AllergoOncology: Expression platform development and functional profiling of an anti-HER2 IgE antibody.
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ABSTRACT:
SUBMITTER: Ilieva KM
PROVIDER: S-EPMC6817356 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Antibody-based drugs are a leading class of biologics used to treat a variety of diseases, including cancer. However, wide antibody implementation is hindered by manufacturing challenges and high production cost. Use of in-vitro-transcribed mRNA (IVT-mRNA) for endogenous protein expression has the potential to circumvent many of the shortcomings of antibody production and therapeutic application. Here, we describe the development of an IVT-mRNA system for in vivo delivery of a humanized anti-HER2 (also known as ERBB2) antibody, trastuzumab, and demonstrate its anticancer activity. We engineered the IVT-mRNA sequence to maximize expression, then formulated the IVT-mRNA into lipid-based nanoparticles (LNPs) to protect the mRNA from degradation and enable efficient in vivo delivery. Systemic delivery of the optimized IVT-mRNA loaded into LNPs resulted in antibody serum concentrations of 45 ± 8.6 ?g/mL for 14 days after LNP injection. Further studies demonstrated an improved pharmacokinetic profile of the produced protein compared to injection of trastuzumab protein. Finally, treatment of tumor-bearing mice with trastuzumab IVT-mRNA LNPs selectively reduced the volume of HER2-positive tumors and improved animal survival. Taken together, the results of our study demonstrate that using IVT-mRNA LNPs to express full-size therapeutic antibodies in the liver can provide an effective strategy for cancer treatment and offers an alternative to protein administration.
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Project description:BackgroundThe efficacy assessment of human anti-IgE monoclonal antibodies (mAbs) in animal models before clinical trials is hampered due to the lack of cross-reactivity of anti-IgE mAbs between species.ObjectiveWe developed CRE-DR (an anti-dog IgE monoclonal antibody), an anti-IgE mouse mAb that recognizes canine and human IgE, and then examined its IgE specificity and cross-reactivity between three animal and human species.MethodsAfter mouse immunization with a synthetic peptide derived from canine IgE (282 NTNDWIEGETYYC294 ), we generated a hybridoma producing CRE-DR. The CRE-DR purified from the ascites of hybridoma-inoculated mice was used for ELISA and Western blot analysis to examine reactivity to dog, human, and rodent IgEs as well as recombinant bovine serum albumin (BSA)-conjugated to canine, human, and rodent IgE amino acid peptides corresponding to the immunizing sequence. We then performed enzyme-linked immunosorbent assays (ELISAs) for dog IgE using sera from dogs with atopic dermatitis (AD) after inhibition with canine IgE and IgG. The amino acid sequence recognized by CRE-DR was identified by ELISA using synthetic peptides.ResultsCRE-DR is a monoclonal mouse IgG1κ specific for dog IgE, and the ELISA values in atopic dog sera were inhibited by dog IgE, but not dog IgG. The binding of CRE-DR to human IgE was relatively maintained, but not to rodent IgEs, which results were confirmed with the BSA-conjugated IgE peptides of the various species. The CRE-DR reactivity was supported by the comparison of amino acid sequence of CRE-DR epitope, DWIEGETYYC, in dog IgE; one, two, and three amino acids were substituted in the human, rat, and mouse IgE epitopes, respectively.Conclusions and clinical relevanceCRE-DR is a mAb cross-reactive to dog and human IgEs, which can allow the use of a dog model of allergy to test the efficacy of a CRE-DR-derived anti-IgE therapeutic mAb before human clinical trials.
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Project description:Targeting of immunoglobulin E (IgE) represents an interesting approach for the treatment of allergic disorders. A high-affinity monoclonal anti-IgE antibody, ligelizumab, has recently been developed to overcome some of the limitations associated with the clinical use of the therapeutic anti-IgE antibody, omalizumab. Here, we determine the molecular binding profile and functional modes-of-action of ligelizumab. We solve the crystal structure of ligelizumab bound to IgE, and report epitope differences between ligelizumab and omalizumab that contribute to their qualitatively distinct IgE-receptor inhibition profiles. While ligelizumab shows superior inhibition of IgE binding to FcεRI, basophil activation, IgE production by B cells and passive systemic anaphylaxis in an in vivo mouse model, ligelizumab is less potent in inhibiting IgE:CD23 interactions than omalizumab. Our data thus provide a structural and mechanistic foundation for understanding the efficient suppression of FcεRI-dependent allergic reactions by ligelizumab in vitro as well as in vivo.
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Project description:Human epidermal growth factor receptor 2 (HER2)-targeted therapies have revolutionized the treatment of HER2-positive breast cancer, both in the metastatic and early stage settings. While trastuzumab and lapatinib had been the mainstays of treatment in combination with chemotherapy, innate and acquired resistance to these therapies occur. More recently, two additional HER2-directed therapies have been approved for HER2-positive breast cancer. Pertuzumab is a humanized monoclonal antibody that binds to the extracellular portion of the receptor on a domain distinct from the binding site of trastuzumab. The addition of pertuzumab to trastuzumab results in synergistic tumor cell inhibition and has been shown to significantly improve clinical outcomes for patients with HER2-positive metastatic breast cancer (MBC) compared to trastuzumab plus chemotherapy alone. In addition, ado-trastuzumab emtansine (T-DM1), a novel antibody-drug conjugate linking trastuzumab with the cytotoxic maytansinoid, DM1, is an effective treatment for HER2-positive breast cancer that has progressed on other HER2-directed therapies. Both pertuzumab and T-DM1 are relatively well tolerated. This review presents the mechanisms of action as well as phase I, II and III clinical data describing the safety and efficacy of pertuzumab and T-DM1 for HER2-positive breast cancer.
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Project description:BACKGROUND:There has been substantial interest in HER2 intratumoral heterogeneity as an explanation for the development of resistance to anti-HER2 therapies in breast cancer, particularly to trastuzumab emtansine (T-DM1). METHODS:Through a literature-based approach, we discuss mechanisms of resistance to HER2-targeting antibody-drug conjugates (ADCs) in breast cancer. RESULTS:We describe results from clinical studies reporting the effect of anti-HER2 strategies particularly ADCs and their mechanistic effect. We review biological findings underlying HER2 heterogeneity and its implication in the development of novel anti-HER2 drugs including new ADCs in clinical development like trastuzumab deruxtecan (DS-8201). CONCLUSIONS:We suggest potential mechanisms to optimize these compounds and their future clinical implementation.
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Project description:Immunoglobulin E (IgE) plays a key role in allergic asthma and is a clinically validated target for monoclonal antibodies. Therapeutic anti-IgE antibodies block the interaction between IgE and the Fc epsilon (Fcε) receptor, which eliminates or minimizes the allergic phenotype but does not typically curtail the ongoing production of IgE by B cells. We generated high-affinity anti-IgE antibodies (MEDI4212) that have the potential to both neutralize soluble IgE and eliminate IgE-expressing B-cells through antibody-dependent cell-mediated cytotoxicity. MEDI4212 variants were generated that contain mutations in the Fc region of the antibody or alterations in fucosylation in order to enhance the antibody's affinity for FcγRIIIa. All MEDI4212 variants bound to human IgE with affinities comparable to the wild-type (WT) antibody. Each variant was shown to inhibit the interaction between IgE and FcεRI, which translated into potent inhibition of FcγRI-mediated function responses. Importantly, all variants bound similarly to IgE at the surface of membrane IgE expressing cells. However, MEDI4212 variants demonstrated enhanced affinity for FcγRIIIa including the polymorphic variants at position 158. The improvement in FcγRIIIa binding led to increased effector function in cell based assays using both engineered cell lines and class switched human IgE B cells. Through its superior suppression of IgE, we anticipate that effector function enhanced MEDI4212 may be able to neutralize high levels of soluble IgE and provide increased long-term benefit by eliminating the IgE expressing B cells before they differentiate and become IgE secreting plasma cells.
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Project description:Autophagy has been emerging as a novel cytoprotective mechanism to increase tumor cell survival under conditions of metabolic stress and hypoxia as well as to escape chemotherapy-induced cell death. To elucidate whether autophagy might also protect cancer cells from the growth inhibitory effects of targeted therapies, we evaluated the autophagic status of preclinical breast cancer models exhibiting auto-acquired resistance to the anti-HER2 monoclonal antibody trastuzumab (Tzb). We first examined the basal autophagic levels in Tzb-naive SKBR3 cells and in two pools of Tzb-conditioned SKBR3 cells (TzbR), which optimally grow in the presence of Tzb doses as high as 200 microg/ml Tzb. Fluorescence microscopic analyses revealed that the number of punctate LC3 structures -a hallmark of autophagy- was drastically higher in Tzb-refractory cells than in Tzb-sensitive SKBR3 parental cells. Immunoblotting analyses confirmed that the lipidation product of the autophagic conversion of LC3 was accumulated to high levels in TzbR cells. High levels of the LC3 lipidated form in Tzb-refractory cells were accompanied by decreased p62/sequestosome-1 protein expression, a phenomenon characterizing the occurrence of increased autophagic flux. Moreover, increased autophagy was actively used to survive Tzb therapy as TzbR pools were exquisitely sensitive to chemical inhibitors of autophagosomal formation/function. Knockdown of LC3 expression via siRNA similarly resulted in reduced TzbR cell proliferation and supra-additively interacted with Tzb to re-sensitize TzbR cells. Sub-groups of Tzb-naive SKBR3 parental cells accumulated LC3 punctate structures and decreased p62 expression after treatment with high-dose Tzb, likely promoting their own resistance. This is the first report showing that HER2-overexpressing breast cancer cells chronically exposed to Tzb exhibit a bona fide up-regulation of the autophagic activity that efficiently works to protect breast cancer cells from the growth-inhibitory effects of Tzb. Therapeutic targeting autophagosome formation/function might represent a novel molecular avenue to reduce the emergence of Tzb resistance in HER2-dependent breast carcinomas.
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Project description:Regulatory agencies establish that a broad physicochemical and biological characterization is necessary for the evaluation of comparability between a biosimilar candidate product and a reference commercial drug. Between them, conformational characterization of proteins is of vital importance to determine its folding and biological functions. In this work, the conformational features of a novel monoclonal antibody (called 5G4) were evaluated by means of circular dichroism spectroscopy and fluorescence. Secondary structure and thermal stability of mAbs were determined by circular dichroism in the far ultraviolet, while three-dimensional folding of proteins was analyzed by both circular dichroism in the near ultraviolet and intrinsic tryptophan fluorescence. In all experiments, Herceptin (Roche) was used as control. Both antibodies showed a composition of secondary structure predominantly of β-sheets (55-56%) and thermal stability of ~ 75°C, suggesting structural similarity. The three-dimensional folding of proteins was also similar due to the absorption spectra of the aromatic residues and the emission wavelength maxima by fluorescence were comparable. The values of the fluorescence attenuation constant (Stern-Volmer constant) for increasing concentrations of acrylamide were also similar, suggesting a degree of exposure of tryptophan residues similar, although it was slightly decreased for Herceptin. Our data permit to consider that 5G4 monoclonal antibody showed similar conformational characteristics when compared with Herceptin.
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