal , Gene Expression , HIV-1/genetics , HIV-1/metabolism , Humans , Immunoglobulin Fragments/metabolism , Immunoglobulin Variable Region/genetics , Immunoglobulin Variable Region/metabolism , Leukocytes, Mononuclear/immunology , Leukocytes, Mononuclear/virology , Mice , Mice, Inbred NOD , Mice, SCID , RNA, Viral/metabolism
7.
Biol Chem ; 403(5-6): 545-556, 2022 04 26.
Article in English | MEDLINE | ID: mdl-34717050

ABSTRACT

Natural killer group 2 member D (NKG2D) plays an important role in the regulation of natural killer (NK) cell cytotoxicity in cancer immune surveillance. With the aim of redirecting NK cell cytotoxicity against tumors, the NKG2D ligand UL-16 binding protein 2 (ULBP2) was fused to a single-chain fragment variable (scFv) targeting the human epidermal growth factor receptor 2 (HER2). The resulting bispecific immunoligand ULBP2:HER2-scFv triggered NK cell-mediated killing of HER2-positive breast cancer cells in an antigen-dependent manner and required concomitant interaction with NKG2D and HER2 as revealed in antigen blocking experiments. The immunoligand induced tumor cell lysis dose-dependently and was effective at nanomolar concentrations. Of note, ULBP2:HER2-scFv sensitized tumor cells for antibody-dependent cell-mediated cytotoxicity (ADCC). In particular, the immunoligand enhanced ADCC by cetuximab, a therapeutic antibody targeting the epidermal growth factor receptor (EGFR) synergistically. No significant improvements were obtained by combining cetuximab and anti-HER2 antibody trastuzumab. In conclusion, dual-dual targeting by combining IgG1 antibodies with antibody constructs targeting another tumor associated antigen and engaging NKG2D as a second NK cell trigger molecule may be promising. Thus, the immunoligand ULBP2:HER2-scFv may represent an attractive biological molecule to promote NK cell cytotoxicity against tumors and to boost ADCC.


Subject(s)
Breast Neoplasms , NK Cell Lectin-Like Receptor Subfamily K , Antibody-Dependent Cell Cytotoxicity , Breast Neoplasms/pathology , Cell Line, Tumor , Cetuximab/pharmacology , Cetuximab/therapeutic use , Female , Humans , NK Cell Lectin-Like Receptor Subfamily K/metabolism , NK Cell Lectin-Like Receptor Subfamily K/therapeutic use , Trastuzumab/pharmacology , Trastuzumab/therapeutic use
8.
Clin Exp Immunol ; 209(1): 22-32, 2022 07 22.
Article in English | MEDLINE | ID: mdl-35325068

ABSTRACT

Natural killer (NK) cells exert an important role in cancer immune surveillance. Recognition of malignant cells and controlled activation of effector functions are facilitated by the expression of activating and inhibitory receptors, which is a complex interplay that allows NK cells to discriminate malignant cells from healthy tissues. Due to their unique profile of effector functions, the recruitment of NK cells is attractive in cancer treatment and a key function of NK cells in antibody therapy is widely appreciated. In recent years, besides the low-affinity fragment crystallizable receptor for immunoglobulin G (FcγRIIIA), the activating natural killer receptors p30 (NKp30) and p46 (NKp46), as well as natural killer group 2 member D (NKG2D), have gained increasing attention as potential targets for bispecific antibody-derivatives to redirect NK cell cytotoxicity against tumors. Beyond modulation of the receptor activity on NK cells, therapeutic targeting of the respective ligands represents an attractive approach. Here, novel therapeutic approaches to unleash NK cells by engagement of activating NK-cell receptors and alternative strategies targeting their tumor-expressed ligands in cancer therapy are summarized.


Subject(s)
Immunotherapy , Neoplasms , Receptors, Natural Killer Cell , Humans , Killer Cells, Natural , Ligands , NK Cell Lectin-Like Receptor Subfamily K/metabolism
9.
J Med Virol ; 94(12): 5780-5789, 2022 12.
Article in English | MEDLINE | ID: mdl-35945627

ABSTRACT

The humoral immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination in patients with chronic inflammatory disease (CID) declines more rapidly with tumor necrosis factor-α (TNF-α) inhibition. Furthermore, the efficacy of current vaccines against Omicron variants of concern (VOC) including BA.2 is limited. Alterations within immune cell populations, changes in IgG affinity, and the ability to neutralize a pre-VOC strain and the BA.2 virus were investigated in these at-risk patients. Serum levels of anti-SARS-CoV-2 IgG, IgG avidity, and neutralizing antibodies (NA) were determined in anti-TNF-α patients (n = 10) and controls (n = 24 healthy individuals; n = 12 patients under other disease-modifying antirheumatic drugs, oDMARD) before and after the second and third vaccination by ELISA, immunoblot and live virus neutralization assay. SARS-CoV-2-specific B- and T cell subsets were analysed by multicolor flow cytometry. Six months after the second vaccination, anti-SARS-CoV-2 IgG levels, IgG avidity and anti-pre-VOC NA titres were significantly reduced in anti-TNF-α recipients compared to controls (healthy individuals: avidity: p ≤ 0.0001; NA: p = 0.0347; oDMARDs: avidity: p = 0.0012; NA: p = 0.0293). The number of plasma cells was increased in anti-TNF-α patients (Healthy individuals: p = 0.0344; oDMARDs: p = 0.0254), while the absolute number of SARS-CoV-2-specific plasma cells 7 days after 2nd vaccination were comparable. Even after a third vaccination, these patients had lower anti-BA.2 NA titres compared to both other groups. We show a reduced SARS-CoV-2 neutralizing capacity in patients under TNF-α blockade. In this cohort, the plasma cell response appears to be less specific and shows stronger bystander activation. While these effects were observable after the first two vaccinations and with older VOC, the differences in responses to BA.2 were enhanced.


Subject(s)
AIDS Vaccines , Antirheumatic Agents , COVID-19 , Influenza Vaccines , Papillomavirus Vaccines , Respiratory Syncytial Virus Vaccines , SAIDS Vaccines , Antibodies, Neutralizing , Antibodies, Viral , BCG Vaccine , COVID-19/prevention & control , Diphtheria-Tetanus Vaccine , Diphtheria-Tetanus-Pertussis Vaccine , Humans , Immunity , Immunoglobulin G , Measles-Mumps-Rubella Vaccine , SARS-CoV-2 , Tumor Necrosis Factor Inhibitors , Tumor Necrosis Factor-alpha , Vaccination
10.
J Allergy Clin Immunol ; 147(5): 1838-1854.e4, 2021 05.
Article in English | MEDLINE | ID: mdl-33326804

ABSTRACT

BACKGROUND: Mast cell and basophil activation by antigen cross-linking of FcεRI-bound IgE is central to allergy pathogenesis. We previously demonstrated global suppression of this process by rapid desensitization with anti-FcεRIα mAbs. OBJECTIVES: We sought to determine whether use of monovalent (mv) anti-FcεRIα mAbs increases desensitization safety without loss of efficacy. METHODS: mv anti-human (hu) FcεRIα mAbs were produced with mouse-derived immunoglobulin variable regions and huIgG1 or huIgG4 C regions and were used to suppress murine IgE-mediated anaphylaxis and food allergy. mAbs were administered as a single dose or as serially increasing doses to mice that express hu instead of mouse FcεRIα; mice that additionally have an allergy-promoting IL-4Rα mutation; and hu cord blood-reconstituted immunodeficient, hu cytokine-secreting, mice that have large numbers of activated hu mast cells. Anaphylaxis susceptibility was sometimes increased by treatment with IL-4 or a ß-adrenergic receptor antagonist. RESULTS: mv anti-hu FcεRIα mAbs are considerably less able than divalent mAbs are to induce anaphylaxis and deplete mast cell and basophil IgE, but mv mAbs still strongly suppress IgE-mediated disease. The mv mAbs can be safely administered as a single large dose to mice with typical susceptibility to anaphylaxis, while a rapid desensitization approach safely suppresses disease in mice with increased susceptibility. Our huIgG4 variant of mv anti-huFcεRIα mAb is safer than our huIgG1 variant is, apparently because reduced interactions with FcεRs decrease ability to indirectly cross-link FcεRI. CONCLUSIONS: mv anti-FcεRIα mAbs more safely suppress IgE-mediated anaphylaxis and food allergy than divalent variants of the same mAbs do. These mv mAbs may be useful for suppression of huIgE-mediated disease.


Subject(s)
Anaphylaxis/drug therapy , Anti-Allergic Agents/therapeutic use , Antibodies, Monoclonal/therapeutic use , Food Hypersensitivity/drug therapy , Immunoglobulin E/immunology , Receptors, IgE/immunology , Anaphylaxis/immunology , Animals , Anti-Allergic Agents/pharmacology , Antibodies, Monoclonal/pharmacology , Female , Food Hypersensitivity/immunology , Immunoglobulin G/immunology , Male , Mast Cells/drug effects , Mast Cells/immunology , Mice, Inbred BALB C , Mice, Transgenic , Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases/immunology , Receptors, IgE/genetics , Syk Kinase/immunology
11.
Cancer Sci ; 112(8): 3029-3040, 2021 Aug.
Article in English | MEDLINE | ID: mdl-34058788

ABSTRACT

Integrin associated protein (CD47) is an important target in immunotherapy, as it is expressed as a "don't eat me" signal on many tumor cells. Interference with its counter molecule signal regulatory protein alpha (SIRPα), expressed on myeloid cells, can be achieved with blocking Abs, but also by inhibiting the enzyme glutaminyl cyclase (QC) with small molecules. Glutaminyl cyclase inhibition reduces N-terminal pyro-glutamate formation of CD47 at the SIRPα binding site. Here, we investigated the impact of QC inhibition on myeloid effector cell-mediated tumor cell killing by epidermal growth factor receptor (EGFR) Abs and the influence of Ab isotypes. SEN177 is a QC inhibitor and did not interfere with EGFR Ab-mediated direct growth inhibition, complement-dependent cytotoxicity, or Ab-dependent cell-mediated cytotoxicity (ADCC) by mononuclear cells. However, binding of a human soluble SIRPα-Fc fusion protein to SEN177 treated cancer cells was significantly reduced in a dose-dependent manner, suggesting that pyro-glutamate formation of CD47 was affected. Glutaminyl cyclase inhibition in tumor cells translated into enhanced Ab-dependent cellular phagocytosis by macrophages and enhanced ADCC by polymorphonuclear neutrophilic granulocytes. Polymorphonuclear neutrophilic granulocyte-mediated ADCC was significantly more effective with EGFR Abs of human IgG2 or IgA2 isotypes than with IgG1 Abs, proposing that the selection of Ab isotypes could critically affect the efficacy of Ab therapy in the presence of QC inhibition. Importantly, QC inhibition also enhanced the therapeutic efficacy of EGFR Abs in vivo. Together, these results suggest a novel approach to specifically enhance myeloid effector cell-mediated efficacy of EGFR Abs by orally applicable small molecule QC inhibitors.


Subject(s)
Aminoacyltransferases/antagonists & inhibitors , Antigens, Differentiation/chemistry , Antineoplastic Agents, Immunological/administration & dosage , CD47 Antigen/metabolism , Neoplasms/drug therapy , Receptors, Immunologic/chemistry , Small Molecule Libraries/administration & dosage , Animals , Antigens, Differentiation/metabolism , Antineoplastic Agents, Immunological/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Cetuximab/administration & dosage , Cetuximab/pharmacology , Drug Synergism , Female , HEK293 Cells , Humans , Male , Mice , Neoplasms/metabolism , Panitumumab/administration & dosage , Panitumumab/pharmacology , Protein Binding/drug effects , Receptors, Immunologic/metabolism , Xenograft Model Antitumor Assays
12.
Haematologica ; 106(7): 1857-1866, 2021 07 01.
Article in English | MEDLINE | ID: mdl-32499243

ABSTRACT

Despite several therapeutic advances, patients with multiple myeloma (MM) require additional treatment options since no curative therapy exists yet. In search of a novel therapeutic antibody, we previously applied phage display with myeloma cell screening and developed TP15, a scFv targeting intercellular adhesion molecule 1 (ICAM-1/CD54). To more precisely evaluate the antibody's modes of action, fully human IgG1 antibody variants were generated bearing wild-type (MSH-TP15) or mutated Fc to either enhance (MSH-TP15 Fc-eng.) or prevent (MSH-TP15 Fc k.o.) Fc gamma receptor binding. Especially MSH-TP15 Fc-eng. induced potent antibody-dependent cell-mediated cytotoxicity (ADCC) against malignant plasma cells by efficiently recruiting NK cells and engaged macrophages for antibody-dependent cellular phagocytosis (ADCP) of tumor cells. Binding studies with truncated ICAM-1 demonstrated MSH-TP15 binding to ICAM-1 domain 1-2. Importantly, MSH-TP15 and MSH-TP15 Fc-eng. both prevented myeloma cell engraftment and significantly prolonged survival of mice in an intraperitoneal xenograft model. In the subcutaneous model MSH-TP15 Fc-eng. was superior to MSH-TP15, whereas MSH-TP15 Fc k.o. was not effective in both models - reflecting the importance of Fc-dependent mechanisms of action also in vivo. The efficient recruitment of immune cells and the potent anti-tumor activity of the Fc-engineered MSH-TP15 antibody hold significant potential for myeloma immunotherapy.


Subject(s)
Multiple Myeloma , Animals , Humans , Mice , Antibody-Dependent Cell Cytotoxicity , Cell Line, Tumor , Immunoglobulin G , Intercellular Adhesion Molecule-1/genetics , Multiple Myeloma/drug therapy , Receptors, IgG/genetics
13.
Blood ; 130(13): 1543-1552, 2017 09 28.
Article in English | MEDLINE | ID: mdl-28698205

ABSTRACT

Antibody therapy constitutes a major advance in the treatment of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). To evaluate the efficacy and the mechanisms of action of CD19 monoclonal antibody therapy in pediatric BCP-ALL, we tested an Fc-engineered CD19 antibody carrying the S239D/I332E mutation for improved effector cell recruitment (CD19-DE). Patient-derived xenografts (PDX) of pediatric mixed-lineage leukemia gene (MLL)-rearranged ALL were established in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. Antibody CD19-DE was efficient in prolonging the survival of NSG mice in a minimal residual disease (MRD) model. The majority of surviving mice remained polymerase chain reaction (PCR)-MRD negative after treatment. When antibody therapy was initiated in overt leukemia, antibody CD19-DE was still efficient in prolonging survival of xenografted mice in comparison with nontreated control animals, but the effects were less pronounced than in the MRD setting. Importantly, the combination of antibody CD19-DE and cytoreduction by chemotherapy (dexamethasone, vincristine, PEG-asparaginase) resulted in significantly improved survival rates in xenografted mice. Antibody CD19-DE treatment was also efficient in a randomized phase 2-like PDX trial using 13 MLL-rearranged BCP-ALL samples. Macrophage depletion by liposomal clodronate resulted in a reversal of the beneficial effects of CD19-DE, suggesting an important role for macrophages as effector cells. In support of this finding, CD19-DE was found to enhance phagocytosis of patient-derived ALL blasts by human macrophages in vitro. Thus, Fc-engineered CD19 antibodies may represent a promising treatment option for infants and children with MLL-rearranged BCP-ALL who have a poor outcome when treated with chemotherapy only.


Subject(s)
Precursor Cell Lymphoblastic Leukemia-Lymphoma/therapy , Adolescent , Animals , Antibodies/genetics , Antibodies/therapeutic use , Antigens, CD19/genetics , Antigens, CD19/immunology , Female , Heterografts , Humans , Immunoglobulin Fc Fragments/genetics , Infant , Macrophages/cytology , Macrophages/immunology , Male , Mice , Mice, Inbred NOD , Neoplasm, Residual/therapy , Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology , Tumor Cells, Cultured
14.
Haematologica ; 102(2): 381-390, 2017 02.
Article in English | MEDLINE | ID: mdl-27658435

ABSTRACT

Interleukin-6 has an important role in the pathophysiology of multiple myeloma where it supports the growth and survival of the malignant plasma cells in the bone marrow. It belongs to a family of cytokines which use the glycoprotein 130 chain for signal transduction, such as oncostatin M or leukemia inhibitory factor. Targeting interleukin-6 in plasma cell diseases is currently evaluated in clinical trials with monoclonal antibodies. Here, efforts were made to elucidate the contribution of interleukin-6 and glycoprotein 130 signaling in malignant plasma cell growth in vivo In the xenograft severe combined immune deficiency model employing our interleukin-6-dependent plasma cell line INA-6, the lack of human interleukin-6 induced autocrine interleukin-6 production and a proliferative response to other cytokines of the glycoprotein 130 family. Herein, mice were treated with monoclonal antibodies against human interleukin-6 (elsilimomab/B-E8), the interleukin-6 receptor (B-R6), and with an antibody blocking glycoprotein 130 (B-R3). While treatment of mice with interleukin-6 and interleukin-6 receptor antibodies resulted in a modest delay in tumor growth, the development of plasmacytomas was completely prevented with the anti-glycoprotein 130 antibody. Importantly, complete inhibition was also achieved using F(ab')2-fragments of monoclonal antibody B-R3. Tumors harbor activated signal transducer and activator of transcription 3, and in vitro, the antibody inhibited leukemia inhibitory factor stimulated signal transducer and activator of transcription 3 phosphorylation and cell growth, while being less effective against interleukin-6. In conclusion, the growth of INA-6 plasmacytomas in vivo under interleukin-6 withdrawal remains strictly dependent on glycoprotein 130, and other glycoprotein 130 cytokines may substitute for interleukin-6. Antibodies against glycoprotein 130 are able to overcome this redundancy and should be explored for a possible therapeutic window.


Subject(s)
Cytokine Receptor gp130/antagonists & inhibitors , Cytokine Receptor gp130/metabolism , Interleukin-6/metabolism , Multiple Myeloma/metabolism , Animals , Antibodies, Monoclonal/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Cytogenetic Analysis , Cytokines/metabolism , Disease Models, Animal , Humans , Mice , Multiple Myeloma/drug therapy , Multiple Myeloma/genetics , Multiple Myeloma/pathology , Receptors, Interleukin-6/metabolism , Signal Transduction/drug effects , Xenograft Model Antitumor Assays
15.
J Immunol ; 195(10): 5077-87, 2015 Nov 15.
Article in English | MEDLINE | ID: mdl-26475927

ABSTRACT

Complement-dependent cytotoxicity (CDC) has been suggested to be an important mechanism of action of tumor-targeting Abs. However, single unmodified epidermal growth factor receptor (EGFR)-targeting IgG1 Abs fail to trigger efficient CDC. For the current study, we generated a CDC-optimized variant of the EGFR Ab matuzumab (H425 wt) by introducing amino acid substitutions K326A/E333A (H425 mt). This Ab was then used to elucidate the impact of complement activation on the capacity of effector cells such as mononuclear cells (MNC) and polymorphonuclear cells (PMN) to exert Ab-dependent cell-mediated cytotoxicity (ADCC). H425 mt, but not H425 wt, significantly induced complement deposition, release of anaphylatoxins, and CDC against distinct tumor cell lines, whereas no differences in ADCC by MNC or PMN were detected. Notably, stronger cytotoxicity was induced by H425 mt than by H425 wt in whole blood assays and in experiments in which MNC or PMN were combined with serum. Although MNC-ADCC was not affected by C5 cleavage, the cytotoxic activity of PMN in the presence of serum strongly depended on C5 cleavage, pointing to a direct interaction between complement and PMN. Strong cell surface expression of C5a receptors was detected on PMN, whereas NK cells completely lacked expression. Stimulation of PMN with C5a led to upregulation of activated complement receptor 3, resulting in enhanced complement receptor 3-dependent PMN-ADCC against tumor cells. In conclusion, complement-optimized EGFR Abs may constitute a promising strategy to improve tumor cell killing by enhancing the interaction between humoral and cellular effector functions in Ab-based tumor therapy.


Subject(s)
Antibodies, Monoclonal, Humanized/pharmacology , Antibodies, Neoplasm/pharmacology , Complement C5a/immunology , ErbB Receptors/antagonists & inhibitors , Leukocytes/immunology , Neoplasms/drug therapy , Antibodies, Monoclonal, Humanized/genetics , Antibodies, Monoclonal, Humanized/immunology , Antibodies, Neoplasm/genetics , Antibodies, Neoplasm/immunology , Cell Line, Tumor , ErbB Receptors/genetics , ErbB Receptors/immunology , Humans , Neoplasms/genetics , Neoplasms/immunology , Neoplasms/pathology , Receptor, Anaphylatoxin C5a/immunology
16.
Transfus Med Hemother ; 44(5): 327-336, 2017 Sep.
Article in English | MEDLINE | ID: mdl-29070978

ABSTRACT

In the last two decades, monoclonal antibodies have revolutionized the therapy of cancer patients. Although antibody therapy has continuously been improved, still a significant number of patients do not benefit from antibody therapy. Therefore, rational optimization of the antibody molecule by Fc engineering represents a major area of translational research to further improve this potent therapeutic option. Monoclonal antibodies are able to trigger a variety of effector mechanisms. Especially Fc-mediated effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement- dependent cytotoxicity (CDC) are considered important in antibody therapy of cancer. Novel mechanistic insights into the action of monoclonal antibodies allowed the development of various Fc engineering approaches to modulate antibodies' effector functions. Strategies in modifying the Fc glycosylation profile (Fc glyco-engineering) or approaches in engineering the protein backbone (Fc protein engineering) have been intensively evaluated. In the current review, Fc engineering strategies resulting in improved ADCC, ADCP and CDC activity are summarized and discussed.

17.
Transfus Med Hemother ; 44(5): 292-300, 2017 Sep.
Article in English | MEDLINE | ID: mdl-29070974

ABSTRACT

BACKGROUND: Engineering of the antibody's fragment crystallizable (Fc) by modifying the amino acid sequence (Fc protein engineering) or the glycosylation pattern (Fc glyco-engineering) allows enhancing effector functions of tumor targeting antibodies. Here, we investigated whether complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) of CD20 antibodies could be improved simultaneously by combining Fc protein engineering and glyco-engineering technologies. METHODS AND RESULTS: Four variants of the CD20 antibody rituximab were generated: a native IgG1, a variant carrying the EFTAE modification (S267E/H268F/S324T/G236A/I332E) for enhanced CDC as well as glyco-engineered, non-fucosylated derivatives of both to boost ADCC. The antibodies bound CD20 specifically with similar affinity. Antibodies with EFTAE modification were more efficacious in mediating CDC, irrespective of fucosylation, than antibodies with wild-type sequences due to enhanced C1q binding. In contrast, non-fucosylated variants had an enhanced affinity to FcγRIIIA and improved ADCC activity. Importantly, the double-engineered antibody lacking fucose and carrying the EFTAE modification mediated both CDC and ADCC with higher efficacy than the native CD20 IgG1 antibody. CONCLUSION: Combining glyco-engineering and protein engineering technologies offers the opportunity to simultaneously enhance ADCC and CDC activities of therapeutic antibodies. This approach may represent an attractive strategy to further improve antibody therapy of cancer and deserves further evaluation.

18.
Blood ; 124(22): 3180-1, 2014 Nov 20.
Article in English | MEDLINE | ID: mdl-25414435

ABSTRACT

In this issue of Blood, Romain et al demonstrate that natural killer (NK) cell­mediated killing of tumor cells coated with the Fc-optimized CD33 antibody DLE-HuM195 reveals a distinct kinetic profile. The presented work gives important novel insights into the mechanism of effector cell­mediated target cell killing triggered by Fc-engineered antibodies and explains how they achieve a higher antibody-dependent cell-mediated cytotoxicity (ADCC) potency than native immunoglobulin G1 (IgG1) antibodies. Using time-lapse imaging microscopy in nanowell grids (TIMING), the authors were able to demonstrate at the single-cell level that antibody Fc engineering improves frequency and promotes kinetic boosting of serial killing mediated by NK cells.


Subject(s)
Antibody-Dependent Cell Cytotoxicity , Immunoglobulin Fc Fragments/immunology , Immunoglobulin G/immunology , Killer Cells, Natural/immunology , Humans
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