Phage Display Antibody Library Research Articles

Abstract 3758: The efficacy of CD133 BiTEs and CAR-T cells in preclinical model of glioblastoma

Abstract Glioblastoma (GBM) is a uniformly fatal primary brain tumor, characterized by a diverse cellular phenotype and genetic heterogeneity. Despite the use of multi-modal treatment including surgical resection, radiotherapy and chemotherapy, the outcome of patients with GBM remains poor. Numerous studies have implicated CD133+ brain tumor initiating cells (BTICs) as drivers of chemo- and radio-resistance in GBM. We recently demonstrated that a CD133-driven gene signature is predictive of poor overall survival and targeting CD133+ treatment-refractory cells may be an effective strategy to block GBM recurrence. Chimeric antigen receptors (CARs) and bispecific T-Cell engaging antibodies (BiTEs) present promising immunotherapeutic approaches that have not yet been validated for recurrent GBM. Using CellectSeq, a novel methodology that combines use of phage-displayed synthetic antibody libraries and DNA sequencing, we developed the CD133-specific monoclonal antibody ‘RW03’. We constructed CD133-specific BiTEs that consist of two arms; one arm recognizes the tumor antigen (CD133) while the second is specific to CD3 antigen. The dual binding specificity was confirmed using flow cytometry. Using CD133high and CD133low primary GBM lines, we validated the binding of BiTEs to CD133+ cells. Further analysis showed binding of BiTEs to human T cells known to express CD3 within a population of healthy donor peripheral blood mononuclear cells. We observed BiTEs redirecting T cells to kill GBMs, with greater efficiency observed in CD133high GBMs, validating BiTE target specificity. Incubating T-cells with BiTEs and the CD133high GBMs resulted in increased expression of T cell activation markers. In parallel, we derived the single chain variable fragment (scFv) from previously generated RW03 and generated a second-generation CAR. Anti-CD133 scFv with a myc tag was cloned in frame with a human CD8 leader sequence, CD8a transmembrane domain, CD28, and hCD3ζ signaling tail in the lentiviral construct pCCL-ΔNGFR. Following lentiviral packaging, the T cells isolated from PBMCs were transduced with CD133 CAR construct. After successful T cell engineering, the expression of ΔNGFR and myc tag was analyzed using flow cytometry to confirm the efficiency of transduction and surface expression of anti-CD133 respectively. CD133-specific CAR-T cells were cytotoxic to CD133+ GBMs. Co-culturing CD133 CAR-T cells with GBMs triggered T cell activation and proliferation. Treatment of GBM tumor-bearing mice with CD133-specific CAR-T cells yielded extended survival in mice and significant reductions in brain tumor burden. The results of this study will establish a translational research program that will form the basis of early phase clinical trials of a promising CD133-based therapeutic strategy for patients with GBM. Citation Format: Parvez Vora, Chirayu Chokshi, Maleeha Qazi, Mohini Singh, Chitra Venugopal, Sujeivan Mahendram, Jarrett Adams, David Bakhshinyan, Max London, Jess Singh, Minomi Subapanditha1,, Nicole McFarlane, James Pan, Jonathan Bramson, Sachdev Sidhu, Jason Moffat, Sheila Singh. The efficacy of CD133 BiTEs and CAR-T cells in preclinical model of glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3758. doi:10.1158/1538-7445.AM2017-3758

Identification of a novel HIV-1-neutralizing antibody from a CRF07_BC-infected Chinese donor.

The identification of human monoclonal antibodies (mAbs) able to neutralize a broad spectrum of primary HIV-1 isolates is highly important for understanding the immune response of HIV-1 infection and developing vaccines and therapeutics. In this study, we isolated a novel human mAb termed Y498 from a phage display antibody library constructed with the PBMC samples of a CRF07_BC-infected Chinese donor whose sera exhibited broadly neutralizing activity. Y498 cross-reacted with diverse Env antigens and neutralized 30% of 70 tested HIV-1 isolates. It efficiently blocked the binding of soluble CD4 to gp120 and competed with the CD4-binding site (CD4bs)-specific mAbs. By combining molecular docking and site-directed mutagenesis, the epitope of Y498 was characterized to contain three antigenic sites on gp120, including the CD4 binding loop in C3, the β23 in C4 and the β24-α5 in C5, which overlap the binding sites of CD4 and CD4bs-directed mAbs (b12, VRC01, A16). Therefore, Y498 is a novel neutralizing human mAb targeting a conformation-dependent CD4bs-based epitope, and its isolation and characterization could provide helpful information for elucidating human immune response to HIV-1 infection and designing effective vaccines and immunotherapeutics.

Utilization of Multi-Immunization and Multiple Selection Strategies for Isolation of Hapten-Specific Antibodies from Recombinant Antibody Phage Display Libraries.

Phage display technology provides a powerful tool for the development of novel recombinant antibodies. In this work, we optimized and streamlined the recombinant antibody discovery process for haptens as an example. A multi-immunization approach was used in order to avoid the need for construction of multiple antibody libraries. Selection methods were developed to utilize the full potential of the recombinant antibody library by applying four different elution conditions simultaneously. High-throughput immunoassays were used to analyse the binding properties of the individual antibody clones. Different carrier proteins were used in the immunization, selection, and screening phases to avoid enrichment of the antibodies for the carrier protein epitopes. Novel recombinant antibodies against mycophenolic acid and ochratoxin A, with affinities up to 39 nM and 34 nM, respectively, were isolated from a multi-immunized fragment antigen-binding (Fab) library.

Next-generation sequencing enables the discovery of more diverse positive clones from a phage-displayed antibody library

Phage display technology provides a powerful tool to screen a library for a binding molecule via an enrichment process. It has been adopted as a critical technology in the development of therapeutic antibodies. However, a major drawback of phage display technology is that because the degree of the enrichment cannot be controlled during the bio-panning process, it frequently results in a limited number of clones. In this study, we applied next-generation sequencing (NGS) to screen clones from a library and determine whether a greater number of clones can be identified using NGS than using conventional methods. Three chicken immune single-chain variable fragment (scFv) libraries were subjected to bio-panning on prostate-specific antigen (PSA). Phagemid DNA prepared from the original libraries as well as from the Escherichia coli pool after each round of bio-panning was analyzed using NGS, and the heavy chain complementarity-determining region 3 (HCDR3) sequences of the scFv clones were determined. Subsequently, through two-step linker PCR and cloning, the entire scFv gene was retrieved and analyzed for its reactivity to PSA in a phage enzyme immunoassay. After four rounds of bio-panning, the conventional colony screening method was performed for comparison. The scFv clones retrieved from NGS analysis included all clones identified by the conventional colony screening method as well as many additional clones. The enrichment of the HCDR3 sequence throughout the bio-panning process was a positive predictive factor for the selection of PSA-reactive scFv clones.

Methods for Selecting Phage Display Antibody Libraries.

The selection process aims sequential enrichment of phage antibody display library in clones that recognize the target of interest or antigen as the library undergoes successive rounds of selection. In this review, selection methods most commonly used for phage display antibody libraries have been comprehensively described.

Anti-cancer Activity of Novel TM4SF5-Targeting Antibodies through TM4SF5 Neutralization and Immune Cell-Mediated Cytotoxicity.

The transmembrane four L6 family member 5 (TM4SF5) protein is a novel molecular target for the prevention and treatment of hepatocellular carcinoma. TM4SF5 is highly expressed in liver, colon, esophageal, and pancreatic cancers and is implicated in tumor progression. Here, we screened monoclonal antibodies that specifically bound to the extracellular loop 2 (EC2) of TM4SF5 from a phage-displayed murine antibody (single-chain variable fragment; scFv) library. We constructed and characterized chimeric antibodies, Ab27 and Ab79, of scFv fused with Fc domain of human IgG1. The affinity (KD) of Ab27 and Ab79 for soluble EC2 was approximately 9.2 nM and 16.9 nM, respectively, as determined by surface plasmon resonance analysis. Ab27 and Ab79 efficiently bound to native TM4SF5 on the cell surface were internalized into the cancer cells, leading to a decrease in cell surface TM4SF5. Ab27 and Ab79 inhibited the proliferation and invasion of TM4SF5-positive liver and colon cancer cells and reduced FAK and c-Src phosphorylation. Ab27 and Ab79 also enhanced anoikis sensitivity and reduced survivin. Ab27 mediated antibody-dependent cell-mediated cytotoxicity in vitro. Ab27 and Ab79 efficiently inhibited tumor growth in a liver cancer xenograft model. These results strongly support the further development of Ab27 as a novel anti-cancer agent in the clinic.

Neutralization of Zika virus by germline-like human monoclonal antibodies targeting cryptic epitopes on envelope domain III

The Zika virus (ZIKV), a flavivirus transmitted by Aedes mosquitoes, has emerged as a global public health concern. Pre-existing cross-reactive antibodies against other flaviviruses could modulate immune responses to ZIKV infection by antibody-dependent enhancement, highlighting the importance of understanding the immunogenicity of the ZIKV envelope protein. In this study, we identified a panel of human monoclonal antibodies (mAbs) that target domain III (DIII) of the ZIKV envelope protein from a very large phage-display naive antibody library. These germline-like antibodies, sharing 98%–100% hoLogy with their corresponding germline IGHV genes, bound ZIKV DIII specifically with high affinities. One mAb, m301, broadly neutralized the currently circulating ZIKV strains and showed a synergistic effect with another mAb, m302, in neutralizing ZIKV in vitro and in a mouse model of ZIKV infection. Interestingly, epitope mapping and competitive binding studies suggest that m301 and m302 bind adjacent regions of the DIII C–C′ loop, which represents a recently identified cryptic epitope that is intermittently exposed in an uncharacterized virus conformation. This study extended our understanding of antigenic epitopes of ZIKV antibodies and has direct implications for the design of ZIKV vaccines.Emerging Microbes & Infections (2017) 6, e89; doi:10.1038/emi.2017.79; published online 11 October 2017

Neutralizing human recombinant antibodies against herpes simplex virus type 1 glycoproteins B from a phage-displayed scFv antibody library

The HSV-1 envelope glycoprotein B (gB) plays a critical role in virus entry into host cells. Neutralizing antibodies can therefore potentially prevent virus entry into target cells and cell-to-cell spread of infection. Our present study focused on the selection of neutralizing single-chain Fv (scFv) antibodies of a phage-displayed nonimmune human scFv antibody library against gB of HSV-1. To enrich specific scFvs, two phage antibodies were isolated against amino acid residues 31–43 derived from the N-terminal part of gB using panning technique. Two scFvs, scFv-gB1 and scFv-gB2, with frequencies of 45% and 20% were obtained from scFv clones after performing PCR and MvaI fingerprinting. In phage ELISA analysis, both gB1 and gB2 scFvs demonstrated high reactivity with the gB peptide. In the neutralization assay, scFv-gB1 and scFv-gB2 represented neutralizing effects of 55% and 59%, respectively. Upon further enhancement of the neutralizing effects of these antibodies, they can be considered as new potential alternatives in the treatment and prophylaxis of HSV-1 infections.

Abstract B079: The efficacy of CD133 BiTEs and CAR-T cells in preclinical model of recurrent glioblastoma

Abstract Glioblastoma (GBM) is a uniformly fatal primary brain tumor, characterized by a diverse cellular phenotype and genetic heterogeneity. Despite the use of aggressive cellular multi-modal treatment including surgical resection, radiotherapy and chemotherapy, the outcome of patients with GBM has failed to improve significantly. Numerous studies have implicated CD133+ brain tumor initiating cells (BTICs) as drivers of chemo- and radio-resistance in GBM. We have recently demonstrated that a CD133-driven gene signature is predictive of poor overall survival and targeting CD133+ treatment-refractory cells may be an effective strategy to block GBM recurrence. Chimeric antigen receptors (CARs) and bispecific T-Cell engaging antibodies (BiTEs) present promising immunotherapeutic approaches that have not yet been validated for recurrent GBM. Using CellectSeq, a novel methodology that combines use of phage-displayed synthetic antibody libraries and DNA sequencing, we developed the CD133-specific monoclonal antibody ‘RW03’. We constructed CD133-specific BiTEs or RW03xCD3 that consist of two arms; one arm recognizes the tumor antigen (CD133) while the second is specific to CD3 antigen. The BiTEs were constructed in four different conformations and dual binding specificity was confirmed using flow cytometry. Using CD133high and CD133low primary GBM lines, we validated the binding of BiTEs to CD133+ cells. Further analysis showed binding of BiTEs to human T cells known to express CD3 within a population of healthy donor peripheral blood mononuclear cells. We observed BiTEs redirecting T cells to kill GBMs, with greater efficiency observed in CD133high GBMs, validating BiTE target specificity. Incubating T-cells with BiTEs and the CD133high GBMs resulted in increased expression of T cell activation markers. In parallel, we derived the single chain variable fragment (scFv) from previously generated RW03 and generated a second-generation CAR. Anti-CD133 scFv with a myc tag was cloned in frame with a human CD8 leader sequence, CD8a transmembrane domain, CD28, and hCD3ζ signaling tail in the lentiviral construct pCCL-ΔNGFR vector in two different orientations: Light chain-linker-Heavy chain (CD133 CAR-LH) and Heavy chain-linker-Light chain (CD133 CAR-HL). Following lentiviral preparation, the T cells isolated from PBMCs were transduced with CD133 CAR-LH and CD133 CAR-VH constructs. After successful T cell engineering, the expression of ΔNGFR and myc tag was analyzed using flow cytometry to confirm the efficiency of transduction and surface expression of anti-CD133 respectively. CD133-specific CAR-T cells were cytotoxic to CD133+ GBMs. Co-culturing CD133 CAR-T cells with GBMs triggered T cell activation and proliferation. Treatment of GBM tumor-bearing mice with CD133-specific CAR-T cells yielded extended survival in mice and significant reductions in brain tumor burden. Furthermore, we uniquely adapted the existing chemoradiotherapy protocol for GBM patients for treatment of immunocompromised mice engrafted with human GBMs. Within this model, we have initiated treatment of recurrent GBM directed against CD133+ BTICs, to allow for a direct prospective comparison of toxicity and efficacy of BiTEs and CAR T cell strategies. Citation Format: Parvez Vora, Chirayu Chokshi, Maleeha Qazi, Chitra Venugopal, Sujeivan Mahendram, Mohini Singh, Jarrett Adams, David Bakhshinyan, Max London, Minomi Subapanditha, Nicole McFarlane, James Pan, Jonathan Bramson, Jason Moffat, Sachdev Sidhu, Sheila Singh. The efficacy of CD133 BiTEs and CAR-T cells in preclinical model of recurrent glioblastoma [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B079.

Tetanus Neurotoxin Neutralizing Antibodies Screened from a Human Immune scFv Antibody Phage Display Library.

Tetanus neurotoxin (TeNT) produced by Clostridium tetani is one of the most poisonous protein substances. Neutralizing antibodies against TeNT can effectively prevent and cure toxicosis. Using purified Hc fragments of TeNT (TeNT-Hc) as an antigen, three specific neutralizing antibody clones recognizing different epitopes were selected from a human immune scFv antibody phage display library. The three antibodies (2-7G, 2-2D, and S-4-7H) can effectively inhibit the binding between TeNT-Hc and differentiated PC-12 cells in vitro. Moreover, 2-7G inhibited TeNT-Hc binding to the receptor via carbohydrate-binding sites of the W pocket while 2-2D and S-4-7H inhibited binding of the R pocket. Although no single mAb completely protected mice from the toxin, they could both prolong survival when challenged with 20 LD50s (50% of the lethal dose) of TeNT. When used together, the mAbs completely neutralized 1000 LD50s/mg Ab, indicating their high neutralizing potency in vivo. Antibodies recognizing different carbohydrate-binding pockets could have higher synergistic toxin neutralization activities than those that recognize the same pockets. These results could lead to further production of neutralizing antibody drugs against TeNT and indicate that using TeNT-Hc as an antigen for screening human antibodies for TeNT intoxication therapy from human immune antibody library was convenient and effective.

High throughput cytotoxicity screening of anti-HER2 immunotoxins conjugated with antibody fragments from phage-displayed synthetic antibody libraries.

Immunotoxins are an important class of antibody-based therapeutics. The potency of the immunotoxins depends on the antibody fragments as the guiding modules targeting designated molecules on cell surfaces. Phage-displayed synthetic antibody scFv libraries provide abundant antibody fragment candidates as targeting modules for the immunoconjugates, but the discovery of optimally functional immunoconjugates is limited by the scFv-payload conjugation procedure. In this work, cytotoxicity screening of non-covalently assembled immunotoxins was developed in high throughput format to discover highly functional synthetic antibody fragments for delivering toxin payloads. The principles governing the efficiency of the antibodies as targeting modules have been elucidated from large volume of cytotoxicity data: (a) epitope and paratope of the antibody-based targeting module are major determinants for the potency of the immunotoxins; (b) immunotoxins with bivalent antibody-based targeting modules are generally superior in cytotoxic potency to those with corresponding monovalent targeting module; and (c) the potency of the immunotoxins is positively correlated with the densities of the cell surface antigen. These findings suggest that screening against the target cells with a large pool of antibodies from synthetic antibody libraries without the limitations of natural antibody responses can lead to optimal potency and minimal off-target toxicity of the immunoconjugates.

Abstract 2972: Development and characterization of synthetic antibodies with a human framework that directly link metabolism to immune system in cancer cells

Abstract Deregulated cellular energetics and evasion of immune system are two hallmark features of cancer cells. In fact, increased uptake of glucose and glutamine by cancer cells elevates flux of a minor glucose pathway known as the hexosamine biosynthetic pathway (HBP), resulting in alteration of glycosylation. Addition of sugar moieties to proteins plays an important role in sensing cellular metabolism, growth factors, signaling molecules, nutrient flux and stress. Since many of these processes are de-regulated in tumorigenesis, global alterations in glycosylation via HBP flux are likely to play an important role in cancer. We hypothesize that cancer cells can “sense” stress through the HBP pathway, and, consequently, reprogram the cell surface and adapt to tougher conditions. Neo-epitopes on the cell surface that emerge from tuning the HBP pathway activity may be prime candidates as readouts that reliably report the status of metabolic reprogramming. In order to advance the understanding of metabolic reprogramming from the perspective of the cell surface, we combined genetics and advanced protein engineering using phage-displayed synthetic human antibody libraries to generate recombinant antibodies (rAbs) that respond to changes in the activity of the HBP. Our method for discovery of surface and nutrient-associated protein sensors (SNAPS) has allowed us to identify multiple human synthetic rAbs whose activity is potentiated under different metabolic states. More specifically, these rAbs recognize cell surface features that discriminate cancer cells with increased HBP flux and effectively recruit components of the immune system to initiate tumor cell lysis. Our findings suggest that the nutrient-dependent cell surface epitopes may have functional roles in defining cancer cell identity and may link multiple cancer hallmarks. Citation Format: Jelena Tomic, Megan McLaughlin, Ron Geyer, Sachdev Sidhu, Jason Moffat. Development and characterization of synthetic antibodies with a human framework that directly link metabolism to immune system in cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2972.

Abstract 1481: Preclinical validation of a novel CD133/CD3 bispecific T-cell engager (BiTE) antibody to target patient-derived glioblastoma cells

Abstract Glioblastoma (GBM), an aggressive primary brain tumor in adults, is feared for its near uniformly fatal prognosis and is characterized by a diverse cellular phenotype and genetic heterogeneity. Despite the use of aggressive multi-modal treatment including surgical resection, radiotherapy and chemotherapy, the outcome of patients with GBM has failed to improve significantly. We developed patient-derived brain tumor initiating cell (BTIC) early passage lines that describe the extent of intertumoral heterogeneity, presenting a powerful preclinical model of GBM. Numerous studies have implicated CD133+ BTICs as drivers of chemo- and radio-resistance in GBM. CD133 expression correlates with disease progression, recurrence, and poor overall survival of GBM patients. Here, we describe the preclinical evaluation of a recombinant RW03xCD3 bispecific T-cell engager (BiTE) antibody that redirects human polyclonal T cells to CD133+ GBM cells, inducing very potent anti-tumor response. Using CellectSeq, a novel methodology that combines use of phage-displayed synthetic antibody libraries and high-throughput DNA sequencing technology, we developed the CD133-specific monoclonal antibody ‘RW03’. We constructed CD133-specific BiTEs or RW03xCD3 that consist of two arms; one arm recognizes the tumor antigen (CD133) while the second is specific to CD3 antigen. The BiTEs were constructed in four different conformations and dual binding specificity was confirmed using flow cytometry. Using CD133high and CD133low primary GBM lines, we validated the binding of BiTEs to CD133+ cells. Further analysis showed binding of BiTEs to human T cells known to express CD3 within a population of healthy donor peripheral blood mononuclear cells. In order to test the ability of BiTEs to functionally elicit CD133-specific cytotoxic responses in vitro, we performed Presto blue-based killing assays. We observed CD133-specific BiTEs redirect T cells to kill CD133-expressing GBM cells in a coculture of T cells and GBM cells. The killing was more efficient in CD133high GBMs compared to CD133low GBMs, validating its specificity to target CD133+ BTICs. Incubating T cells with BiTEs and GBMs resulted in increased surface expression of T-cell activation markers CD69 and CD25 in both, CD4+ and CD8+ T cells populations. Treatment with BiTEs yielded extended survival in mice and significant reductions in brain tumor burden. This rigorously obtained data offers compelling evidence that BiTE-mediated cytotoxicity against treatment-resistant and evasive CD133+ GBM BTICs could provide a very potent, specific and novel therapeutic strategy for GBM patients. Citation Format: Parvez Vora, Chitra Venugopal, Jarrett Adams, James Pan, Chirayu Chokshi, Maleeha Qazi, Minomi Subapanditha, Mohini Singh, David Bakhshinyan, Ksenia Bezverbnaya, Nicole McFarlane, Jonathan Bramson, Sachdev Sidhu, Jason Moffat, Sheila Singh. Preclinical validation of a novel CD133/CD3 bispecific T-cell engager (BiTE) antibody to target patient-derived glioblastoma cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1481.

Abstract 3805: A novel anti-CD24 monoclonal antibody, humanized and affinity maturated for targeting gastrointestinal cancers

Abstract Background: CD24 is a cell-surface heavily glycosylated GPI-anchored protein. We had previously shown that CD24 in an important player in the multistep process of GI carcinogenesis (Gastro 2006, Clin Can Res 2007, Can Res 2008). The creation of chimeric, humanized or fully human antibodies was a major breakthrough and led to a wave of US FDA-approved antibodies. Aim: To further improve the efficacy of the humanized anti-CD24 mAb by increasing its binding strength and thereby generating a novel therapy tool for GI malignancy Methods: From murine to humanized, unarmed and conjugated, small derivatives and full IgG antibodies were recombinantly engineered. The antibody genes were recovered, amplified and cloned into appropriate vectors. Then the vectors were introduced into a host (mammalian and E.coli) and adequate amounts of functional antibody were achieved. Sequence analysis of the CDR loops was the base for library designing. Affinity maturation was performed in two-steps selection (CDR walking) and by using phage display technique. The binding of the different derivatives were evaluated on full Glycan array in which more than 70 sugar moieties were printed. Results: In vivo antibody targeting and accumulation within a CD24 positive tumor and its excess clearance was clearly demonstrated using live imaging device (Maestro Cri device). High-affinity antibodies were selected and created from combinatorial phage-displayed antibody libraries that contain varying degrees of diversity at randomized positions. A chosen matured clone was isolated and showed higher binding strength (1.8×10-8), compared to the parental murine and humanized Abs. The matured antibody showed selective recognition and binding to the CD24 antigen which proves that the genetic manipulations carried out did not affect its properties. Its stability was enhanced following the maturation process,as well as its pharmacokinetics parameters which showed a long serum half-life.The matured antibody mediates ADCC (antibody-dependent cell cytotoxicity), 75% of target cell lysis was demonstrated. Combined treatment with standard chemotherapy and natural products, such as monoterpenes (terpinen-4-ol), showed significant reduction in cell viability (90% cell death). Binding of anti-CD24 Ab to glycan microarray could not be detected while high binding intensities were observed where the whole CD24 protein was printed, indicating that the antibodies bind to the core peptide and not to its sugar residues. Conclusion: Targeting CD24 may be a promising treatment for GI malignancies in combination with chemotherapy and natural agents. The resulted matured humanized anti-CD24 mAb proved to be more effective than the murine parental Ab. The long serum half-life is desirable as it would decrease the need for repetitive injections. Citation Format: Shiran Shapira, Dina Kazanov, Vered Padler Karavani, Itai Benhar, Nadir Arber. A novel anti-CD24 monoclonal antibody, humanized and affinity maturated for targeting gastrointestinal cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3805.

Isolation and characterization of a novel neutralizing antibody targeting the CD4-binding site of HIV-1 gp120

Isolation and characterization of novel HIV-1 neutralizing antibodies assists the development of effective AIDS vaccines and immune therapeutics. In this study, we constructed a phage display antibody library by using the PBMC samples of a clade B′ HIV-1-infected long-term nonprogressor (LTNP) whose sera exhibited broadly neutralizing activity. A novel human monoclonal antibody (hMAb), termed A16, was identified by panning the library with two clades of HIV-1 Env glycoproteins. We demonstrated that A16 neutralized 32% of 73 tested HIV-1 isolates and it targeted the CD4-binding site (CD4bs) of gp120 with high affinity. By selecting the peptide mimotopes in combination with computational algorithms and site-directed mutagenesis, the epitope of A16 was mapped to the structurally conserved sites located within the β1-α1, loop D, β20-β21 (bridging sheet) and β24-α5 of gp120, which critically determine the CD4 binding and are involved in the epitopes of CD4bs-directed antibodies. Our studies have shed new insights for the immune response of HIV-1 infection and offered a new tool for designing vaccine immunogens and antibody-based immune therapy.

Screening and identification of human ZnT8-specific single-chain variable fragment (scFv) from type 1 diabetes phage display library.

Zinc transporter 8 (ZnT8) is a major autoantigen and a predictive marker in type 1 diabetes (T1D). To investigate ZnT8-specific antibodies, a phage display library from T1D was constructed and single-chain antibodies against ZnT8 were screened and identified. Human T1D single-chain variable fragment (scFv) phage display library consists of approximately 1×10(8) clones. After four rounds of bio-panning, seven unique clones were positive by phage ELISA. Among them, C27 and C22, which demonstrated the highest affinity to ZnT8, were expressed in Escherichia coli Top10F' and then purified by affinity chromatography. C27 and C22 specifically bound ZnT8 N/C fusion protein and ZnT8 C terminal dimer with one Arg325Trp mutation. The specificity to human islet cells of these scFvs were further confirmed by immunohistochemistry. In conclusion, we have successfully constructed a T1D phage display antibody library and identified two ZnT8-specific scFv clones, C27 and C22. These ZnT8-specific scFvs are potential agents in immunodiagnostic and immunotherapy of T1D.

Construction and characterization of a highly reactive chicken-derived single-chain variable fragment (scFv) antibody against Staphylococcus aureus developed with the T7 phage display system

The purpose of this study was to construct a single-chain variable fragment (scFv) antibody from chicken egg yolk immunoglobulin (IgY) by means of genetic engineering and subsequent panning for a specific antibody against Staphylococcus aureus. Methods and resultsWe amplified the scFv using blood and spleen obtained from 100-day-old Roman chickens immunized with inactivated S. aureus and subsequently constructed a T7 phage display antibody library using phage display technology. Four non-repeated blood scFv and 6 spleen scFv were obtained following 3 rounds of panning of the T7 phage display antibody library, enzyme-linked immunosorbent assay and sequencing. These 10 scFv were cloned into the prokaryotic expression vector pCold I with expression induced at a low temperature. Four soluble proteins were obtained. Among them, soluble protein SFV6 derived from the spleen showed good reactivity against S. aureus using indirect ELISA and produced a particularly strong antibacterial effect in vitro. ConclusionWe were successful in isolating a highly specific scFv antibody against S. aureus from the spleen phage display library. This study provides a simple and rapid method for the quick preparation of a large number of antibodies against S. aureus and provides the foundation for the positioning of antibodies in the organism and the study of the antibacterial mechanism through which the antibody functions.

Specific Noncompetitive Immunoassay for HT-2 Mycotoxin Detection.

Here we demonstrate a novel homogeneous one-step immunoassay, utilizing a pair of recombinant antibody antigen-binding fragments (Fab), that is specific for HT-2 toxin and has a positive readout. Advantages over the conventional competitive immunoassay formats such as enzyme-linked immunosorbent assay (ELISA) are the specificity, speed, and simplicity of the assay. Recombinant antibody HT2-10 Fab recognizing both HT-2 and T-2 toxins was developed from a phage display antibody library containing 6 × 10(7) different antibody clones. Specificity of the immunoassay was introduced by an anti-immune complex (IC) antibody binding the primary antibody-HT-2 toxin complex. When the noncompetitive immune complex assay was compared to the traditional competitive assay, an over 10-fold improvement in sensitivity was observed. Although the HT2-10 antibody has 100% cross-reactivity for HT-2 and T-2 toxins, the immune complex assay is highly specific for HT-2 alone. The assay performance with real samples was evaluated using naturally contaminated wheat reference material. The half-maximal effective concentration (EC50) value of the time-resolved fluorescence resonance energy transfer (TR-FRET) assay was 9.6 ng/mL, and the limit of detection (LOD) was 0.38 ng/mL (19 μg/kg). The labeled antibodies can be predried to the assay vials, e.g., microtiter plate wells, and readout is ready in 10 min after the sample application.

A Human Anti-Toll Like Receptor 4 Fab Fragment Inhibits Lipopolysaccharide-Induced Pro-Inflammatory Cytokines Production in Macrophages.

The results of clinical and experimental studies suggest that endotoxin/toll-like receptor 4 (TLR4)-mediated proinflammatory and profibrotic signaling activation is critical in the development of hepatic fibrosis. However, studies examining the role of specific TLR4 inhibitor are still lacking. The present study was aimed to prepare a human anti-TLR4 Fab fragment, named hTLR4-Fab01, and to explore its immune activity. We screened the positive clone of anti-human TLR4 phagemid from a human phage-display antibody library using recombinant TLR4 protein, which was used as template cDNA for the amplification of variable regions of the heavy (VH) chain and light chain (VL), then coupled with highly conserved regions of the heavy chain domain 1 (CH1) and the light chain (CL), respectively. Thus, the prokaryotic expression vector pETDuet-1 of hTLR4-Fab01 was constructed and transformed into Escherichia coli (E. coli) BL21. The characteristic of hTLR4-Fab01 was examined by SDS-PAGE, Western blotting, ELISA, affinity and kinetics assay. Further, our data demonstrate that hTLR4-Fab01 could specifically bind to TLR4, and its treatment obviously attenuated the proinflammatory effect, characterized by less LPS-induced TNF-α, IL-1, IL-6 and IL-8 production in human macrophages. In conclusion, we have successfully prepared the hTLR4-Fab01 with efficient activity for blocking LPS-induced proinflammatory cytokines production, suggesting that the hTLR4-Fab01 may be a potential candidate for the treatment of hepatic fibrosis.

Antibody-Mediated Pathogen Resistance in Plants.

The methods described in this chapter were developed in order to produce transgenic plants expressing pathogen-specific single-chain variable fragment (scFv) antibodies fused to antifungal peptides (AFPs), conferring resistance against fungal pathogens. We describe the selection from a phage display library of avian scFv antibodies that recognize cell surface proteins on fungi from the genus Fusarium, and the construction of scFv-AFP fusion protein constructs followed by their transient expression in tobacco (Nicotiana spp.) plants and stable expression in Arabidopsis thaliana plants. Using these techniques, the antibody fusion with the most promising in vitro activity can be used to generate transgenic plants that are resistant to pathogens such as Fusarium oxysporum f. sp. matthiolae.