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1.
Haematologica ; 104(9): 1841-1852, 2019 09.
Article in English | MEDLINE | ID: mdl-30792198

ABSTRACT

CD20 monoclonal antibody therapies have significantly improved the outlook for patients with B-cell malignancies. However, many patients acquire resistance, demonstrating the need for new and improved drugs. We previously demonstrated that the natural process of antibody hexamer formation on targeted cells allows for optimal induction of complement-dependent cytotoxicity. Complement-dependent cytotoxicity can be potentiated by introducing a single point mutation such as E430G in the IgG Fc domain that enhances intermolecular Fc-Fc interactions between cell-bound IgG molecules, thereby facilitating IgG hexamer formation. Antibodies specific for CD37, a target that is abundantly expressed on healthy and malignant B cells, are generally poor inducers of complement-dependent cytotoxicity. Here we demonstrate that introduction of the hexamerization-enhancing mutation E430G in CD37-specific antibodies facilitates highly potent complement-dependent cytotoxicity in chronic lymphocytic leukemia cells ex vivo Strikingly, we observed that combinations of hexamerization-enhanced CD20 and CD37 antibodies cooperated in C1q binding and induced superior and synergistic complement-dependent cytotoxicity in patient-derived cancer cells compared to the single agents. Furthermore, CD20 and CD37 antibodies colocalized on the cell membrane, an effect that was potentiated by the hexamerization-enhancing mutation. Moreover, upon cell surface binding, CD20 and CD37 antibodies were shown to form mixed hexameric antibody complexes consisting of both antibodies each bound to their own cognate target, so-called hetero-hexamers. These findings provide novel insights into the mechanisms of synergy in antibody-mediated complement-dependent cytotoxicity and provide a rationale to explore Fc-engineering and antibody hetero-hexamerization as a tool to enhance the cooperativity and therapeutic efficacy of antibody combinations.


Subject(s)
Antibodies, Monoclonal, Humanized/pharmacology , Antigens, CD20/immunology , Antigens, Neoplasm/immunology , Complement System Proteins/immunology , Immunoglobulin Fc Fragments/immunology , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Tetraspanins/immunology , Antibody-Dependent Cell Cytotoxicity/drug effects , Cell Line, Tumor , Complement C1q/immunology , Fluorescence Resonance Energy Transfer , Humans , Immunoglobulin G/immunology , Leukemia, Lymphocytic, Chronic, B-Cell/blood , Mutation , Protein Binding , Rituximab/pharmacology
2.
Proc Natl Acad Sci U S A ; 115(24): E5467-E5476, 2018 06 12.
Article in English | MEDLINE | ID: mdl-29844189

ABSTRACT

T cell-engaging bispecific antibodies (biAbs) present a promising strategy for cancer immunotherapy, and numerous bispecific formats have been developed for retargeting cytolytic T cells toward tumor cells. To explore the therapeutic utility of T cell-engaging biAbs targeting the receptor tyrosine kinase ROR1, which is expressed by tumor cells of various hematologic and solid malignancies, we used a bispecific ROR1 × CD3 scFv-Fc format based on a heterodimeric and aglycosylated Fc domain designed for extended circulatory t1/2 and diminished systemic T cell activation. A diverse panel of ROR1-targeting scFv derived from immune and naïve rabbit antibody repertoires was compared in this bispecific format for target-dependent T cell recruitment and activation. An ROR1-targeting scFv with a membrane-proximal epitope, R11, revealed potent and selective antitumor activity in vitro, in vivo, and ex vivo and emerged as a prime candidate for further preclinical and clinical studies. To elucidate the precise location and engagement of this membrane-proximal epitope, which is conserved between human and mouse ROR1, the 3D structure of scFv R11 in complex with the kringle domain of ROR1 was determined by X-ray crystallography at 1.6-Å resolution.


Subject(s)
Antibodies, Bispecific/immunology , Antineoplastic Agents/immunology , Epitopes/immunology , Receptor Tyrosine Kinase-like Orphan Receptors/immunology , T-Lymphocytes, Cytotoxic/immunology , Animals , CD3 Complex/immunology , Cell Line, Tumor , Crystallography, X-Ray/methods , Humans , Immunotherapy/methods , Jurkat Cells , K562 Cells , Mice , Rabbits , Single-Chain Antibodies/immunology , Xenograft Model Antitumor Assays/methods
3.
Blood ; 132(5): 521-532, 2018 08 02.
Article in English | MEDLINE | ID: mdl-29743179

ABSTRACT

The Bruton tyrosine kinase inhibitor ibrutinib induces high rates of clinical response in chronic lymphocytic leukemia (CLL). However, there remains a need for adjunct treatments to deepen response and to overcome drug resistance. Blinatumomab, a CD19/CD3 bispecific antibody (bsAb) designed in the BiTE (bispecific T-cell engager) format, is approved by the US Food and Drug Administration for the treatment of relapsed or refractory B-cell precursor acute lymphoblastic leukemia. Because of its short half-life of 2.1 hours, blinatumomab requires continuous intravenous dosing for efficacy. We developed a novel CD19/CD3 bsAb in the single-chain Fv-Fc format (CD19/CD3-scFv-Fc) with a half-life of ∼5 days. In in vitro experiments, both CD19/CD3-scFv-Fc and blinatumomab induced >90% killing of CLL cells from treatment-naïve patients. Antileukemic activity was associated with increased autologous CD8 and CD4 T-cell proliferation, activation, and granzyme B expression. In the NOD/SCID/IL2Rγnull patient-derived xenograft mouse model, once-weekly treatment with CD19/CD3-scFv-Fc eliminated >98% of treatment-naïve CLL cells in blood and spleen. By contrast, blinatumomab failed to induce a response, even when administered daily. We next explored the activity of CD19/CD3-scFv-Fc in the context of ibrutinib treatment and ibrutinib resistance. CD19/CD3-scFv-Fc induced more rapid killing of CLL cells from ibrutinib-treated patients than those from treatment-naïve patients. CD19/CD3-scFv-Fc also demonstrated potent activity against CLL cells from patients with acquired ibrutinib-resistance harboring BTK and/or PLCG2 mutations in vitro and in vivo using patient-derived xenograft models. Taken together, these data support investigation of CD19/CD3 bsAb's and other T cell-recruiting bsAb's as immunotherapies for CLL, especially in combination with ibrutinib or as rescue therapy in ibrutinib-resistant disease.


Subject(s)
Antibodies, Bispecific/therapeutic use , Antigens, CD19/immunology , CD3 Complex/immunology , Drug Resistance, Neoplasm/drug effects , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Pyrazoles/pharmacology , Pyrimidines/pharmacology , Salvage Therapy , Adenine/analogs & derivatives , Animals , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/immunology , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Mice , Mice, Inbred NOD , Mice, SCID , Piperidines , Single-Chain Antibodies/immunology , Tumor Cells, Cultured , Xenograft Model Antitumor Assays
4.
Clin Immunol ; 181: 24-28, 2017 08.
Article in English | MEDLINE | ID: mdl-28578024

ABSTRACT

We examined complement-dependent cytotoxicity (CDC) by hexamer formation-enhanced CD20 mAb Hx-7D8 of patient-derived chronic lymphocytic leukemia (CLL) cells that are relatively resistant to CDC. CDC was analyzed in normal human serum (NHS) and serum from an individual genetically deficient for C9. Hx-7D8 was able to kill up to 80% of CLL cells in complete absence of C9. We conclude that the narrow C5b-8 pores formed without C9 are sufficient for CDC due to efficient antibody-mediated hexamer formation. In the absence of C9, we observed transient intracellular increases of Ca2+ during CDC (as assessed with FLUO-4) that were extended in time. This suggests that small C5b-8 pores allow Ca2+ to enter the cell, while dissipation of the fluorescent signal accompanying cell disintegration is delayed. The Ca2+ signal is retained concomitantly with TOPRO-3 (viability dye) staining, thereby confirming that Ca2+ influx represents the most proximate mediator of cell death by CDC.


Subject(s)
Complement C9/deficiency , Complement System Proteins/immunology , Immunologic Deficiency Syndromes/immunology , Leukemia, Lymphocytic, Chronic, B-Cell , Rituximab/pharmacology , Calcium/metabolism , Cell Survival/drug effects , Complement C9/immunology , Complement Membrane Attack Complex/immunology , Complement Membrane Attack Complex/metabolism , Complement System Proteins/metabolism , Hereditary Complement Deficiency Diseases , Humans , Immunotherapy , Polymerization
5.
Clin Immunol ; 171: 32-35, 2016 Oct.
Article in English | MEDLINE | ID: mdl-27546448

ABSTRACT

During malarial anemia, 20 uninfected red blood cells (RBCs) are destroyed for every RBC infected by Plasmodium falciparum (Pf). Increasing evidence indicates an important role for complement in destruction of uninfected RBCs. Products of RBC lysis induced by Pf, including the digestive vacuole and hematin, activate complement and promote C3 fragment deposition on uninfected RBCs. C3-opsonized cells are then subject to extravascular destruction mediated by fixed tissue macrophages which express receptors for C3 fragments. The Compstatin family of cyclic peptides blocks complement activation at the C3 cleavage step, and is under investigation for treatment of complement-mediated diseases. We demonstrate, that under a variety of stringent conditions, second-generation Compstatin analogue Cp40 completely blocks hematin-mediated deposition of C3 fragments on naïve RBCs. Our findings indicate that prophylactic provision of Compstatin for malaria-infected individuals at increased risk for anemia may provide a safe and inexpensive treatment to prevent or substantially reduce malarial anemia.


Subject(s)
Complement C3b/metabolism , Erythrocytes/drug effects , Hemin/metabolism , Peptides, Cyclic/pharmacology , Anemia/drug therapy , Erythrocytes/metabolism , Humans , Malaria, Falciparum/drug therapy , Plasmodium falciparum
6.
J Immunol ; 197(5): 1762-75, 2016 09 01.
Article in English | MEDLINE | ID: mdl-27474078

ABSTRACT

Recently, we demonstrated that IgG Abs can organize into ordered hexamers after binding their cognate Ags expressed on cell surfaces. This process is dependent on Fc:Fc interactions, which promote C1q binding, the first step in classical pathway complement activation. We went on to engineer point mutations that stimulated IgG hexamer formation and complement-dependent cytotoxicity (CDC). The hexamer formation-enhanced (HexaBody) CD20 and CD38 mAbs support faster, more robust CDC than their wild-type counterparts. To further investigate the CDC potential of these mAbs, we used flow cytometry, high-resolution digital imaging, and four-color confocal microscopy to examine their activity against B cell lines and primary chronic lymphocytic leukemia cells in sera depleted of single complement components. We also examined the CDC activity of alemtuzumab (anti-CD52) and mAb W6/32 (anti-HLA), which bind at high density to cells and promote substantial complement activation. Although we observed little CDC for mAb-opsonized cells reacted with sera depleted of early complement components, we were surprised to discover that the Hexabody mAbs, as well as ALM and W6/32, were all quite effective at promoting CDC in sera depleted of individual complement components C6 to C9. However, neutralization studies conducted with an anti-C9 mAb verified that C9 is required for CDC activity against cell lines. These highly effective complement-activating mAbs efficiently focus activated complement components on the cell, including C3b and C9, and promote CDC with a very low threshold of MAC binding, thus providing additional insight into their enhanced efficacy in promoting CDC.


Subject(s)
ADP-ribosyl Cyclase 1/metabolism , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Antibody-Dependent Cell Cytotoxicity , Antigens, CD20/metabolism , Antigens/immunology , Binding Sites, Antibody , Complement C9/metabolism , Membrane Glycoproteins/metabolism , ADP-ribosyl Cyclase 1/immunology , Alemtuzumab , Antibodies, Monoclonal, Humanized/immunology , Antigens, CD20/immunology , B-Lymphocytes/immunology , Cell Line, Tumor , Complement Activation , Complement C3b/metabolism , Complement C9/immunology , Complement System Proteins/immunology , Complement System Proteins/metabolism , Humans , Membrane Glycoproteins/immunology
7.
Mol Immunol ; 70: 13-23, 2016 Feb.
Article in English | MEDLINE | ID: mdl-26690706

ABSTRACT

Complement-dependent cytotoxicity is an important mechanism of action of certain mAbs used in cancer immunotherapy, including ofatumumab and rituximab. However, the detailed sequence of cellular changes that occur in nucleated cells attacked by mAb and complement has not been delineated. Recently developed CD20 mAbs, engineered to form hexamers on binding to cells, react with B-cells in serum, chelate C1q, and then activate complement and promote cell killing considerably more effectively than their wild-type precursors. We used these engineered mAbs as a model to investigate the sequence of events that occur when mAbs bind to B-cell lines and to primary cells from patients with chronic lymphocytic leukemia and then activate complement. Based on four-color confocal microscopy real-time movies and high resolution digital imaging, we find that after CD20 mAb binding and C1q uptake, C3b deposits on cells, followed by Ca(2+) influx, revealed by bright green signals generated on cells labeled with FLUO-4, a Ca(2+) indicator. The bright FLUO-4/Ca(2+) signal fades, replaced by punctate green signals in mitochondria, indicating Ca(2+) localization. This step leads to mitochondrial poisoning followed by cell death. The entire sequence is completed in <2 min for hexamerization-enhanced CD20 mAb-mediated killing. To our knowledge this is the first time the entire process has been characterized in detail in real time. By identifying multiple discrete steps in the cytotoxic pathway for nucleated cells our findings may inform future development and more effective application of complement-fixing mAbs to cancer treatment.


Subject(s)
Antibodies, Monoclonal/immunology , Antibody-Dependent Cell Cytotoxicity/immunology , Antineoplastic Agents/immunology , B-Lymphocytes/immunology , Leukemia, Lymphocytic, Chronic, B-Cell/immunology , Antibodies, Monoclonal/pharmacology , Antibodies, Monoclonal, Humanized , Antineoplastic Agents/pharmacology , Cell Line , Cell Line, Tumor , Complement Activation/immunology , Humans , Microscopy, Confocal , Rituximab/immunology , Rituximab/pharmacology
8.
MAbs ; 7(4): 672-80, 2015.
Article in English | MEDLINE | ID: mdl-26037225

ABSTRACT

Human IgG is produced with C-terminal lysines that are cleaved off in circulation. The function of this modification was unknown and generally thought not to affect antibody function. We recently reported that efficient C1q binding and complement-dependent cytotoxicity (CDC) requires IgG hexamerization at the cell surface. Here we demonstrate that C-terminal lysines may interfere with this process, leading to suboptimal C1q binding and CDC of cells opsonized with C-terminal lysine-containing IgG. After we removed these lysines with a carboxypeptidase, maximal complement activation was observed. Interestingly, IgG1 mutants containing either a negative C-terminal charge or multiple positive charges lost CDC almost completely; however, CDC was fully restored by mixing C-terminal mutants of opposite charge. Our data indicate a novel post-translational control mechanism of human IgG: human IgG molecules are produced in a pro-form in which charged C-termini interfere with IgG hexamer formation, C1q binding and CDC. To allow maximal complement activation, C-terminal lysine processing is required to release the antibody's full cytotoxic potential.


Subject(s)
Antibodies, Monoclonal/immunology , Complement Activation/immunology , Complement C1q/immunology , Cytotoxicity, Immunologic , Immunoglobulin G/immunology , Mutation, Missense , Amino Acid Substitution , Antibodies, Monoclonal/genetics , Complement Activation/genetics , Complement C1q/genetics , HEK293 Cells , Humans , Immunoglobulin G/genetics , Lysine/genetics , Lysine/immunology
9.
J Chem Inf Model ; 47(5): 1906-12, 2007.
Article in English | MEDLINE | ID: mdl-17715910

ABSTRACT

In this study, we have developed a two model system to mimic the active and inactive states of a G-protein coupled receptor specifically the alpha1A adrenergic receptor. We have docked two agonists, epinephrine (phenylamine type) and oxymetazoline (imidazoline type), as well as two antagonists, prazosin and 5-methylurapidil, into two alpha1A receptor models, active and inactive. The best docking complexes for both agonists had hydrophilic interactions with D106, while neither antagonist did. Prazosin and oxymetazoline had hydrophobic interactions with F308 and F312. We predict from our study that the active state is stabilized by the interaction of F193 with I114, L197, V278, F281, and V282. The active state is further stabilized by the interaction of F312 with L75, V79, and L80. We also predict that the inactive state of the receptor is stabilized by the interaction of F312 with W102, F288, and M292.


Subject(s)
Adrenergic alpha-Antagonists/pharmacology , Receptors, Adrenergic, alpha-1/drug effects , Receptors, Adrenergic, alpha-1/metabolism , Adrenergic alpha-Antagonists/chemistry , Epinephrine/chemistry , Epinephrine/pharmacology , Ligands , Models, Molecular , Nitrogen/chemistry , Oxymetazoline/chemistry , Oxymetazoline/pharmacology , Piperazines/chemistry , Piperazines/pharmacology , Prazosin/chemistry , Prazosin/pharmacology , Protein Conformation , Rhodopsin/chemistry , Structure-Activity Relationship
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