Human inhalable antibody fragments neutralizing SARS-CoV-2 variants for COVID-19 therapy.

As of December 2021, coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), remains a global emergency, and novel therapeutics are urgently needed. Here we describe human single-chain variable fragment (scFv) antibodies (76clAbs) that block an epitope of the SARS-CoV-2 spike protein essential for ACE2-mediated entry into cells. 76clAbs neutralize the Delta variant and other variants being monitored (VBMs) and inhibit spike-mediated pulmonary cell-cell fusion, a critical feature of COVID-19 pathology. In two independent animal models, intranasal administration counteracted the infection. Because of their high efficiency, remarkable stability, resilience to nebulization, and low cost of production, 76clAbs may become a relevant tool for rapid, self-administrable early intervention in SARS-CoV-2-infected subjects independently of their immune status.

Antibody drug conjugates with hydroxamic acid cargos for histone deacetylase (HDAC) inhibition.

Antitumor hydroxamates SAHA and Dacinostat have been linked to cetuximab and trastuzumab through a non-cleavable linker based on the p-mercaptobenzyl alcohol structure. These antibody drug conjugates (ADCs) were able to inhibit HDAC in several tumour cell lines. The cetuximab based ADCs block human lung adenocarcinoma cell proliferation, demonstrating that bioconjugation with antibodies is a suitable approach for targeted therapy based on hydroxamic acid-containing drugs. This work also shows that ADC-based delivery might be used to overcome the classical pharmacokinetic problems of hydroxamic acids.

Synthesis and preliminary in vitro evaluation of DOTA-Tenatumomab conjugates for theranostic applications in tenascin expressing tumors.

Tenatumomab is an anti-tenascin murine monoclonal antibody previously used in clinical trials for delivering radionuclides to tumors by both pre-targeting (biotinylated Tenatumomab within PAGRIT) and direct 131Iodine labeling approaches. Here we present the synthesis and in vitro characterization of three Tenatumomab conjugates to bifunctional chelating agents (NHS-DOTA, NCS-DOTA and NCS-DTPA). Results indicate ST8198AA1 (Tenatumomab-DOTAMA, derived by conjugation of NHS-DOTA), as the most promising candidate in terms of conjugation rate and yield, stability, antigen immunoreactivity and affinity. Labeling efficiency of the different chelators was investigated with a panel of cold metals indicating DOTAMA as the best chelator. Labeling of Tenatumomab-DOTAMA was then optimized with several metals and stability performed confirms suitability of this conjugate for further development. ST8198AA1 represents an improvement of the previous antibody forms because the labeling with radionuclides like 177Lu or 64Cu would allow theranostic applications in patients bearing tenascin expressing tumors.

ErbB2 Targeted Epigenetic Modulation: Anti-tumor Efficacy of the ADC Trastuzumab-HDACi ST8176AA1.

Targeted therapy using monoclonal antibodies conjugated to toxins is gaining space in the treatment of cancer. Here, we report the anti-tumor effect of a new antibody drug conjugate (ADC) delivering a HDAC inhibitor to ErbB2+ solid tumors. Trastuzumab was partially reduced with tris [2-carboxyethyl] phosphine (TCEP) and conjugated to ST7464AA1, the active form of the prodrug HDAC inhibitor ST7612AA1, through a maleimide-thiol linker to obtain the Antibody Drug Conjugate (ADC) ST8176AA1. The average drug/antibody ratio (DAR) was 4.5 as measured by hydrophobic interaction chromatography (HIC). Binding of ST8176AA1 to ErbB2 receptor and internalization in tumor cells were investigated by enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), cytofluorimetry, and High Content Screening (HCS) Imaging. The biological activity of the ADC was evaluated in vitro and in vivo by measuring cell proliferation/cell cycle, apoptosis/DNA damage, tubulin, and histone acetylation and modulation of Epithelial/Mesenchymal Transition (EMT) markers. Receptor binding and internalization of ST8176AA1 were confirmed to be similar to trastuzumab. Higher anti-tumor activity of ST8176AA1 compared to trastuzumab was observed in vitro in tumor cell lines. Such higher activity correlated with increased acetylation of histones and alfa-tubulin as a consequence of HDAC inhibitor-mediated epigenetic modulation that also induced increased expression of ErbB2 and estrogen receptor in triple negative breast cancer cells. Consistently with in vitro data, ST8176AA1 exhibited higher tumor growth inhibition than trastuzumab in xenograft models of ovary and colon carcinoma and in two patient-derived xenograft (PDX) models of pancreatic carcinoma. Immunohistochemistry analysis of tumor masses showed lower expression of the proliferation marker Ki67 and higher expression of cleaved caspase-3 in mice treated with the ADC compared to those treated with trastuzumab and results correlated with increased acetylation of both histones and tubulin. Collectively, present data indicate that ADC ST8176AA1 can target epigenetic modulation to ErbB2+ tumors. Interestingly, the amount of HDACi estimated to be delivered at the ST8176AA1 effective dose would correspond to ~1/1,000 of ST7612AA1 effective dose. Therefore, ST8176AA1 is an attractive new therapeutic candidate because it exhibits increased anti-tumor potency compared to trastuzumab by exerting epigenetic modulation at a much safer dose compared to standard HDACi-based therapeutic protocols.

Antibody drug conjugates (ADCs) charged with HDAC inhibitor for targeted epigenetic modulation.

We describe here two novel antibody-drug conjugates loaded with the HDAC inhibitor ST7612AA1 (IC50 equal to 0.07 µM on NCI-H460 cells), a thiol-based molecule with a moderate toxicity in vivo. Two payloads were prepared using cleavable and non-cleavable linkers. After anchoring to cetuximab through amide bond with lysines, the resulting HDAC inhibitor-antibody conjugates showed ability to recognize EGFR and efficient internalization in tumor cells. Both ADCs induced sensible increment of histones 3 and 4 and alpha-tubulin acetylation. Animal models of human solid tumors showed high anti-tumor efficacy of the conjugates without the toxicity generally observed with traditional ADCs delivering highly potent cytotoxic drugs. These compounds, the first ADCs charged with not highly cytotoxic warheads, are potentially suitable for epigenetic modulation, extending the ADC strategy to the targeted delivery of HDAC inhibitors with many possible therapeutic applications beyond cancer.

AvidinOX-anchored biotinylated trastuzumab and pertuzumab induce down-modulation of ErbB2 and tumor cell death at concentrations order of magnitude lower than not-anchored antibodies.

The oxidized version of Avidin, known as AvidinOX, was previously shown to link to tissue proteins upon injection or nebulization, thus becoming a stable receptor for biotinylated therapeutics. AvidinOX is currently under clinical investigation to target radioactive biotin to inoperable tumor lesions (ClinicalTrials.gov NCT02053324). Presently, we show that the anti-ErbB2 monoclonal antibodies Trastuzumab and Pertuzumab can be chemically biotinylated while maintaining their biochemical and biological properties. By using several and diverse experimental conditions, we show that when AvidinOX is conjugated to tumor cells, low antibody concentrations of biotinylated Trastuzumab (bTrast) or Pertuzumab (bPert) prevent internalization of ErbB2, induce endoplasmic reticulum stress, cell cycle arrest and apoptosis leading to inhibition of proliferation and ErbB2 signaling. Moreover, we found that the treatment is able to induce down-modulation of ErbB2 thus bypassing the known resistance of this receptor to degradation. Interestingly, we show that AvidinOX anchorage is a way to counteract agonistic activities of Trastuzumab and Pertuzumab. Present data are in agreement with previous observations from our group indicating that the engagement of the Epidermal Growth Factor Receptor (EGFR) by AvidinOX-bound biotinylated Cetuximab or Panitumumab, leads to potent tumor inhibition both in vitro and in animal models. All results taken together encourage further investigation of AvidinOX-based treatments with biotinylated antibodies directed to the members of the EGFR family.

Intra-tumor AvidinOX allows efficacy of low dose systemic biotinylated Cetuximab in a model of head and neck cancer.

For locally advanced and metastatic head and neck squamous cell carcinoma (HNSCC), the current clinical use of Cetuximab in chemo/radiotherapy protocols is often associated to severe systemic toxicity. Here we report in vitro data in human FaDu pharynx SCC cells, showing that inactive concentrations of biotinylated Cetuximab (bCet) become active upon anchorage to AvidinOX on the surface of tumor cells. AvidinOX-anchored bCet induces apoptosis and DNA damage as well as specific inhibition of signaling, degradation and abrogation of nuclear translocation of EGFR. In the mouse model of FaDu cancer, we show that intra-tumor injection of AvidinOX allows anti-tumor activity of an otherwise inactive, intraperitoneally delivered, low dose bCet. Consistently with in vitro data, in vivo tumor inhibition is associated to induction of apoptosis, DNA damage and reduced angiogenesis. AvidinOX is under clinical investigation for delivering radioactive biotin to inoperable tumors (ClinicalTrials.gov NCT02053324) and present data support its use for the local treatment of HNSCC in combination with systemic administration of low dose bCet.

Efficacy of aerosol therapy of lung cancer correlates with EGFR paralysis induced by AvidinOX-anchored biotinylated Cetuximab.

Lung cancer, as well as lung metastases from distal primary tumors, could benefit from aerosol treatment. Unfortunately, because of lung physiology, clearance of nebulized drugs is fast, paralleled by unwanted systemic exposure. Here we report that nebulized AvidinOX can act as an artificial receptor for biotinylated drugs. In nude and SCID mice with advanced human KRAS-mutated A549 metastatic lung cancer, pre-nebulization with AvidinOX enables biotinylated Cetuximab to control tumor growth at a dose lower than 1/25,000 the intravenous effective dose. This result correlates with a striking, specific and unpredictable effect of AvidinOX-anchored biotinylated Cetuximab, as well as Panitumumab, observed on a panel of tumor cell lines, leading to inhibition of dimerization and signalling, blockade of endocytosis, induction of massive lysosomal degradation and abrogation of nuclear translocation of EGFR. Excellent tolerability, together with availability of pharmaceutical-grade AvidinOX and antibodies, will allow rapid clinical translation of the proposed therapy.

Optimized selection of anti-tumor recombinant antibodies from phage libraries on intact cells.

Generation of human recombinant antibody libraries displayed on the surface of the filamentous phage and selection of specific antibodies against desirable targets allows production of fully human antibodies usable for repeated administration in humans. Various lymphoid tissues from immunized donors, such as lymph nodes or peripheral blood lymphocytes from individuals with tumor or lymphocytes infiltrating tumor masses may serve as a source of specific anti-tumor antibody repertoire for generation of tumor-focused phage display libraries. In the case of lack of tumor-associated antigens in the purified form, high affinity anti-tumor antibodies can be isolated through library panning on whole cells expressing these antigens. However, affinity selection against cell surface specific antigens within highly heterogeneous population of molecules is not a very efficient process that often results in the selection of unspecific antibodies or antibodies against intracellular antigens that are generally useless for targeted immunotherapy. In this work, we developed a new cell-based antibody selection protocol that, by eliminating the contamination of dead cells from the cell suspension, dramatically improves the selection frequency of anti-tumor antibodies recognizing cell surface antigens.

Chemical linkage to injected tissues is a distinctive property of oxidized avidin.

We recently reported that the oxidized avidin, named AvidinOX®, resides for weeks within injected tissues as a consequence of the formation of Schiff's bases between its aldehyde groups and tissue protein amino groups. We also showed, in a mouse pre-clinical model, the usefulness of AvidinOX for the delivery of radiolabeled biotin to inoperable tumors. Taking into account that AvidinOX is the first oxidized glycoprotein known to chemically link to injected tissues, we tested in the mouse a panel of additional oxidized glycoproteins, with the aim of investigating the phenomenon. We produced oxidized ovalbumin and mannosylated streptavidin which share with avidin glycosylation pattern and tetrameric structure, respectively and found that neither of them linked significantly to cells in vitro nor to injected tissues in vivo, despite the presence of functional aldehyde groups. The study, extended to additional oxidized glycoproteins, showed that the in vivo chemical conjugation is a distinctive property of the oxidized avidin. Relevance of the high cationic charge of avidin into the stable linkage of AvidinOX to tissues is demonstrated as the oxidized acetylated avidin lost the property. Plasmon resonance on matrix proteins and cellular impedance analyses showed in vitro that avidin exhibits a peculiar interaction with proteins and cells that allows the formation of highly stable Schiff's bases, after oxidation.

Therapeutic use of avidin is not hampered by antiavidin antibodies in humans.

Hen egg white avidin is increasingly used in the clinic as part of multifactor treatments such as pretargeted radionuclide therapy of cancer or as an antidote of biotinylated drugs. Taking into account that naturally occurring human antiavidin antibodies (HAVA) are common in humans, the present work investigates avidin immunogenicity as part of risk/benefit evaluations. Sera from 139 oncology patients naive to avidin were confirmed to exhibit HAVA with lognormally distributed titers. HAVA were boosted after avidin treatment, with no correlation with the avidin dose or with the basal titer. No antibody-related clinical symptoms were observed in 21 HAVA-positive patients treated with avidin. In mouse models, high mouse antiavidin antibody titers, induced to simulate the worst human condition, neither reduced the biotin uptake of intratissue-injected avidin nor affected the capacity of intravenously injected avidin to clear a biotinylated drug from circulation. In both models the avidin treatment was well tolerated. Results indicate that avidin immunogenicity does not affect its safety and efficacy, thus encouraging its further use in clinical applications.

Biochemical and biological characterization of a new oxidized avidin with enhanced tissue binding properties.

Chicken avidin and bacterial streptavidin are widely employed in vitro for their capacity to bind biotin, but their pharmacokinetics and immunological properties are not always optimal, thereby limiting their use in medical treatments. Here we investigate the biochemical and biological properties of a new modified avidin, obtained by ligand-assisted sodium periodate oxidation of avidin. This method allows protection of biotin-binding sites of avidin from inactivation caused by the oxidation step and delay of avidin clearance from injected tissue by generation of aldehyde groups from avidin carbohydrate moieties. Oxidized avidin shows spectroscopic properties similar to that of native avidin, indicating that tryptophan residues are spared from oxidation damage. In strict agreement with these results, circular dichroism and isothermal titration calorimetry analyses confirm that the ligand-assisted oxidation preserves the avidin protein structure and its biotin binding capacity. In vitro cell binding and in vivo tissue residence experiments demonstrate that aldehyde groups provide oxidized avidin the property to bind cellular and interstitial protein amino groups through Schiff's base formation, resulting in a tissue half-life of 2 weeks, compared with 2 h of native avidin. In addition, the efficient uptake of the intravenously injected (111)In-BiotinDOTA (ST2210) in the site previously treated with modified avidin underlines that tissue-bound oxidized avidin retains its biotin binding capacity in vivo. The results presented here indicate that oxidized avidin could be employed to create a stable artificial receptor in diseased tissues for the targeting of biotinylated therapeutics.

Tumor-infiltrating B lymphocytes as an efficient source of highly specific immunoglobulins recognizing tumor cells.

BACKGROUND: There is much evidence that tumor cells elicit a humoral immune response in patients. In most cases, the presence of antibodies in peripheral blood is detected only in small proportion of patients with tumors overexpressing the corresponding antigen. In the present study, we analyzed the significance of local humoral response provided by tumor-infiltrating lymphocytes in breast cancer patients. METHODS: The ability of a patient's immune system to produce specific antibodies inside tumor tissue, capable of recognizing tumor cells, was explored through analysis of the oligoclonality of antibodies derived from tumor-infiltrating lymphocytes and construction of a series of recombinant antibody libraries in scFv format, derived from breast tumor-infiltrating B lymphocytes. These libraries and one from peripheral blood lymphocytes of a single breast cancer patient were panned against three purified surface tumor antigens, such as CEA, MUC1 and ED-B domain, and against intact MCF7 breast carcinoma cells. RESULTS: Application of novel display vector, pKM19, allowed isolation of a large panel of breast cancer-specific antibodies against known tumor antigens, as well as against breast carcinoma cells. Reactivity of novel scFvs was confirmed by ELISA, immunohistochemistry, fluorescence staining and flow cytometry. We demonstrated that seven of ten primary breast tumor specimens, obtained using discarded surgical material, could be exploited as an appropriate source for generation of phage display libraries, giving highly specific antitumor antibodies which recognize heterologous tumor cells. CONCLUSION: Local humoral immune response within tumor tissue in breast cancer patients frequently has an oligoclonal character. Efficient selection of specific antitumor antibodies from recombinant antibody libraries, derived from such oligoclonal tumor-infiltrated B lymphocytes, indicates the presence of natural immune response against tumor antigens in these patients. The described method is very promising for development of antitumor antibodies, potentially useful for diagnostic and therapeutic approaches.

Low and high tenascin-expressing tumors are efficiently targeted by ST2146 monoclonal antibody.

ST2146biot is a biotinylated anti-tenascin monoclonal antibody (mAb) to be used for Pretargeted Antibody Guided Radioimmunotherapy (PAGRIT) of solid tumors. In vivo biodistribution studies of (125)I-labeled ST2146biot were done in nude mice transplanted with human HT-29 colon carcinoma and/or human U-118MG glioblastoma cells characterized for low and high tenascin expression, respectively. In vitro results show that ST2146 retains immunoreactivity upon biotinylation, in contrast to other anti-tenascin mAbs. In vivo biodistribution of ST2146 shows specific tumor accumulation up to 10 days after the i.v. injection, with no relevant differences between biotinylated and nonbiotinylated ST2146. A dose of 4 microg/mouse saturates the low tenascin-expressing human colon carcinoma HT-29, whereas the high tenascin-expressing human glioblastoma U-118MG seems to be saturated at a ST2146biot dose between 320 and 640 microg/mouse. The percentage of injected dose per gram of tumor ranges from 10% to 30%, corresponding to an amount of ST2146biot/g of tumor of approximately 400 ng/g and >200 microg/g for HT-29 and U-118MG, respectively. Tumor to normal organs uptake ratios are between 15 and 60, confirming high tumor selectivity of ST2146biot despite its cross-reactivity with the tenascin expressed at low level in the normal mouse organs. The ST2146biot localization data are substantially confirmed even when both low and high tenascin-expressing tumors are implanted in the same animal. To our knowledge, the absolute amount of ST2146biot, specifically localized in xenotransplanted human tumors, is the highest thus far described and supports the clinical use of this mAb in PAGRIT(R).

Selection, affinity maturation, and characterization of a human scFv antibody against CEA protein.

BACKGROUND: CEA is a tumor-associated antigen abundantly expressed on several cancer types, including those naturally refractory to chemotherapy. The selection and characterization of human anti-CEA single-chain antibody fragments (scFv) is a first step toward the construction of new anticancer monoclonal antibodies designed for optimal blood clearance and tumor penetration. METHODS: The human MA39 scFv, selected for its ability to recognize a CEA epitope expressed on human colon carcinomas, was first isolated from a large semi-synthetic ETH-2 antibody phage library, panned on human purified CEA protein. Subsequently, by in vitro mutagenesis of a gene encoding for the scFv MA39, a new library was established, and new scFv antibodies with improved affinity towards the CEA cognate epitope were selected and characterized. RESULTS: The scFv MA39 antibody was affinity-maturated by in vitro mutagenesis and the new scFv clone, E8, was isolated, typed for CEA family member recognition and its CEACAM1, 3 and 5 shared epitope characterized for expression in a large panel of human normal and tumor tissues and cells. CONCLUSION: The binding affinity of the scFv E8 is in a range for efficient, in vivo, antigen capture in tumor cells expressing a shared epitope of the CEACAM1, 3 and 5 proteins. This new immunoreagent meets all criteria for a potential anticancer compound: it is human, hence poorly or not at all immunogenic, and it binds selectively and with good affinity to the CEA epitope expressed by metastatic melanoma and colon and lung carcinomas. Furthermore, its small molecular size should provide for efficient tissue penetration, yet give rapid plasma clearance.

Identification and refinement of a peptide affinity ligand with unique specificity for a monoclonal anti-tenascin-C antibody by screening of a phage display library.

Using phage display technology, a 22-mer peptide was selected as a ligand with unique specificity for the murine monoclonal ST2146 antibody that recognizes the EGF repeats region of the human tumor-associated antigen tenascin-C. This peptide, synthesized in an 8-branched form to enhance its binding properties, is useful in replacing the native antigen in the affinity and immunoreactivity characterization of the ST2146 antibody and its biotinylated derivatives. Affinity resins, prepared by immobilizing the mimotope or its shorter 10-mer binding unit on a chromatographic support, were able to capture ST2146 directly from the hybridoma supernatant, with antibody recovery and host cell protein (HCP) reduction similar to or better than protein A sorbent, a purity degree exceeding 95%, and full recovery of antibody activity. The affinity constants of both peptides, as determined by frontal analysis of broad-zone elution affinity chromatography and BiaCore measurements, were very similar and included in a range suitable for affinity ligands. Column capacity, determined by applying a large excess of purified ST2146 to 1 mL of column bed volume, was close to 50 mg/mL for both resins. These matrices retain their ST2146 binding properties after various treatments, including sanitization, thus indicating very high stability in terms of ligand leakage and degradation. Moreover, the short form shows higher enzymatic stability, thus proving more suitable as ligand for ST2146 affinity purification.

Improved tumor targeting by combined use of two antitenascin antibodies.

PURPOSE: In the pretargeted antibody-guided radioimmunotherapy (PAGRIT) system, the combined use of two different antibodies directed against the same tumor antigen could represent a valid approach for improving tumor targeting and therapeutic efficacy. We developed a novel monoclonal antitenascin antibody, ST2485, and studied its biochemical and functional properties by in vitro and in vivo assays. We then investigated the first of the three-step therapy combining ST2485 with another antitenascin antibody, ST2146, previously described, to increase accumulation of biotinylated antibodies at the tumor site. EXPERIMENTAL DESIGN: Studies of immunoreactivity, affinity, immunohistochemistry, and biodistribution in xenograft model were carried out on ST2485. Analysis of the ST2485 and ST2146 combination was preliminary carried out by ELISA and BiaCore tests and then by in vivo distribution studies after administration of the radiolabeled biotinylated antibodies, followed by a chase with avidin as clearing agent. RESULTS: ST2485 was found to be a suitable antibody for therapeutic applications. Indeed, for its behavior in all tests, it was comparable with ST2146 and better than BC2, an antibody already used for clinical trials. The additivity of ST2146 and ST2485 in tenascin C binding, shown by in vitro tests, was confirmed by biodistribution studies in a xenograft model where tumor localization of the antibodies was near the sum of each antibody alone, with a tumor-to-blood ratio higher than 24. CONCLUSION: The results reported in this study suggest that a monoclonal antitenascin antibody mixture can improve tumor targeting. This strategy could represent progress for therapeutic approaches such as PAGRIT.