ABSTRACT
The epidermal growth factor receptor (EGFR) plays an essential role in cellular signaling pathways that regulate cell growth, proliferation and survival, and is often found dysregulated in cancer. Several monoclonal IgG antibodies have been clinically tested over the years which exert their function via blocking the ligand binding domain (thereby inhibiting downstream signaling) and induction of Fc-related effector functions, such as antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). However, these IgG antibodies do not optimally recruit neutrophils, by far the most abundant white blood cell population in humans. Therefore, we reformatted six therapeutic EGFR antibodies (cetuximab, panitumumab, nimotuzumab, necitumumab, zalutumumab, and matuzumab) into the IgA3.0 format, which is an IgA2 isotype that has been adapted for clinical application. Reformatting these antibodies preserved Fab-mediated functions such as EGFR binding, growth inhibition and ligand blockade. Additionally, whole leukocyte ADCC was significantly increased when using this panel of IgA3.0 antibodies compared to their respective IgG counterparts, with no major differences between IgA3.0 antibodies. In vivo, IgA3.0 matuzumab outperformed the other antibodies, resulting in the strongest suppression of tumor outgrowth in a long intraperitoneal model. We show that neutrophils are important for the suppression of tumor outgrowth. IgA3.0 matuzumab exhibited reduced receptor internalization compared to the other antibodies, possibly accounting for its superior in vivo Fc-mediated tumor cell killing efficacy. In conclusion, reformatting EGFR antibodies into an IgA3.0 format increased Fc-mediated killing while retaining Fab-mediated functions and could therefore be a good alternative for the currently available antibody therapies.
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 , PhagocytosisABSTRACT
Rituximab (RTX) plus chemotherapy (R-CHOP) applied as a first-line therapy for lymphoma leads to a relapse in approximately 40% of the patients. Therefore, novel approaches to treat aggressive lymphomas are being intensively investigated. Several RTX-resistant (RR) cell lines have been established as surrogate models to study resistance to R-CHOP. Our study reveals that RR cells are characterized by a major downregulation of CD37, a molecule currently explored as a target for immunotherapy. Using CD20 knockout (KO) cell lines, we demonstrate that CD20 and CD37 form a complex, and hypothesize that the presence of CD20 stabilizes CD37 in the cell membrane. Consequently, we observe a diminished cytotoxicity of anti-CD37 monoclonal antibody (mAb) in complement-dependent cytotoxicity in both RR and CD20 KO cells that can be partially restored upon lysosome inhibition. On the other hand, the internalization rate of anti-CD37 mAb in CD20 KO cells is increased when compared to controls, suggesting unhampered efficacy of antibody drug conjugates (ADCs). Importantly, even a major downregulation in CD37 levels does not hamper the efficacy of CD37-directed chimeric antigen receptor (CAR) T cells. In summary, we present here a novel mechanism of CD37 regulation with further implications for the use of anti-CD37 immunotherapies.
Subject(s)
Antigens, CD20 , Immunotherapy , Lymphoma, B-Cell , Rituximab , Tetraspanins , Humans , Antigens, CD20/immunology , Antigens, CD20/metabolism , Antigens, CD20/genetics , Rituximab/pharmacology , Rituximab/therapeutic use , Tetraspanins/genetics , Tetraspanins/metabolism , Cell Line, Tumor , Lymphoma, B-Cell/immunology , Lymphoma, B-Cell/therapy , Lymphoma, B-Cell/genetics , Lymphoma, B-Cell/drug therapy , Immunotherapy/methods , Antigens, Neoplasm/immunology , Antigens, Neoplasm/genetics , Drug Resistance, Neoplasm/drug effects , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Doxorubicin/pharmacology , Doxorubicin/administration & dosage , Cyclophosphamide/pharmacology , Cyclophosphamide/therapeutic use , Vincristine/pharmacology , Vincristine/therapeutic use , Antibodies, Monoclonal/pharmacology , Antibodies, Monoclonal/therapeutic use , Receptors, Chimeric Antigen/immunology , Receptors, Chimeric Antigen/genetics , Receptors, Chimeric Antigen/metabolism , Gene Expression Regulation, NeoplasticABSTRACT
Antibodies that specifically bind to individual human fragment crystallizable γ receptors (FcγRs) are of interest as research tools in studying immune cell functions, as well as components in bispecific antibodies for immune cell engagement in cancer therapy. Monoclonal antibodies for human low-affinity FcγRs have been successfully generated by hybridoma technology and are widely used in pre-clinical research. However, the generation of monoclonal antibodies by hybridoma technology that specifically bind to the high-affinity receptor FcγRI is challenging. Monomeric mouse IgG2a, IgG2b, and IgG3 bind human FcγRI with high affinity via the Fc part, leading to an Fc-mediated rather than a fragment for antigen binding (Fab)-mediated selection of monoclonal antibodies. Blocking the Fc-binding site of FcγRI with an excess of human IgG or Fc during screening decreases the risk of Fc-mediated interactions but can also block the potential epitopes of new antibody candidates. Therefore, we replaced hybridoma technology with phage display of a single-chain fragment variable (scFv) antibody library that was generated from mice immunized with FcγRI-positive cells and screened it with a cellular panning approach assisted by next-generation sequencing (NGS). Seven new FcγRI-specific antibody sequences were selected with this methodology, which were produced as Fc-silent antibodies showing FcγRI-restricted specificity.
Subject(s)
Antibodies, Monoclonal , Receptors, IgG , Receptors, IgG/immunology , Receptors, IgG/metabolism , Animals , Mice , Humans , Antibodies, Monoclonal/immunology , Immunoglobulin G/immunology , Immunization , Single-Chain Antibodies/immunology , Single-Chain Antibodies/genetics , Peptide Library , Cell Surface Display Techniques , Hybridomas , Antibody Specificity , Female , Mice, Inbred BALB CABSTRACT
CD19-directed immunotherapy has become a cornerstone in the therapy of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). CD19-directed cellular and antibody-based therapeutics have entered therapy of primary and relapsed disease and contributed to improved outcomes in relapsed disease and lower therapy toxicity. However, efficacy remains limited in many cases due to a lack of therapy response, short remission phases, or antigen escape. Here, BCP-ALL cell lines, patient-derived xenograft (PDX) samples, human macrophages, and an in vivo transplantation model in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice were used to examine the therapeutic potency of a CD19 antibody Fc-engineered for improved effector cell recruitment (CD19-DE) and antibody-dependent cellular phagocytosis (ADCP), in combination with a novel modified CD47 antibody (Hu5F9-IgG2σ). For the in vivo model, only samples refractory to CD19-DE monotherapy were chosen. Hu5F9-IgG2σ enhanced ADCP by CD19-DE in various BCP-ALL cell line models with varying CD19 surface expression and cytogenetic backgrounds, two of which contained the KMT2A-AFF1 fusion. Also, the antibody combination was efficient in inducing ADCP by human macrophages in pediatric PDX samples with and adult samples with and without KMT2A-rearrangement in vitro. In a randomized phase 2-like PDX trial using seven KMT2A-rearranged BCP-ALL samples in NSG mice, the CD19/CD47 antibody combination proved highly efficient. Our findings support that the efficacy of Fc-engineered CD19 antibodies may be substantially enhanced by a combination with CD47 blockade. This suggests that the combination may be a promising therapy option for BCP-ALL, especially in relapsed patients and/or patients refractory to CD19-directed therapy.
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 â¼85% 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 useABSTRACT
Natural killer (NK) cells emerged as a promising effector population that can be harnessed for anti-tumor therapy. In this work, we constructed NK cell engagers (NKCEs) based on NKp30-targeting single domain antibodies (sdAbs) that redirect the cytotoxic potential of NK cells toward epidermal growth factor receptor (EGFR)-expressing tumor cells. We investigated the impact of crucial parameters such as sdAb location, binding valencies, the targeted epitope on NKp30, and the overall antibody architecture on the redirection capacity. Our study exploited two NKp30-specific sdAbs, one of which binds a similar epitope on NKp30 as its natural ligand B7-H6, while the other sdAb addresses a non-competing epitope. For EGFR-positive tumor targeting, humanized antigen-binding domains of therapeutic antibody cetuximab were used. We demonstrate that NKCEs bivalently targeting EGFR and bivalently engaging NKp30 are superior to monovalent NKCEs in promoting NK cell-mediated tumor cell lysis and that the architecture of the NKCE can substantially influence killing capacities depending on the NKp30-targeting sdAb utilized. While having a pronounced impact on NK cell killing efficacy, the capabilities of triggering antibody-dependent cellular phagocytosis or complement-dependent cytotoxicity were not significantly affected comparing the bivalent IgG-like NKCEs with cetuximab. However, the fusion of sdAbs can have a slight impact on the NK cell release of immunomodulatory cytokines, as well as on the pharmacokinetic profile of the NKCE due to unfavorable spatial orientation within the molecule architecture. Ultimately, our findings reveal novel insights for the engineering of potent NKCEs triggering the NKp30 axis.
Subject(s)
Epidermal Growth Factor , Killer Cells, Natural , Cetuximab/metabolism , Epidermal Growth Factor/metabolism , Binding Sites, Antibody , ErbB Receptors/metabolism , Epitopes/metabolismABSTRACT
The activating receptor natural killer group 2, member D (NKG2D) represents an attractive target for immunotherapy as it exerts a crucial role in cancer immunosurveillance by regulating the activity of cytotoxic lymphocytes. In this study, a panel of novel NKG2D-specific single-chain fragments variable (scFv) were isolated from naïve human antibody gene libraries and fused to the fragment antigen binding (Fab) of rituximab to obtain [CD20×NKG2D] bibodies with the aim to recruit cytotoxic lymphocytes to lymphoma cells. All bispecific antibodies bound both antigens simultaneously. Two bibody constructs, [CD20×NKG2D#3] and [CD20×NKG2D#32], efficiently activated natural killer (NK) cells in co-cultures with CD20+ lymphoma cells. Both bibodies triggered NK cell-mediated lysis of lymphoma cells and especially enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) by CD38 or CD19 specific monoclonal antibodies suggesting a synergistic effect between NKG2D and FcγRIIIA signaling pathways in NK cell activation. The [CD20×NKG2D] bibodies were not effective in redirecting CD8+ T cells as single agents, but enhanced cytotoxicity when combined with a bispecific [CD19×CD3] T cell engager, indicating that NKG2D signaling also supports CD3-mediated T cell activation. In conclusion, engagement of NKG2D with bispecific antibodies is attractive to directly activate cytotoxic lymphocytes or to support their activation by monoclonal antibodies or bispecific T cell engagers. As a perspective, co-targeting of two tumor antigens may allow fine-tuning of antibody cancer therapies. Our proposed combinatorial approach is potentially applicable for many existing immunotherapies but further testing in different preclinical models is necessary to explore the full potential.
Subject(s)
Antibodies, Bispecific , Lymphoma , Neoplasms , Humans , Antibodies, Bispecific/pharmacology , Antibodies, Bispecific/metabolism , NK Cell Lectin-Like Receptor Subfamily K/metabolism , Killer Cells, Natural , Lymphoma/metabolism , Antibodies, Monoclonal/pharmacology , Antibodies, Monoclonal/metabolism , Antigens, CD19ABSTRACT
Here, we generated bispecific antibody (bsAb) derivatives that mimic the function of interleukin (IL)-18 based on single domain antibodies (sdAbs) specific to IL-18 Rα and IL-18 Rß. For this, camelids were immunized, followed by yeast surface display (YSD)-enabled discovery of VHHs targeting the individual receptor subunits. Upon reformatting into a strictly monovalent (1 + 1) bispecific sdAb architecture, several bsAbs triggered dose-dependent IL-18 R downstream signaling on IL-18 reporter cells, as well as IFN-γ release by peripheral blood mononuclear cells in the presence of low-dose IL-12. However, compared with IL-18, potencies and efficacies were considerably attenuated. By engineering paratope valencies and the spatial orientation of individual paratopes within the overall design architecture, we were able to generate IL-18 mimetics displaying significantly augmented functionalities, resulting in bispecific cytokine mimetics that were more potent than IL-18 in triggering proinflammatory cytokine release. Furthermore, generated IL-18 mimetics were unaffected from inhibition by IL-18 binding protein decoy receptor. Essentially, we demonstrate that this strategy enables the generation of IL-18 mimetics with tailor-made cytokine functionalities.
Subject(s)
Antibodies, Bispecific , Single-Domain Antibodies , Interleukin-18 , Leukocytes, Mononuclear , Binding Sites, AntibodyABSTRACT
The majority of therapeutic antibodies, bispecific antibodies, and chimeric antigen receptor (CAR) T cells in cancer therapy are based on an antibody or antibody fragment that specifically binds a target present on the surface of a tumor cell. Suitable antigens that can be used for immunotherapy are ideally tumor-specific or tumor-associated and stably expressed on the tumor cell. The identification of new target structures to further optimize immunotherapies could be realized by comparing healthy and tumor cells using "omics" methods to select promising proteins. However, differences in post-translational modifications and structural alterations that can be present on the tumor cell surface are difficult to identify or even not accessible by these techniques. In this chapter, we describe an alternative approach to potentially identify antibodies targeting novel tumor-associated antigens (TAA) or epitopes by using cellular screening and phage display of antibody libraries. Isolated antibody fragments can be further converted into chimeric IgG or other antibody formats to investigate the anti-tumor effector functions and finally identify and characterize the respective antigen.
Subject(s)
Bacteriophages , Neoplasms , Humans , Antigens, Surface , Peptide Library , Neoplasms/therapy , Antigens , Antigens, NeoplasmABSTRACT
In recent years, the development of bispecific antibodies (bsAbs) has experienced tremendous progress for disease treatment, and consequently, a plethora of bsAbs is currently scrutinized in clinical trials. Besides antibody scaffolds, multifunctional molecules referred to as immunoligands have been developed. These molecules typically harbor a natural ligand entity for the engagement of a specific receptor, while binding to the additional antigen is facilitated by an antibody-derived paratope. Immunoligands can be exploited to conditionally activate immune cells, e.g., natural killer (NK) cells, in the presence of tumor cells, ultimately causing target-dependent tumor cell lysis. However, many ligands naturally show only moderate affinities toward their cognate receptor, potentially hampering killing capacities of immunoligands. Herein, we provide protocols for yeast surface display-based affinity maturation of B7-H6, the natural ligand of NK cell-activating receptor NKp30.
Subject(s)
Neoplasms , Saccharomyces cerevisiae , Humans , Saccharomyces cerevisiae/metabolism , Ligands , Natural Cytotoxicity Triggering Receptor 3/chemistry , Natural Cytotoxicity Triggering Receptor 3/metabolism , B7 Antigens/chemistry , B7 Antigens/metabolism , Neoplasms/metabolism , Killer Cells, NaturalABSTRACT
Upregulation of surface expressed sialoglycans on tumor cells is one of the mechanisms which promote tumor growth and progression. Specifically, the interactions of sialic acids with sialic acid-binding immunoglobulin-like lectins (Siglecs) on lymphoid or myeloid cells transmit inhibitory signals and lead to suppression of anti-tumor responses. Here, we show that neutrophils express among others Siglec-9, and that EGFR and HER2 positive breast tumor cells express ligands for Siglec-9. Treatment of tumor cells with neuraminidases or a sialyl transferase inhibitor significantly reduced binding of a soluble recombinant Siglec-9-Fc fusion protein, while EGFR and HER2 expression remained unchanged. Importantly, the cytotoxic activity of neutrophils driven by therapeutic EGFR or HER2 antibodies in vitro was increased by blocking the sialic acid/Siglec interaction, either by reducing tumor cell sialylation or by a Siglec-9 blocking antibody containing an effector silenced Fc domain. In vivo a short-term xenograft mouse model confirmed the improved therapeutic efficacy of EGFR antibodies against sialic acid depleted, by a sialyltransferase inhibitor, tumor cells compared to untreated cells. Our studies demonstrate that sialic acid/Siglec interactions between tumor cells and myeloid cells can impair antibody dependent tumor cell killing, and that Siglec-9 on polymorphonuclear cells (PMN) is critically involved. Considering that PMN are often a highly abundant cell population in the tumor microenvironment, Siglec-9 constitutes a promising target for myeloid checkpoint blockade to improve antibody-based tumor immunotherapy.
Subject(s)
N-Acetylneuraminic Acid , Neoplasms , Humans , Mice , Animals , N-Acetylneuraminic Acid/metabolism , Neutrophils/metabolism , Sialic Acid Binding Immunoglobulin-like Lectins/metabolism , Antibodies , Sialic Acids/metabolism , ErbB Receptors , Tumor MicroenvironmentABSTRACT
BACKGROUND: Chimeric antigen receptor (CAR) T cell therapy has proven to be successful against hematological malignancies. However, exploiting CAR T cells to treat solid tumors is more challenging for various reasons including the lack of suitable target antigens. Here, we identify the transmembrane protein CD317 as a novel target antigen for CAR T cell therapy against glioblastoma, one of the most aggressive solid tumors. METHODS: CD317-targeting CAR T cells were generated by lentivirally transducing human T cells from healthy donors. The anti-glioma activity of CD317-CAR T cells toward various glioma cells was assessed in vitro in cell lysis assays. Subsequently, we determined the efficacy of CD317-CAR T cells to control tumor growth in vivo in clinically relevant mouse glioma models. RESULTS: We generated CD317-specific CAR T cells and demonstrate strong anti-tumor activity against several glioma cell lines as well as primary patient-derived cells with varying CD317 expression levels in vitro. A CRISPR/Cas9-mediated knockout of CD317 protected glioma cells from CAR T cell lysis, demonstrating the target specificity of the approach. Silencing of CD317 expression in T cells by RNA interference reduced fratricide of engineered T cells and further improved their effector function. Using orthotopic glioma mouse models, we demonstrate the antigen-specific anti-tumor activity of CD317-CAR T cells, which resulted in prolonged survival and cure of a fraction of CAR T cell-treated animals. CONCLUSIONS: These data reveal a promising role of CD317-CAR T cell therapy against glioblastoma, which warrants further evaluation to translate this immunotherapeutic strategy into clinical neuro-oncology.
Subject(s)
Glioblastoma , Glioma , Receptors, Chimeric Antigen , Mice , Animals , Humans , Receptors, Chimeric Antigen/genetics , Glioblastoma/pathology , T-Lymphocytes , Cell Line, Tumor , Immunotherapy, Adoptive/methods , Glioma/pathology , Xenograft Model Antitumor AssaysABSTRACT
Herein, we describe the generation of potent NK cell engagers (NKCEs) based on single domain antibodies (sdAbs) specific for NKp46 harboring the humanized Fab version of Cetuximab for tumor targeting. After immunization of camelids, a plethora of different VHH domains were retrieved by yeast surface display. Upon reformatting into Fc effector-silenced NKCEs targeting NKp46 and EGFR in a strictly monovalent fashion, the resulting bispecific antibodies elicited potent NK cell-mediated killing of EGFR-overexpressing tumor cells with potencies (EC50 killing) in the picomolar range. This was further augmented via co-engagement of Fcγ receptor IIIa (FcγRIIIa). Importantly, NKp46-specific sdAbs enabled the construction of various NKCE formats with different geometries and valencies which displayed favorable biophysical and biochemical properties without further optimization. By this means, killing capacities were further improved significantly. Hence, NKp46-specific sdAbs are versatile building blocks for the construction of different NKCE formats.
Subject(s)
Antibodies, Bispecific , Neoplasms , Single-Domain Antibodies , Humans , Killer Cells, Natural , Antibodies, Bispecific/chemistry , ErbB Receptors , Cell Line, TumorABSTRACT
Ovarian carcinomas have the highest lethality amongst gynecological tumors. A problem after primary resection is the recurrence of epithelial ovarian carcinomas which is often associated with chemotherapy resistance. To improve the clinical outcome, it is of high interest to consider alternative therapy strategies. Due to their pronounced plasticity, γδ T cells are attractive for T-cell-based immunotherapy. However, tumors might escape by the release of lectin galectin-3, which impairs γδ T-cell function. Hence, we tested the effect of galectin-3 on the different γδ T-cell subsets. After coculture between ovarian tumor cells and Vδ1 or Vδ2 T cells enhanced levels of galectin-3 were released. This protein did not affect the cytotoxicity of both γδ T-cell subsets, but differentially influenced the proliferation of the two γδ T-cell subsets. While increased galectin-3 levels and recombinant galectin-3 inhibited the proliferation of Vδ2 T cells, Vδ1 T cells were unaffected. In contrast to Vδ1 T cells, the Vδ2 T cells strongly upregulated the galectin-3 binding partner α3ß1-integrin after their activation correlating with the immunosuppressive properties of galectin-3. In addition, galectin-3 reduced the effector memory compartment of zoledronate-activated Vδ2 T cells. Therefore, our data suggest that an activation of Vδ1 T-cell proliferation as part of a T-cell-based immunotherapy can be of advantage.
Subject(s)
Galectin 3 , Ovarian Neoplasms , Female , Humans , Galectin 3/genetics , Carcinoma, Ovarian Epithelial , Cell Proliferation , Coculture Techniques , Immunosuppressive Agents , Ovarian Neoplasms/therapyABSTRACT
BACKGROUND: Acute myeloid leukemia (AML) is a fatal clonal hematopoietic malignancy, which results from the accumulation of several genetic aberrations in myeloid progenitor cells, with a worldwide 5-year survival prognosis of about 30%. Therefore, the development of more effective therapeutics with novel mode of action is urgently demanded. One common mutated gene in the AML is the DNA-methyltransferase DNMT3A whose function in the development and maintenance of AML is still unclear. To specifically target "undruggable" oncogenes, we initially invented an RNAi-based targeted therapy option that uses the internalization capacity of a colorectal cancer specific anti-EGFR-antibody bound to cationic protamine and the anionic siRNA. Here, we present a new experimental platform technology of molecular oncogene targeting in AML. METHODS: Our AML-targeting system consists of an internalizing anti-CD33-antibody-protamine conjugate, which together with anionic molecules such as siRNA or ibrutinib-Cy3.5 and cationic free protamine spontaneously assembles into vesicular nanocarriers in aqueous solution. These nanocarriers were analyzed concerning their physical properties and relevant characteristics in vitro in cell lines and in vivo in xenograft tumor models and patient-derived xenograft leukemia models with the aim to prepare them for translation into clinical application. RESULTS: The nanocarriers formed depend on a balanced electrostatic combination of the positively charged cationic protamine-conjugated anti-CD33 antibody, unbound cationic protamine and the anionic cargo. This nanocarrier transports its cargo safely into the AML target cells and has therapeutic activity against AML in vitro and in vivo. siRNAs directed specifically against two common mutated genes in the AML, the DNA-methyltransferase DNMT3A and FLT3-ITD lead to a reduction of clonal growth in vitro in AML cell lines and inhibit tumor growth in vivo in xenotransplanted cell lines. Moreover, oncogene knockdown of DNMT3A leads to increased survival of mice carrying leukemia patient-derived xenografts. Furthermore, an anionic derivative of the approved Bruton's kinase (BTK) inhibitor ibrutinib, ibrutinib-Cy3.5, is also transported by this nanocarrier into AML cells and decreases colony formation. CONCLUSIONS: We report important results toward innovative personalized, targeted treatment options via electrostatic nanocarrier therapy in AML.
Subject(s)
Leukemia, Myeloid, Acute , Protamines , Humans , Mice , Animals , Static Electricity , RNA, Small Interfering/therapeutic use , Leukemia, Myeloid, Acute/drug therapy , Methyltransferases , DNAABSTRACT
Antibody-dependent cellular phagocytosis (ADCP) by macrophages, an important effector function of tumor targeting antibodies, is hampered by 'Don´t Eat Me!' signals such as CD47 expressed by cancer cells. Yet, human leukocyte antigen (HLA) class I expression may also impair ADCP by engaging leukocyte immunoglobulin-like receptor subfamily B (LILRB) member 1 (LILRB1) or LILRB2. Analysis of different lymphoma cell lines revealed that the ratio of CD20 to HLA class I cell surface molecules determined the sensitivity to ADCP by the combination of rituximab and an Fc-silent variant of the CD47 antibody magrolimab (CD47-IgGσ). To boost ADCP, Fc-silent antibodies against LILRB1 and LILRB2 were generated (LILRB1-IgGσ and LILRB2-IgGσ, respectively). While LILRB2-IgGσ was not effective, LILRB1-IgGσ significantly enhanced ADCP of lymphoma cell lines when combined with both rituximab and CD47-IgGσ. LILRB1-IgGσ promoted serial engulfment of lymphoma cells and potentiated ADCP by non-polarized M0 as well as polarized M1 and M2 macrophages, but required CD47 co-blockade and the presence of the CD20 antibody. Importantly, complementing rituximab and CD47-IgGσ, LILRB1-IgGσ increased ADCP of chronic lymphocytic leukemia (CLL) or lymphoma cells isolated from patients. Thus, dual checkpoint blockade of CD47 and LILRB1 may be promising to improve antibody therapy of CLL and lymphomas through enhancing ADCP by macrophages.
Subject(s)
CD47 Antigen , Leukemia, Lymphocytic, Chronic, B-Cell , Humans , CD47 Antigen/metabolism , Leukocyte Immunoglobulin-like Receptor B1/metabolism , Rituximab/pharmacology , Rituximab/therapeutic use , Rituximab/metabolism , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Cell Line, Tumor , Phagocytosis , Macrophages , Antibodies/metabolism , Antigens, CD/metabolismABSTRACT
Targeting CD19 represents a promising strategy for the therapy of B-cell malignancies. Although non-engineered CD19 antibodies are poorly effective in mediating complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP), these effector functions can be enhanced by Fc-engineering. Here, we engineered a CD19 antibody with the aim to improve effector cell-mediated killing and CDC activity by exchanging selected amino acid residues in the Fc domain. Based on the clinically approved Fc-optimized antibody tafasitamab, which triggers enhanced ADCC and ADCP due to two amino acid exchanges in the Fc domain (S239D/I332E), we additionally added the E345K amino acid exchange to favor antibody hexamerization on the target cell surface resulting in improved CDC. The dual engineered CD19-DEK antibody bound CD19 and Fcγ receptors with similar characteristics as the parental CD19-DE antibody. Both antibodies were similarly efficient in mediating ADCC and ADCP but only the dual optimized antibody was able to trigger complement deposition on target cells and effective CDC. Our data provide evidence that from a technical perspective selected Fc-enhancing mutations can be combined (S239D/I332E and E345K) allowing the enhancement of ADCC, ADCP and CDC with isolated effector populations. Interestingly, under more physiological conditions when the complement system and FcR-positive effector cells are available as effector source, strong complement deposition negatively impacts FcR engagement. Both effector functions were simultaneously active only at selected antibody concentrations. Dual Fc-optimized antibodies may represent a strategy to further improve CD19-directed cancer immunotherapy. In general, our results can help in guiding optimal antibody engineering strategies to optimize antibodies' effector functions.
Subject(s)
Antibody-Dependent Cell Cytotoxicity , Receptors, IgG , Amino Acids , Antigens, CD19 , Complement System Proteins , Immunoglobulin Fc Fragments , Receptors, IgG/genetics , Receptors, IgG/metabolismABSTRACT
Pemphigoid diseases are autoimmune chronic inflammatory skin diseases, which are characterized by blistering of the skin and/or mucous membranes, and circulating and tissue-bound autoantibodies. The well-established pathomechanisms comprise autoantibodies targeting various structural proteins located at the dermal-epidermal junction, leading to complement factor binding and activation. Several effector cells are thus attracted and activated, which in turn inflict characteristic tissue damage and subepidermal blistering. Moreover, the detection of linear complement deposits in the skin is a diagnostic hallmark of all pemphigoid diseases. However, recent studies showed that blistering might also occur independently of complement. This review reassesses the importance of complement in pemphigoid diseases based on current research by contrasting and contextualizing data from in vitro, murine and human studies.