Phage Display Antibody Library Research Articles

Tumor-Targeting Ability of Novel Anti-Prostate-Specific Membrane Antigen Antibodies.

Patients with prostate-specific membrane antigen (PSMA)-positive tumors can benefit from PSMA-targeted therapy; thus, we have constructed a phage-displayed synthetic antibody library for the production of novel PSMA antibodies with superior PSMA-targeting ability, favoring clinical management. The binding affinities of anti-PSMA antibodies were verified by an enzyme-linked immunosorbent assay (ELISA). Several in vitro and in vivo experiments, including cellular uptake, internalization, and cytotoxicity studies, micro single photon emission computed tomography (microSPECT)/CT, and biodistribution studies, were performed to select the most promising antibody among six different antibodies. The results showed the target affinities of our antibodies in the ELISA assays (7A, 8C, 8E, and 11A) were comparable to the existing antibodies (J591). The half-maximal effective concentrations of 7A, 8C, 8E, 11A, and J591 were 2.95, 6.64, 5.50, 2.08, and 4.79, respectively. The radiochemical yield of 111In-labeled antibodies ranged from 30% to 50% with high radiochemical purity (>90%). In the cellular uptake studies, the accumulated radioactivity of 111In-J591, 111In-7A, and 111In-11A increased over time. The internalized percentage of 111In-11A was the highest (32.14% ± 2.06%) at 48 h after incubation, whereas that of 111In-J591 peaked at 22.43% ± 4.38% at 24 h and dropped to 13.52% ± 3.03% at 48 h postincubation. Twenty-four hours after injection, radioactivity accumulation appeared in the LNCaP xenografts of the mice injected with 111In-11A, 111In-8E, 111In-7A, and 111In-J591 but not in the xenografts of the 111In-8C-injected group. Marked liver uptake was noticed in all groups except the 111In-11A-injected group. Moreover, the killing effect of 177Lu-11A was superior to that of 177Lu-J591 at low concentrations. In conclusion, we successfully demonstrated that 11A IgG owned the most optimal biological characteristics among several new anti-PSMA antibodies and it can be an excellent PSMA-targeting component for the clinical use.

Generation of Human Domain Antibody Fragments as Potential Insecticidal Agents against Helicoverpa armigera by Cadherin-Based Screening.

New insecticidal genes and approaches for pest control are a hot research area. In the present study, we explored a novel strategy for the generation of insecticidal proteins. The midgut cadherin of Helicoverpa armigera (H. armigera) was used as a target to screen materials that have insecticidal activity. After three rounds of panning, the phage-displayed human domain antibody B1F6, which not only binds to the H. armigera cadherin CR9-CR11 but also significantly inhibits Cry1Ac toxins from binding to CR9-CR11, was obtained from a phage-displayed human domain antibody (DAb) library. To better analyze the relevant activity of B1F6, soluble B1F6 protein was expressed by Escherichia coli BL21 (DE3). The cytotoxicity assays demonstrated that soluble B1F6 induced Sf9 cell death when expressing H. armigera cadherin on the cell membrane. The insect bioassay results showed that soluble B1F6 protein (90 μg/cm2) caused 49.5 ± 3.3% H. armigera larvae mortality. The midgut histological results showed that soluble B1F6 caused damage to the midgut epithelium of H. armigera larvae. The present study explored a new strategy and provided a basic material for the generation of new insecticidal materials.

Antibody CDR amino acids underlying the functionality of antibody repertoires in recognizing diverse protein antigens

Antibodies recognize protein antigens with exquisite specificity in a complex aqueous environment, where interfacial waters are an integral part of the antibody–protein complex interfaces. In this work, we elucidate, with computational analyses, the principles governing the antibodies’ specificity and affinity towards their cognate protein antigens in the presence of explicit interfacial waters. Experimentally, in four model antibody–protein complexes, we compared the contributions of the interaction types in antibody–protein antigen complex interfaces with the antibody variants selected from phage-displayed synthetic antibody libraries. Evidently, the specific interactions involving a subset of aromatic CDR (complementarity determining region) residues largely form the predominant determinant underlying the specificity of the antibody–protein complexes in nature. The interfacial direct/water-mediated hydrogen bonds accompanying the CDR aromatic interactions are optimized locally but contribute little in determining the epitope location. The results provide insights into the phenomenon that natural antibodies with limited sequence and structural variations in an antibody repertoire can recognize seemingly unlimited protein antigens. Our work suggests guidelines in designing functional artificial antibody repertoires with practical applications in developing novel antibody-based therapeutics and diagnostics for treating and preventing human diseases.

A Novel Synthetic Antibody Library with Complementarity-Determining Region Diversities Designed for an Improved Amplification Profile.

Antibody discovery by phage display consists of two phases, i.e., the binding phase and the amplification phase. Ideally, the selection process is dominated by the former, and all the retrieved clones are amplified equally during the latter. In reality, the amplification efficiency of antibody fragments varies widely among different sequences and, after a few rounds of phage display panning, the output repertoire often includes rapidly amplified sequences with low or no binding activity, significantly diminishing the efficiency of antibody isolation. In this work, a novel synthetic single-chain variable fragment (scFv) library with complementarity-determining region (CDR) diversities aimed at improved amplification efficiency was designed and constructed. A previously reported synthetic scFv library with low, non-combinatorial CDR diversities was panned against protein A superantigen, and the library repertoires before and after the panning were analyzed by next generation sequencing. The enrichment or depletion patterns of CDR sequences after panning served as the basis for the design of the new library. Especially for CDR-H3 with a higher and more random diversity, a machine learning method was applied to predict potential fast-amplified sequences among a simulated sequence repertoire. In a direct comparison with the previous generation library, the new library performed better against a panel of antigens in terms of the number of binders isolated, the number of unique sequences, and/or the speed of binder enrichment. Our results suggest that the amplification-centric design of sequence diversity is a valid strategy for the construction of highly functional phage display antibody libraries.

Monoclonal Antibodies Targeting Surface-Exposed Epitopes of Candida albicans Cell Wall Proteins Confer In Vivo Protection in an Infection Model.

ABSTRACTMonoclonal antibody (mAb)-based immunotherapies targeting systemic and deep-seated fungal infections are still in their early stages of development, with no licensed antifungal mAbs currently being available for patients at risk. The cell wall glycoproteins of Candida albicans are of particular interest as potential targets for therapeutic antibody generation due to their extracellular location and key involvement in fungal pathogenesis. Here, we describe the generation of recombinant human antibodies specifically targeting two key cell wall proteins (CWPs) in C. albicans: Utr2 and Pga31. These antibodies were isolated from a phage display antibody library using peptide antigens representing the surface-exposed regions of CWPs expressed at elevated levels during in vivo infection. Reformatted human-mouse chimeric mAbs preferentially recognized C. albicans hyphal forms compared to yeast cells, and increased binding was observed when the cells were grown in the presence of the antifungal agent caspofungin. In J774.1 macrophage interaction assays, mAb pretreatment resulted in the faster engulfment of C. albicans cells, suggesting a role of the CWP antibodies as opsonizing agents during phagocyte recruitment. Finally, in a series of clinically predictive mouse models of systemic candidiasis, our lead mAb achieved improved survival (83%) and a several-log reduction of the fungal burden in the kidneys, similar to the levels achieved for the fungicidal drug caspofungin and superior to the therapeutic efficacy of any anti-Candida mAb reported to date.

Synthetic antibodies block receptor binding and current-inhibiting effects of α-cobratoxin from Naja kaouthia.

Each year, thousands of people fall victim to envenomings caused by cobras. These incidents often result in death due to paralysis caused by α-neurotoxins from the three-finger toxin (3FTx) family, which are abundant in elapid venoms. Due to their small size, 3FTxs are among the snake toxins that are most poorly neutralized by current antivenoms, which are based on polyclonal antibodies of equine or ovine origin. While antivenoms have saved countless lives since their development in the late 18th century, an opportunity now exists to improve snakebite envenoming therapy via the application of new biotechnological methods, particularly by developing monoclonal antibodies against poorly neutralized α-neurotoxins. Here, we describe the use of phage-displayed synthetic antibody libraries and the development and characterization of six synthetic antibodies built on a human IgG framework and developed against α-cobratoxin - the most abundant long-chain α-neurotoxin from Naja kaouthia venom. The synthetic antibodies exhibited sub-nanomolar affinities to α-cobratoxin and neutralized the curare-mimetic effect of the toxin in vitro. These results demonstrate that phage display technology based on synthetic repertoires can be used to rapidly develop human antibodies with drug-grade potencies as inhibitors of venom toxins.

Preparation of a Single-Chain Antibody against Nucleocapsid Protein of Porcine Deltacoronavirus by Phage Display Technology.

Porcine deltacoronavirus (PDCoV) mainly causes severe diarrhea and intestinal pathological damage in piglets and poses a serious threat to pig farms. Currently, no effective reagents or vaccines are available to control PDCoV infection. Single-chain fragment variable (scFv) antibodies can effectively inhibit virus infection and may be a potential therapeutic reagent for PDCoV treatment. In this study, a porcine phage display antibody library from the peripheral blood lymphocytes of piglets infected with PDCoV was constructed and used to select PDCoV-specific scFv. The library was screened with four rounds of biopanning using the PDCoV N protein, and the colony with the highest affinity to the PDCoV N protein was obtained (namely, N53). Then, the N53-scFv gene fragment was cloned into plasmid pFUSE-hIgG-Fc2 and expressed in HEK-293T cells. The scFv-Fc antibody N53 (namely, scFv N53) was purified using Protein A-sepharose. The reactive activity of the purified antibody with the PDCoV N protein was confirmed by indirect enzyme-linked immunosorbent assay (ELISA), western blot and indirect immunofluorescence assay (IFA). Finally, the antigenic epitopes that the scFv N53 recognized were identified by a series of truncated PDCoV N proteins. The amino acid residues 82GELPPNDTPATTRVT96 of the PDCoV N protein were verified as the minimal epitope that can be recognized by the scFv-Fc antibody N53. In addition, the interaction between the scFv-Fc antibody N53 and the PDCoV N protein was further analyzed by molecule docking. In conclusion, our research provides some references for the treatment and prevention of PDCoV.

Identification of single domain antibodies with insect cytotoxicity using phage-display antibody library screening and Plutella xylostella ATP-binding cassette transporter subfamily C member 2 (ABCC2) -based insect cell expression system

It is extremely imminent to study a new strategy to manage agricultural pest like Plutella xylostella (P. xylostella) which is currently resistant to most of pesticides, including three domain-Cry toxins from Bacillus thuringiensis (Bt). In this study, we reported a phage displayed single domain antibody screening from human domain antibody (DAb) library targeted on Spodoptera frugiperda 9 (Sf9) cells expressed Cry1Ac toxin receptor, ATP-dependent binding cassette transporter C2 in P. xylostella (PxABCC2). After three rounds of panning, three cytotoxic antibodies (1D2, 2B7, 3C4) were obtained from thirty-eight antibodies and displayed high binding ability towards PxABCC2-expressed Sf9 cells. Through homology modeling and molecular docking, the interaction mode indicated that the most cytotoxic 1D2 of the three antibodies presented the lowest binding free energy required and had the most hydrogen bond formed with PxABCC2 in molecular docking analysis. Functional assay of key regions in 1D2 via Alanine replacement indicated that complementarity-determining region (CDR) 3 played a crucial role in antibody exerts binding activity and cytotoxicity. This study provides the first trial for discovering of potential cytotoxic antibodies from the human antibody library via specific receptor-expressed insect cell system biopanning.

Therapeutic effects of TM4SF5-targeting chimeric and humanized monoclonal antibodies in hepatocellular and colon cancer models

The transmembrane 4 L six family member 5 (TM4SF5) is aberrantly expressed in hepatocellular and colorectal cancers, and has been implicated in tumor progression, suggesting that it could serve as a novel therapeutic target. Previously, we screened a murine antibody phage-display library to generate a novel monoclonal antibody, Ab27, that is specific to the extracellular loop 2 of TM4SF5. In this study, we evaluated the effects of chimeric Ab27 using cancer cells expressing endogenous TM4SF5 or stably overexpressing TM4SF5 in vivo and in vitro. Monotherapy with Ab27 significantly decreased tumor growth in liver and colon cancer xenograft models, including a sorafenib-resistant model, and decreased the phosphorylation of focal adhesion kinase (FAK), p27Kip1, and signal transducer and activator of transcription 3 (STAT3). No general Ab27 toxicity was observed in vivo. Combination treatment with Ab27 and sorafenib or doxorubicin exerted higher antitumor activity than monotherapy. In addition, we humanized the Ab27 sequence by the complementarity-determining region (CDR) grafting method. The humanized antibody Ab27-hz9 had reduced immunogenicity but exhibited target recognition and antitumor activity comparable with those of Ab27. Both Ab27 and Ab27-hz9 efficiently targeted tumor cells expressing TM4SF5 in vivo. These observations strongly support the further development of Ab27-hz9 as a novel therapeutic agent against liver and colorectal cancers.

A new single-chain variable fragment (scFv) antibody provides sensitive and specific detection of citrus tristeza virus

Citrus tristeza virus (CTV) is the most economically important virus disease of citrus worldwide. To develop a specific serological assay for CTV, a Tomlinson phage display antibody library of single chain variable fragments (scFv) was screened with a recombinant CTV coat protein (CTV-CP) heterologously expressed in Escherichia coli. The phage clones were checked by ELISA to identify clones with high specificity for CTV-CP. Eight clones were strongly reactive with CTV-CP. Nucleotide sequencing of these clones revealed that all of them contained the same sequence. Thus, the phage-displayed scFv antibody was termed scFvF10. Evaluation of scFvF10 binding to CTV-CP by plate-trapped antigen ELISA (PTA-ELISA) and immunoblotting, showed that it was specific and allowed sensitive detection of CTV-CP. Homology-based molecular modeling and docking analysis confirmed that the interaction between CTV-CP and scFvF10, with a binding energy of -738 kj mol-1, occurred mainly by 12 intermolecular hydrogen bonds. Moreover, triple-antibody sandwich (TAS)-ELISA using scFvF10 as second antibody showed high sensitivity in the detection of CTV infected samples. The CTV detection limit of scFvF10 by PTA-ELISA and TAS-ELISA were 0.05 and 0.01 μg CP/mL, respectively. Our results with different diagnostic assays demonstrated that scFvF10 has the potential to be used as an efficient tool for CTV-infected plant diagnosis.

Generation of Anti-THSD7A Antibodies Using a Human Antibody Phage Display Library

Generation of Anti-THSD7A Antibodies Using a Human Antibody Phage Display Library

Anti-IL1RAP/CD3 Bispecific Antibody (BsAb) Is a Promising Novel and Effective Therapy for Acute Myeloid Leukemia (AML)

Despite increasing insight of the mechanisms of leukemogenesis, implementation of risk-stratification treatment strategies and use of molecular targeting therapeutics, only a minority of AML patients are cured with chemotherapy-based regimens. Most patients are either primary refractory to induction therapy or subsequently relapse following a brief remission likely due to persistence of chemo-resistant leukemia stem cell (LSC)-enriched subpopulations. Therefore there are urgent unmet therapeutic needs. The principle of CD3 based BsAb therapy is to recruit and activate T cell effector function upon engaging target cells expressing a tumor associated antigen (TAA). Several TAA (e.g.CD33, CD123) have been found to be differentially overexpressed on the surface of AML blasts. Recently, the expression of IL-1 receptor accessory protein (IL1RAP) was reported to be higher on LSCs and more differentiated leukemic progenitor cells (LPCs) than on normal hematopoietic stem cells (HSC), making it an attractive TAA target for immunotherapy. Thus, we hypothesize that an anti-IL1RAP/CD3 BsAb could preferentially target LSCs and LPCs while sparing normal HSCs, thereby offering a window of therapeutic opportunity for AML patients.To test this hypothesis we first determined the expression pattern of IL-1RAP. IL1RAP was differentially expressed on AML cell lines: HL60 and MV4-11 (high), THP-1 (intermediate), and KG-1a (low). Its expression levels were variable on primary blasts from AML patients (n=10). CD34+ peripheral blood mononuclear cells (PBMC) from healthy donors showed little or no IL1RAP expression. Next, we screened 12 anti-IL1RAP monoclonal antibodies from our human phage display antibody library. Based on the results of the BIAcore and antibody dependent cellular cytotoxity (ADCC) assays performed on MV4-11 cells, we selected one lead candidate with binding affinity of ~20nM and EC50 4.9nM for further development. Using knobs-into-holes strategy, we paired this IL1RAP antibody with an anti-CD3 antibody. The dual binding ability of this BsAb was confirmed by flow cytometric analysis. We subsequently proved T cell dependent cytotoxicity (TDCC) of this BsAb using both AML cell lines and AML primary blasts as targets. The anti-IL1RAP/CD3 BsAb induced over 95% of specific killing on all tested AML samples at E: T (Effector : Target) ratio of 1:1 compared with no cytotoxicity effect on the IL-1RAP-negative Raji cells in the 48-hour long term killing (LTK) assay. In line with the cytotoxic data, there was also substantial release of cytokines including IFN-γ, TNF-α and Granzyme B from the AML cells but not Raji cells. TDCC reported over 90% of IL1-RAP specific killing in all but one (only 60%) patient samples (n=10), which had the lowest IL1RAP expression level. The killing of leukemia cells was also observed by time-lapse live cell microscope imaging using luciferase-expressing MV4-11luci+ cells with addition of 100ng/ml BsAb and T cells at E:T ratio of 5:1, which documented effective T cell recruitment and cytolytic activities against MV4-11luci+ cells. We also tested the anti-leukemic activity of this BsAb in vivo using a NSG xenograft mouse model. NSG mice transplanted with 1x106 MV4-11luci+ cells were treated with 200µg anti-IL1RAP/CD3 BsAb plus 3x106 unstimulated T cells, vehicle, T cells alone or anti-CD19/CD3 BsAb (control Ab) plus T cells twice a week for 30 days. In vivo imaging demonstrated effective elimination of the leukemia signal (P<0.001) in MV4-11luci+-engrafted NSG mice treated with T cells and anti-IL1RAP/CD3 BsAb (photon flux (PF) =5.5x105) compared with intense leukemia infiltration in controls treated with either vehicle (PF=3.5x109), T cells alone (PF=4x109), or control anti-CD19/CD3 BsAb plus T cells (PF=2x109). In a separate experiment, treatment with anti-IL1RAP/CD3 BsAb plus T cells of NSG mice engrafted with primary AML blasts led to significant decrease in disease burden than treatment with isotype antibody plusT cells (p=0.004). Further characterization and determination of disease response in these animals is ongoing.In conclusion, we have successfully engineered and characterized an anti-IL1RAP/CD3 BsAb derived from our human naïve phage display library. Our data demonstrated that this BsAb can effectively eradicate AML cells in vitro and in vivo, supporting the clinical translation of this novel immunotherapeutic agent for treatment of AML patients. DisclosuresKwak:InnoLifes: Consultancy, Equity Ownership; Pepromene Bio: Consultancy, Equity Ownership; Celltrion, Inc: Consultancy. Stein:Stemline: Consultancy; Amgen: Consultancy, Speakers Bureau.

Novel human neutralizing mAbs specific for Spike-RBD of SARS-CoV-2

Among the therapies against the pandemic SARS-CoV-2 virus, monoclonal Antibodies (mAbs) targeting the Spike glycoprotein represent good candidates to interfere in the Spike/ACE2 interaction, preventing virus cell entry. Since anti-spike mAbs, used individually, might be unable to block the virus entry in the case of resistant mutations, we designed an innovative strategy for the isolation of multiple novel human scFvs specific for the binding domain (RBD) of Spike. By panning a large phage display antibody library on immobilized RBD, we obtained specific binders by eluting with ACE2 in order to identify those scFvs recognizing the epitope of Spike interacting with its receptor. We converted the novel scFvs into full size IgG4, differently from the previously isolated IgG1 mAbs, to avoid unwanted potential side effects of IgG1 potent effector functions on immune system. The novel antibodies specifically bind to RBD in a nanomolar range and interfere in the interaction of Spike with ACE2 receptor, either used as purified protein or when expressed on cells in its native conformation. Furthermore, some of them have neutralizing activity for virus infection in cell cultures by using two different SARS-CoV-2 isolates including the highly contagious VOC 202012/01 variant and could become useful therapeutic tools to fight against the SARS-CoV-2 virus.

Construction of a full-length antibody phage display vector

Antibody phage display technology plays an important role in the development of monoclonal antibodies, humanization, and affinity evolution of antibodies. Thus far, antibody phage display mainly focuses on the display of antibody variable region or antigen-binding fragments. In this study, we constructed a new phage display system that can display full-length IgG antibodies on M13 phage. The phage display vector contains open reading frames (ORFs) encoding full-length the heavy and light chains of the antibody. NcoI/XhoI restriction enzyme sites were used to clone the variable region of the heavy chain into the heavy chain ORF, and SalI/NotI sites were used to clone the light chain variable region. SnaBI and SbfI restriction enzyme sites were designed between the cloning sites of heavy and light chains, respectively, to increase the cloning efficiency. The full-length antibodies of nivolumab against programmed death factor 1, trastuzumab against human epidermal growth factor 2, diL2K against the cluster of differentiation 3 epsilon, and adalimumab against tumor necrosis factor- alpha were displayed on phage with the vector. Phage-displayed antibodies showed their original antigen-binding activity. An amber codon shifted the vector to express IgG in non-suppressed Escherichia coli. The heavy and light chains of the E. coli-expressed antibodies could be detected through western blotting, and the antigen-binding activity was confirmed using an enzyme-linked immunosorbent assay. Biopanning was carried out with a model phage display antibody library, and the results showed that the novel phage system could be used for antibody library construction and highly efficient antibody screening. The reported system is the first full-length antibody phage display system.

Identification of a novel protective human monoclonal antibody, LXY8, that targets the key neutralizing epitopes of staphylococcal enterotoxin B

Staphylococcal enterotoxin B (SEB), one of the exotoxins produced by Staphylococcus aureus, is the key toxin that causes poisoning reactions and toxic shock syndrome. In the current research work, a novel human antibody named LXY8 was screened from a human phage display antibody library, and LXY8 blocked the interaction between SEB and the T cell receptor (TCR). The binding activity between LXY8 and SEB was 0.525 nM. Furthermore, LXY8 could effectively inhibit the SEB-induced activation of peripheral blood mononuclear cells and release of cytokines. In the BALB/c mouse model, LXY8 effectively neutralized SEB toxicity in vivo. Finally, based on computer-guided molecular modeling, we designed a series of SEB mutation sites; these sites facilitated the determination of the key residues (i.e.176EFNN179) of SEB recognized by LXY8. The research revealed that the 176EFNN179 residues of SEB are important for specific antibody-antigen recognition. The results may be helpful for the development of antibody-based therapy for SEB-induced toxic shock syndrome.

Preparation of Anti Toll-Like Receptor-4 Nano-Antibody and Its Effect on Gram Negative Sepsis.

The essence of sepsis is the excessive immune response caused by infectious factors, which leads to the release of a large number of inflammatory factors and the injury of tissues and organs. Once the reaction is triggered, it will develop according to its own rules, and it does not depend on infection factors. The occurrence and severity of sepsis depend entirely on the reaction of the body. When pathogens invade the body, they rely on pattern recognition receptors to play a defensive role, TLR4 is the main switch of innate immunity. The current research shows that LPS can induce the up-regulation of TLR4 gene expression in monocytes, neutrophils and other immune cells. The upregulated TLR4, as a new receptor, mediates more cell activation and the release of inflammatory mediators. Under this positive feedback, the inflammatory response is constantly amplified, which eventually leads to the development of sepsis. In this study, TLR4 extracellular domain antigen was obtained by protein expression and purification, and anti-TLR4 C-terminal and intermediate domain nano-antibody was obtained by phage display antibody library preparation technology. In vitro and in vivo experiments confirmed that anti-TLR4 nano-antibody can effectively reduce the release of inflammatory factors and improve the survival rate of animals, while the C-terminal and intermediate domain are closed at the same time, the effect is more obvious. The clinical treatment of sepsis provides new ideas and strategies.

Phage Display Technology, Phage Display System, Antibody Library, Prospects and Challenges

Phage display technology is a unique gene recombination expression technology, and it is also a simple and effective screening tool. Through panning, a protein or peptide with high affinity and selectivity to the target is obtained. Antibody phage display has become the first and most widely used in vitro screening technology. Phage display derivatives play an important role in the diagnosis and treatment of diseases. This article reviews the phage display system of phage display technology, the size and classification of antibody libraries and their applications, and discusses the application prospects and challenges of phage display technology. This thesis lays the foundation for the theoretical and experimental research of bacteriophages.

A novel human anti-TIGIT monoclonal antibody with excellent function in eliciting NK cell-mediated antitumor immunity

TIGIT is an emerging novel checkpoint target that is expressed on both tumor-infiltrating T cells and NK cells. Some current investigational antibodies targeting TIGIT have also achieved dramatic antitumor efficacy in late clinical research. Most recently, the relevance of NK cell–associated TIGIT signaling pathway to tumors’ evasion of the immune system has been clearly revealed, which endows NK cells with a pivotal role in the therapeutic effects of TIGIT blockade. In this article, we describe a novel anti-TIGIT monoclonal antibody, AET2010, which was acquired from a phage-displayed human single-chain antibody library through a cell panning strategy. With emphasis on its regulation of NK cells, we confirmed the excellent ex vivo and in vivo antitumor immunity of AET2010 mediated by the NK-92MI cells. Intriguingly, our work also revealed that AET2010 displays a lower affinity but parallel avidity and activity relative to MK7684, an investigational monoclonal antibody from MSD, implying a reasonable balance of potency and potential side effects for AET2010. Together, these results are promising and warrant further development of AET2010.

Recent advances in therapeutic applications of neutralizing antibodies for virus infections: an overview.

Antibodies are considered as an excellent foundation to neutralize pathogens and as highly specific therapeutic agents. Antibodies are generated in response to a vaccine but little use as immunotherapy to combat virus infections. A new generation of broadly cross-reactive and highly potent antibodies has led to a unique chance for them to be used as a medical intervention. Neutralizing antibodies (monoclonal and polyclonal antibodies) are desirable for pharmaceutical products because of their ability to target specific epitopes with their variable domains by precise neutralization mechanisms. The isolation of neutralizing antiviral antibodies has been achieved by Phage displayed antibody libraries, transgenic mice, B cell approaches, and hybridoma technology. Antibody engineering technologies have led to efficacy improvements, to further boost antibody in vivo activities. “Although neutralizing antiviral antibodies have some limitations that hinder their full development as therapeutic agents, the potential for prevention and treatment of infections, including a range of viruses (HIV, Ebola, MERS-COV, CHIKV, SARS-CoV, and SARS-CoV2), are being actively pursued in human clinical trials.”

Development of Anti-Yersinia pestis Human Antibodies with Features Required for Diagnostic and Therapeutic Applications.

BackgroundYersinia pestis is a category A infective agent that causes bubonic, septicemic, and pneumonic plague. Notably, the acquisition of antimicrobial or multidrug resistance through natural or purposed means qualifies Y. pestis as a potential biothreat agent. Therefore, high-quality antibodies designed for accurate and sensitive Y. pestis diagnostics, and therapeutics potentiating or replacing traditional antibiotics are of utmost need for national security and public health preparedness.MethodsHere, we describe a set of human monoclonal immunoglobulins (IgG1s) targeting Y. pestis fraction 1 (F1) antigen, previously derived from in vitro evolution of a phage-display library of single-chain antibodies (scFv). We extensively characterized these antibodies and their effect on bacterial and mammalian cells via: ELISA, flow cytometry, mass spectrometry, spectroscopy, and various metabolic assays.ResultsTwo of our anti-F1 IgG (αF1Ig 2 and αF1Ig 8) stood out for high production yield, specificity, and stability. These two antibodies were additionally attractive in that they displayed picomolar affinity, did not compete when binding Y. pestis, and retained immunoreactivity upon chemical derivatization. Most importantly, these antibodies detected <1,000 Y. pestis cells in sandwich ELISA, did not harm respiratory epithelial cells, induced Y. pestis agglutination at low concentration (350 nM), and caused apparent reduction in cell growth when radiolabeled at a nonagglutinating concentration (34 nM).ConclusionThese antibodies are amenable to the development of accurate and sensitive diagnostics and immuno/radioimmunotherapeutics.