Evaluation of Phage Display Biopanning Strategies for the Selection of Anti-Cell Surface Receptor Antibodies

Monoclonal antibodies (mAbs) are one of the most successful and versatile protein-based pharmaceutical products used to treat multiple pathological conditions. The remarkable specificity of mAbs and their affinity for biological targets has led to the implementation of mAbs in the therapeutic regime of oncogenic, chronic inflammatory, cardiovascular, and infectious diseases. Thus, the discovery of novel mAbs with defined functional activities is of crucial importance to expand our ability to address current and future clinical challenges. In vitro, antigen-driven affinity selection employing phage display biopanning is a commonly used technique to isolate mAbs. The success of biopanning is dependent on the quality and the presentation format of the antigen, which is critical when isolating mAbs against membrane protein targets. Here, we provide a comprehensive investigation of two established panning strategies, surface-tethering of a recombinant extracellular domain and cell-based biopanning, to examine the impact of antigen presentation on selection outcomes with regards to the isolation of positive mAbs with functional potential against a proof-of-concept type I cell surface receptor. Based on the higher sequence diversity of the resulting antibody repertoire, presentation of a type I membrane protein in soluble form was more advantageous over presentation in cell-based format. Our results will contribute to inform and guide future antibody discovery campaigns against cell surface proteins.     

Phage Display Approaches for the Isolation of Monoclonal Antibodies Against Dengue Virus Envelope Domain III from Human and Mouse Derived Libraries

Domain III of the dengue virus envelope protein (EDIII, aa295-395) has an immunoglobulin fold and is the proposed receptor-binding domain of the virus. Previous studies have shown that monoclonal antibodies against EDIII can be neutralizing and have therapeutic potential. Here, cloned Fab-phage libraries of human and mouse origin were screened for DENV specific antibodies. Firstly, bacterially expressed EDIII or whole virus particles were used as bait in biopanning against a large naïve human Fab-phage library (>10 billion independent clones). Multiple panning strategies were employed, and in excess of 1000 clones were screened, but all of the antibodies identified bound the envelope in regions outside EDIII suggesting EDIII antibodies are virtually absent from the naïve human repertoire. Next, a chimeric Fab-phage library was constructed from a panel of EDIII specific mouse hybridomas by pooling the VH and VL chain sequences from the hybridomas and cloning these into the pComb3X phagemid vector with human CH and CL encoding sequences. Biopanning against EDIII identified a unique antibody (C9) that cross-reacts with EDIII from DENV1-3 and, in the IgG format, binds and neutralizes DENV2 in cell-based assays. Sequence analysis and saturation mutagenesis of complementary determining regions (CDR) in the C9 light chain suggest an antigen recognition model in which the LCDR3 is a key determinant of EDIII specificity, while modifications in LCDR1 and LCDR2 affect DENV serotype cross-reactivity. Overall, this study supports the current prevailing opinion that neutralizing anti-EDIII monoclonal antibodies can be readily generated in murine systems, but in humans the anti-DENV immune response is directed away from domain III.     

New Anti-Flavivirus Fusion Loop Human Antibodies with Zika Virus-Neutralizing Potential

Zika virus infections exhibit recurrent outbreaks and can be responsible for disease complications such as congenital Zika virus syndrome. Effective therapeutic interventions are still a challenge. Antibodies can provide significant protection, although the antibody response may fail due to antibody-dependent enhancement reactions. The choice of the target antigen is a crucial part of the process to generate effective neutralizing antibodies. Human anti-Zika virus antibodies were selected by phage display technology. The antibodies were selected against a mimetic peptide based on the fusion loop region in the protein E of Zika virus, which is highly conserved among different flaviviruses. Four rounds of selection were performed using the synthetic peptide in two strategies: the first was using the acidic elution of bound phages, and the second was by applying a competing procedure. After panning, the selected VH and VL domains were determined by combining NGS and bioinformatic approaches. Three different human monoclonal antibodies were expressed as scFvs and further characterized. All showed a binding capacity to Zika (ZIKV) and showed cross-recognition with yellow fever (YFV) and dengue (DENV) viruses. Two of these antibodies, AZ1p and AZ6m, could neutralize the ZIKV infection in vitro. Due to the conservation of the fusion loop region, these new antibodies can potentially be used in therapeutic intervention against Zika virus and other flavivirus illnesses.     

Candida Cell-Surface-Specific Monoclonal Antibodies Protect Mice against Candida auris Invasive Infection

Candida auris is a multidrug-resistant fungal pathogen that can cause disseminated bloodstream infections with up to 60% mortality in susceptible populations. Of the three major classes of antifungal drugs, most C. auris isolates show high resistance to azoles and polyenes, with some clinical isolates showing resistance to all three drug classes. We reported in this study a novel approach to treating C. auris disseminated infections through passive transfer of monoclonal antibodies (mAbs) targeting cell surface antigens with high homology in medically important Candida species. Using an established A/J mouse model of disseminated infection that mimics human candidiasis, we showed that C3.1, a mAb that targets β-1,2-mannotriose (β-Man₃), significantly extended survival and reduced fungal burdens in target organs, compared to control mice. We also demonstrated that two peptide-specific mAbs, 6H1 and 9F2, which target hyphal wall protein 1 (Hwp1) and phosphoglycerate kinase 1 (Pgk1), respectively, also provided significantly enhanced survival and reduction of fungal burdens. Finally, we showed that passive transfer of a 6H1+9F2 cocktail induced significantly enhanced protection, compared to treatment with either mAb individually. Our data demonstrate the utility of β-Man₃- and peptide-specific mAbs as an effective alternative to antifungals against medically important Candida species including multidrug-resistant C. auris.     

Extraction of Human IgG4 Monoclonal Antibodies Using AOT- and HDEHP-Isooctane Reverse Micelles

The extraction of 0.05 and 1 mg mL^?1 human IgG4 using reverse micelles (RMs) formed with anionic surfactants AOT or HDEHP in isooctane was evaluated. For both surfactants the use of 1 mg mL^?1 IgG4 resulted in higher forward extraction (FE), and generally better backward extraction (BE) yields than 0.05 mg mL^?1 IgG4, achieving optimum FE and BE yields at FE pHs of 5 at 3.13 mM AOT and 6 at 1.56 mM HDEHP. IgG4 precipitation at the interface was observed at the lower pHs during FE which appeared to cause low overall extraction yields. Water content analysis revealed AOT-RMs were much bigger than HDEHP-RMs.     

Principles and Current Clinical Landscape of Multispecific Antibodies against Cancer

Building upon the resounding therapeutic success of monoclonal antibodies, and supported by accelerating progress in engineering methods, the field of multispecific therapeutic antibodies is growing rapidly. Over 140 different molecules are currently in clinical testing, with excellent results in recent phase 1–3 clinical trials for several of them. Multivalent bispecific IgG-modified formats predominate today, with a clear tendency for more target antigens and further increased valency in newer constructs. The strategies to augment anticancer efficacy are currently equally divided between disruption of multiple surface antigens, and additional redirection of cytotoxic T or NK lymphocytes against the tumor. Both effects complement other modern modalities, such as tyrosine kinase inhibitors and adoptive cell therapies, with which multispecifics are increasingly applied in combination or merged, for example, in the form of antibody producing CAR-T cells and oncolytics. While mainly focused on B-cell malignancies early on, the contemporary multispecific antibody sector accommodates twice as many trials against solid compared to hematologic cancers. An exciting emerging prospect is the targeting of intracellular neoantigens using T-cell receptor (TCR) fusion proteins or TCR-mimic antibody fragments. Considering the fact that introduction of PD-(L)1 inhibitors only a few years ago has already facilitated 5-year survival rates of 30–50% for per se highly lethal neoplasms, such as metastatic melanoma and non-small-cell lung carcinoma, the upcoming enforcement of current treatments with “next-generation” immunotherapeutics, offers a justified hope for the cure of some advanced cancers in the near future.     

从噬菌体表面展示肽库中筛选边缘无浆体膜表面蛋白5单克隆抗体识别的抗原表位

目的从噬菌体表面展示肽库中筛选边缘无浆体膜表面蛋白5(Membrane surface protein5,MSP5)单克隆抗体识别的抗原表位。方法用MSP5单克隆抗体1D8作为靶标,对噬菌体展示随机12肽库进行筛选,通过ELISA和竞争抑制ELISA鉴定筛选克隆的结合特性,并提取阳性克隆的单链DNA,进行测序分析。结果从表面展示随机肽序列的噬菌体文库中筛选到与MSP5单抗1D8特异结合的噬菌体克隆,其一致序列为LING。竞争抑制试验表明,含特异序列的克隆能与MSP5重组蛋白抗原竞争。结论初步确定MSP5单克隆抗体1D8的抗原表位为线性表位,为进一步研究其在边缘无浆体诊断及新型疫苗研制中的作用奠定了基础。     

Expression and characterisation of recombinant human CD48 and isolation of a human anti‐CD48 monoclonal antibody by phage display

Human CD48, a membrane‐bound, glycosylphosphatidylinositol (GPI)‐linked glycoprotein, is a potential tumour target for the treatment of leukaemias and lymphomas. CD48 is expressed on T‐ and B‐cells, however <5% of CD34⁺ progenitor cells express CD48. A truncated, 45 kDa soluble form of the full length CD48 was expressed in Chinese hamster ovary (CHO) cells, and was shown to consist of a broad range of charge isoforms, with the most abundant isoforms between pI 4.5 and 5.0. The truncated form of CD48 was shown to bind to antibodies raised against native, GPI‐linked CD48 by surface plasmon resonance analysis. A synthetic, human, scFv immunoglobulin gene library was screened against recombinant CD48 by phage display, and an scFv antibody fragment, (designated N2A) was isolated after four rounds of biopanning. N2A was reassembled as a human IgG1 human monoclonal antibody, expressed in CHO cells and the binding of IgG1‐N2A to recombinant CD48 was confirmed by surface plasmon resonance. Flow cytometry studies of IgG1‐N2A binding to Raji cells showed the specificity of N2A for GPI‐linked CD48 was conserved, and presents the potential for IgG1‐N2A as a lead antibody candidate for the treatment of white blood cell malignancies. Copyright © 2005 Society of Chemical Industry     

New Frontiers in Monoclonal Antibodies for the Targeted Therapy of Acute Myeloid Leukemia and Myelodysplastic Syndromes

Acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) represent an unmet clinical need whose prognosis is still dismal. Alterations of immune response play a prominent role in AML/MDS pathogenesis, revealing novel options for immunotherapy. Among immune system regulators, CD47, immune checkpoints, and toll-like receptor 2 (TLR2) are major targets. Magrolimab antagonizes CD47, which is overexpressed by AML and MDS cells, thus inducing macrophage phagocytosis with clinical activity in AML/MDS. Sabatolimab, an inhibitor of T-cell immunoglobulin and mucin domain-containing protein 3 (TIM3), which disrupts its binding to galectin-9, has shown promising results in AML/MDS, enhancing the effector functions of lymphocytes and triggering tumor cell death. Several other surface molecules, namely CD33, CD123, CD45, and CD70, can be targeted with monoclonal antibodies (mAbs) that exert different mechanisms of action and include naked and conjugated antibodies, bispecific T-cell engagers, trispecific killer engagers, and fusion proteins linked to toxins. These novel mAbs are currently under investigation for use as monotherapy or in combination with hypomethylating agents, BCL2 inhibitors, and chemotherapy in various clinical trials at different phases of development. Here, we review the main molecular targets and modes of action of novel mAb-based immunotherapies, which can represent the future of AML and higher risk MDS treatment.     

Antibody Libraries as Tools to Discover Functional Antibodies and Receptor Pleiotropism

Most antibodies currently in use have been selected based on their binding affinity. However, nowadays, antibodies that can not only bind but can also alter the function of cell surface signaling components are increasingly sought after as therapeutic drugs. Therefore, the identification of such functional antibodies from a large antibody library is the subject of intensive research. New methods applied to combinatorial antibody libraries now allow the isolation of functional antibodies in the cellular environment. These selected agonist antibodies have provided new insights into important issues of signal transduction. Notably, when certain antibodies bind to a given receptor, the cell fate induced by them may be the same or different from that induced by natural agonists. In addition, combined with phenotypic screening, this platform allows us to discover unexpected experimental results and explore various phenomena in cell biology, such as those associated with stem cells and cancer cells.     

Phage Display Screening of Bovine Antibodies to Foot-and-Mouth Disease Virus and Their Application in a Competitive ELISA for Serodiagnosis

For serodiagnosis of foot-and-mouth disease virus (FMDV), monoclonal antibody (MAb)-based competitive ELISA (cELISA) is commonly used since it allows simple and reproducible detection of antibody response to FMDV. However, the use of mouse-origin MAb as a detection reagent is questionable, as antibody responses to FMDV in mice may differ in epitope structure and preference from those in natural hosts such as cattle and pigs. To take advantage of natural host-derived antibodies, a phage-displayed scFv library was constructed from FMDV-immune cattle and subjected to two separate pannings against inactivated FMDV type O and A. Subsequent ELISA screening revealed high-affinity scFv antibodies specific to a serotype (O or A) as well as those with pan-serotype specificity. When BvO17, an scFv antibody specific to FMDV type O, was tested as a detection reagent in cELISA, it successfully detected FMDV type O antibodies for both serum samples from vaccinated cattle and virus-challenged pigs with even higher sensitivity than a mouse MAb-based commercial FMDV type O antibody detection kit. These results demonstrate the feasibility of using natural host-derived antibodies such as bovine scFv instead of mouse MAb in cELISA for serological detection of antibody response to FMDV in the susceptible animals.     

A Patent Review on the Therapeutic Application of Monoclonal Antibodies in COVID-19

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) contains spike proteins that assist the virus in entering host cells. In the absence of a specific intervention, efforts are afoot throughout the world to find an effective treatment for SARS-CoV-2. Through innovative techniques, monoclonal antibodies (MAbs) are being designed and developed to block a particular pathway of SARS-CoV-2 infection. More than 100 patent applications describing the development of MAbs and their application against SARS-CoV-2 have been registered. Most of them target the receptor binding protein so that the interaction between virus and host cell can be prevented. A few monoclonal antibodies are also being patented for the diagnosis of SARS-CoV-2. Some of them, like Regeneron^® have already received emergency use authorization. These protein molecules are currently preferred for high-risk patients such as those over 65 years old with compromised immunity and those with metabolic disorders such as obesity. Being highly specific in action, monoclonal antibodies offer one of the most appropriate interventions for both the prevention and treatment of SARS-CoV-2. Technological advancement has helped in producing highly efficacious MAbs. However, these agents are known to induce immunogenic and non-immunogenic reactions. More research and testing are required to establish the suitability of administering MAbs to all patients at risk of developing a severe illness. This patent study is focused on MAbs as a therapeutic option for treating COVID-19, as well as their invention, patenting information, and key characteristics.     

采用基因拼接方法构建人源特异性抗乙型肝炎病毒表面抗原Fab噬菌体抗体库

目的　构建人源特异性抗乙型肝炎病毒表面抗原Fab噬菌体抗体库。方法　从抗乙型肝炎病毒表面抗体高滴度 (1:10 2 4 )的人全血中分离外周血单个核细胞 (PBMC) ,经RT PCR分别扩增出轻链可变区和重链可变区 ,再以噬菌体质粒为模板分别扩增出轻链恒定区 (Cκ)和重链恒定区 (CH1) ,将轻链可变区和轻链恒定区 (Cκ)及重链可变区和重链恒定区 (CH1)进行第 1次基因拼接 ,分别形成κ轻链和Fd重链 ,再以κ轻链和Fd重链作为模板进行第 2次基因拼接 ,形成完整的Fab基因 ,与pComb3H SS噬菌体质粒连接后 ,电穿孔转化大肠杆菌XL1 Blue。结果　通过多次电穿孔转化 ,获得总容量为 4× 10 5库容的噬菌体抗体库。结论　构建的Fab抗体库可用于筛选特异性抗乙型肝炎病毒表面抗原Fab抗体分子     

The Use of Distinctive Monoclonal Antibodies in FMD VLP- and P1-Based Blocking ELISA for the Seromonitoring of Vaccinated Swine

The serum neutralization (SN) test has been regarded as the “gold standard” for seroconversion following foot-and-mouth disease virus (FMDV) vaccination, although a high-level biosafety laboratory is necessary. ELISA is one alternative, and its format is constantly being improved. For instance, standard polyclonal antisera have been replaced by monoclonal antibodies (MAbs) for catching and detecting antibodies, and inactive viruses have been replaced by virus-like particles (VLPs). To the best of current knowledge, however, no researchers have evaluated the performances of different MAbs as tracers. In previous studies, we successfully identified site 1 and site 2 MAbs Q10E and P11A. In this study, following the established screening platform, the VLPs of putative escape mutants from sites 1 to 5 were expressed and used to demonstrate that S11B is a site 3 MAb. Additionally, the vulnerability of VLPs prompted us to assess another diagnostic antigen: unprocessed polyprotein P1. Therefore, we established and evaluated the performance of blocking ELISA (bELISA) systems based on VLPs and P1, pairing them with Q10E, P11A, S11B, and the non-neutralizing TSG MAb as tracers. The results indicated that the VLP paired with S11B demonstrated the highest correlation with the SN titers (R² = 0.8071, n = 63). Excluding weakly positive serum samples (SN = 16–32, n = 14), the sensitivity and specificity were 95.65% and 96.15% (kappa = 0.92), respectively. Additionally, the P1 pairing with Q10E also demonstrated a high correlation (R² = 0.768). We also discovered that these four antibodies had steric effects on one another to varying degrees, despite recognizing distinct antigenic sites. This finding indicated that MAbs as tracers could not accurately detect specific antibodies, possibly because MAbs are bulky compared to a protomeric unit. However, our results still provide convincing support for the application of two pairs of bELISA systems: VLP:S11B-HRP and P1:Q10E-HRP.     

从半合成噬菌体抗体库筛选抗狂犬病毒人单链抗体

目的　应用纯化的狂犬病毒抗原从半合成噬菌体抗体库中筛选针对狂犬病毒的人单链抗体(ScFv)。方法　用固相化的狂犬病毒抗原对半合成抗体库进行 3轮“吸附 洗脱 扩增”的筛选 ,从第 3轮洗脱下来的克隆中获得一株有可溶性表达且特异性结合狂犬病毒抗原的ScFv ,并进行基因序列测定。结果　所获氨基酸序列经blast数据库搜索 ,与一种抗狂犬病毒免疫球蛋白的氨基酸序列同源性最高 ( 82 % )。经检索kabat数据库 ,发现其轻、重链可变区分别属于VkⅠ型、VHⅢ型。结论　从噬菌体抗体库可以方便快捷地分离到针对狂犬病毒的单链抗体 ,对狂犬病毒的预防具有重要意义     

Generation of Potent Anti-Vascular Endothelial Growth Factor Neutralizing Antibodies from Mouse Phage Display Library for Cancer Therapy

Vascular endothelial growth factor (VEGF) is an important stimulator for angiogenesis in solid tumors. Blocking VEGF activity is an effective therapeutic strategy to inhibit tumor growth and metastasis. Avastin, a humanized monoclonal antibody recognizes VEGF, has been approved by the US Food and Drug Administration. To generate potential VEGF-recognizing antibodies with better tumor regression ability than that of Avastin, we have designed a systematic antibody selection plan. From mice immunized with recombinant human VEGF, we generated three phage display libraries, scFv-M13KO7, Fab-M13KO7, and scFv-Hyperphage, in single-chain Fv (scFv) or Fab format, displayed using either M13KO7 helper phage or Hyperphage. Solid-phase and solution-phase selection strategies were then applied to each library, generating six panning combinations. A total of sixty-four antibodies recognizing VEGF were obtained. Based on the results of epitope mapping, binding affinity, and biological functions in tumor inhibition, eight antibodies were chosen to examine their abilities in tumor regression in a mouse xenograft model using human COLO 205 cancer cells. Three of them showed improvement in the inhibition of tumor growth (328%–347% tumor growth ratio (% of Day 0 tumor volume) on Day 21 vs. 435% with Avastin). This finding suggests a potential use of these three antibodies for VEGF-targeted therapy.     

重组人源抗狂犬病病毒单克隆抗体的质控方法及质量标准的建立

目的建立重组人源抗狂犬病病毒单克隆抗体(rhRMcAb)的质控方法和质量标准。方法采用小鼠中和试验(MNT)和快速荧光灶抑制试验(RFFIT)分别测定6批rhRMcAb样品的中和活性;还原、非还原SDS-PAGE检测rhRMcAb样品的纯度;还原烷基化胰蛋白酶酶切和反相高效液相色谱法分析rhRMcAb的肽图;焦谷氨酸肽酶去除N-端焦谷氨酸封闭后,氨基酸序列分析仪测定rhRMcAb的N-端氨基酸序列;表面等离子共振法(Surface plasmon resonance,SPR)测定rhRMcAb与狂犬病病毒糖蛋白的亲和常数;按《中国药典》三部(2005版)要求检测rhRMcAb的各项其他指标,并建立其质量标准。结果采用MNT和RFFIT2种方法检测6批rhRMcAb的中和活性,批间活性基本一致;3批rhRMcAb原液的还原SDS-PAGE纯度大于99.0%,非还原SDS-PAGE除抗体主带外,在高、低相对分子质量处分别存在一些次带;3批原液样品的肽图图谱与理化测定对照品一致;N-端氨基酸序列与理论序列一致;结合狂犬病病毒糖蛋白抗原的亲和常数为1.86×108/M;其他各项指标均符合《中国药典》三部(2005版)要求;建立的质量标准中,中和活性应不低于500IU/mg,SDS-PAGE及HPLC纯度应不低于98.0%。结论已建立了rhRMcAb的质控方法和质量标准,可用于rhRMcAb产品的检定。     

Two Synthetic Antibodies that Recognize and Neutralize Distinct Proteolytic Forms of the Ebola Virus Envelope Glycoprotein

Ebola virus (EBOV) is a highly pathogenic member of the Filoviridae family of viruses that causes severe hemorrhagic fever. Infection proceeds through fusion of the host cell and viral membranes, a process that is mediated by the viral envelope glycoprotein (GP). Following endosomal uptake, a key step in viral entry is the proteolytic cleavage of GP by host endosomal cysteine proteases. Cleavage exposes a binding site for the host cell receptor Niemann‐Pick C1 (NPC1) and may induce conformational changes in GP leading to membrane fusion. However, the precise details of the structural changes in GP associated with proteolysis and the role of these changes in viral entry have not been established. Here, we have employed synthetic antibody technology to identify antibodies targeting EBOV GP prior to and following proteolysis (i.e. in the “uncleaved” [GP_UNCL] and “cleaved” [GP_CL] forms). We identified antibodies with distinct recognition profiles: Fab_CL bound preferentially to GP_CL (EC₅₀=1.7 nM ), whereas Fab_UNCL bound specifically to GP_UNCL (EC₅₀=75 nM ). Neutralization assays with GP‐containing pseudotyped viruses indicated that these antibodies inhibited GP_CL‐ or GP_UNCL‐mediated viral entry with specificity matching their recognition profiles (IC₅₀: 87 nM for IgG_CL; 1 μM for Fab_UNCL). Competition ELISAs indicate that Fab_CL binds an epitope distinct from that of KZ52, a well‐characterized EBOV GP antibody, and from that of the luminal domain of NPC1. The binding epitope of Fab_UNCL was also distinct from that of KZ52, suggesting that Fab_UNCL binds a novel neutralization epitope on GP_UNCL. Furthermore, the neutralizing ability of Fab_CL suggests that there are targets on GP_CL available for neutralization. This work showcases the applicability of synthetic antibody technology to the study of viral membrane fusion, and provides new tools for dissecting intermediates of EBOV entry.     

Binding of the Phage Display Derived Peptide CaIX-P1 on Human Colorectal Carcinoma Cells Correlates with the Expression of Carbonic Anhydrase IX

Phage display represents an attractive screening strategy for the identification of novel, specific binding ligands that could be used for tumor targeting. Recently, a new peptide (CaIX-P1) with affinity for human carbonic anhydrase IX (CAIX) was identified and evaluated. The aim of the present study is to characterize the properties of CaIX-P1 for targeting human colorectal carcinoma and investigate the correlation of peptide binding with the expression of carbonic anhydrase IX. Human colorectal carcinoma HCT116 and HT29 cells were investigated for CAIX expression using Western Blot analysis. Binding and competition studies of ¹²⁵I-radiolabeled CaIX-P1 were performed on HCT116 cells in vitro. FACS analysis and fluorescence microscopy studies were carried out after cell incubation with fluorescein-labeled CaIX-P1 and rhodamine-labeled anti-human CAIX-mAb. Our studies revealed an enhanced in vitro expression of carbonic anhydrase IX in HCT116 and HT29 cells with increasing cell density. Binding of ¹²⁵I-labeled-CaIX-P1 on HCT116 cells increased with increasing cell density and correlated to the CAIX expression. FACS analysis demonstrated a correlation of cell labeling between FITC-CaIX-P1 and rhodamine-labeled anti-CAIX-mAb in both HCT116 and HT29 cells. The results of our study indicate that the phage display identified peptide CaIX-P1 might be an attractive candidate for the development of a ligand targeting CAIX in colorectal cancer.     

Establishment of Murine Hybridoma Cells Producing Antibodies against Spike Protein of SARS-CoV-2

In 2020 the world faced the pandemic of COVID-19 severe acute respiratory syndrome caused by a new type of coronavirus named SARS-CoV-2. To stop the spread of the disease, it is crucial to create molecular tools allowing the investigation, diagnoses and treatment of COVID-19. One of such tools are monoclonal antibodies (mAbs). In this study we describe the development of hybridoma cells that can produce mouse mAbs against receptor binding domain of SARS-CoV-2 spike (S) protein. These mAbs are able to specifically detect native and denatured S proteins in all tested applications, including immunoblotting, enzyme-linked immunosorbent assay, immunofluorescence staining of cells and immunohistochemical staining of paraffin embedded patients’ tissue samples. In addition, we showed that the obtained mAbs can efficiently block SARS-CoV-2 infection in in vitro experiments. Finally, we determined the amino acid sequence of light and heavy chains of the mAbs. This information will allow the use of corresponding peptides to establish genetically engineered therapeutic antibodies. To date multiple mAbs against SARS-CoV-2 proteins have been established, however, bigger sets of various antibodies will allow the detection and neutralization of SARS-CoV-2, even if the virus acquires novel mutations.