Phage-displayed Single Chain Variable Fragment Research Articles

Development of a [89Zr]Zr-labeled Human Antibody using a Novel Phage-displayed Human scFv Library.

Tax-interacting protein 1 (TIP1) is a cancer-specific radiation-inducible cell surface antigen that plays a role in cancer progression and resistance to therapy. This study aimed to develop a novel anti-TIP1 human antibody for noninvasive PET imaging in patients with cancer. A phage-displayed single-chain variable fragment (scFv) library was created from healthy donors' blood. High-affinity anti-TIP1 scFvs were selected from the library and engineered to human IgG1. Purified Abs were characterized by size exclusion chromatography high-performance liquid chromatography (SEC-HPLC), native mass spectrometry (native MS), ELISA, BIAcore, and flow cytometry. The labeling of positron emitter [89Zr]Zr to the lead Ab, L111, was optimized using deferoxamine (DFO) chelator. The stability of [89Zr]Zr-DFO-L111 was assessed in human serum. Small animal PET studies were performed in lung cancer tumor models (A549 and H460). We obtained 95% pure L111 by SEC-HPLC. Native MS confirmed the intact mass and glycosylation pattern of L111. Conjugation of three molar equivalents of DFO led to the optimal DFO-to-L111 ratio of 1.05. Radiochemical purity of 99.9% and specific activity of 0.37 MBq/μg was obtained for [89Zr]Zr-DFO-L111. [89Zr]Zr-DFO-L111 was stable in human serum over 7 days. The immunoreactive fraction in cell surface binding studies was 96%. In PET, preinjection with 4 mg/kg cold L111 before [89Zr]Zr-DFO-L111 (7.4 MBq; 20 μg) significantly (P < 0.01) enhanced the tumor-to-muscle standard uptake values (SUVmax) ratios on day 5 compared with day 2 postinjection. L111 Ab targets lung cancer cells in vitro and in vivo. [89Zr]Zr-DFO-L111 is a human antibody that will be evaluated in the first in-human study of safety and PET imaging.

A tetravalent TREM2 agonistic antibody reduced amyloid pathology in a mouse model of Alzheimer's disease.

Triggering receptor expressed on myeloid cells 2 (TREM2) plays crucial roles in Alzheimer's disease (AD) by regulating microglia migration toward, and phagocytosis of oligomeric amyloid-β (oAβ) and amyloid plaques. Studies in rodent models of AD have shown that mice with increased TREM2 expression have reduced amyloid pathology. Here, we identified a TREM2 agonist monoclonal Ab (Ab18) by panning a phage-displayed single-chain variable fragment Ab library. By engineering the bivalent immunoglobulin G1 (IgG1) to tetra-variable domain immunoglobulin (TVD-Ig), we further increased the TREM2 activation by 100-fold. Stronger TREM2 activation led to enhanced microglia phagocytosis of the oAβ-lipid complex, migration toward oAβ, and improved microglia survival in vitro. Mechanistic studies showed increased TREM2 clustering on microglia by the tetravalent Ab18 TVD-Ig without altering microglial TREM2 amount. An engineered bispecific Ab targeting TREM2 and transferrin receptor (TfR; Ab18 TVD-Ig/αTfR) improved Ab brain entry by more than 10-fold with a broad brain parenchyma distribution. Weekly treatment of 5XFAD mice (a model of AD) with Ab18 TVD-Ig/αTfR showed a considerable reduction of amyloid burden with increased microglia migration to and phagocytosis of amyloid plaques, improved synaptic and neuronal marker intensity, improved cognitive functions, reduced endogenous tau hyperphosphorylation, and decreased phosphorylated neurofilament H immunostaining. This study demonstrated the feasibility of engineering multivalent TREM2 agonistic Ab coupled with TfR-mediated brain delivery to enhance microglia functions and reduce amyloid pathology in vitro and in vivo. This Ab engineering approach enables the development of effective TREM2-targeting therapies for AD.

Effective killing of cells expressing CD276 (B7-H3) by a bispecific T cell engager based on a new fully human antibody

The pancaner molecule CD276 (B7-H3) is an attractive target for antibody based therapy. We identified from a large (1011) phage-displayed single-chain variable fragment (scFv) library, a fully human antibody, B11, which bound with high avidity (KD=0.4 nM) to CD276. B11 specifically bound to the V1/V2 domain of CD276 and competed with the antibody 8H9 (Omburtamab). It was used to design an IgG-format bispecific T cell engager B11-BiTE, which was more effective than 8H9-BiTE in 14 different cancer cell lines. B11-BiTE also exhibited strong ADCC/ADCP. Therefore, the fully human B11-BiTE is a promising candidate for treatment of tumors expressing CD276.

Intercellular Adhesion Molecule-1 as Target for CAR-T-Cell Therapy of Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) comprises lethal malignancies with limited treatment options. Chimeric antigen receptor T (CAR-T) cell therapy is an effective immunotherapeutic strategy that has demonstrated unprecedented efficacy in the treatment of hematological malignancies but has shown limited success in the management of some solid tumors. Many malignant tumors are related to increased expression of intercellular adhesion molecule-1 (ICAM1), providing a rationale for ICAM1-specific immunotherapies for the treatment of cancer. Here, we validated the expression of ICAM1 in TNBC tissues. Subsequently, we generated a phage-displayed single-chain variable fragment (scFv) library using splenocytes from ICAM1-immunized mice and selected a novel ICAM1-specific scFv, mG2-scFv. Using mG2-scFv as the extracellular antigen binding domain, we constructed ICAM1-specific CAR-T cells and demonstrated the robust and specific killing of TNBC cell lines in vitro. Most importantly, in the TNBC mouse model, ICAM1-specific CAR-T cells significantly reduced the growth of the TNBC tumor, resulting in long-term remission and improved survival. Together, these results indicated that ICAM1-specific CAR-T cells have high therapeutic potential against ICAM1-positive TNBC tumors.

Production of a phage-displayed single chain variable fragment antibody against infectious bursal disease virus

Purpose : To develop specific single chain variable fragments (scFv) against infectious bursal disease virus (IBDV) via phage display technology. Methods : Purified viruses were initially applied for iterative panning rounds of scFv phage display libraries. The binding ability of the selected scFv antibody fragments against the IBDV particles was analyzed by indirect enzyme-linked immunosorbent assay (ELISA) followed by blotting assays. Threedimensional (3D) structure of the selected scFv antibody fragment and VP3 protein were predicted through in silico analysis. Structural characterization of the antibody-antigen complexes was carried out by computational docking analysis. Results : The serological results obtained from the ELISA and blotting analysis showed that the selected clones produced specific scFv antibody fragments that were capable of effectively detecting infectious bursal disease (IBD) in the infected animal tissue. Biodiversity analysis by BstNI finger printing and nucleotide sequencing revealed that there was no major difference in nucleotide sequences of the selected clones. Further analysis demonstrated that this recombinant fragment of the antibody was able to bind to VP3 structural protein of IBDV with a molecular weight of ~30 kDa. Molecular docking results revealed that the binding energy of scFv to IBDV-VP3 was 545 kj/mol. Conclusion : The developed scFv antibody fragments possess great potentials for the diagnosis of IBD. The findings of the present study confirm the feasibility of using phage display technology for rapid production of antibodies against IBD diseases by applying naive scFv libraries. Keywords : Antibody, Molecular docking, Phage display technology, Single chain variable fragments

Identification of tumor-binding scFv derived from clonally related B cells in tumor and lymph node of a patient with breast cancer.

The purpose of this study was to determine the clonal relationship between B cells within a breast cancer and the B cells in the tumor-draining lymph node (TDLN). We also determined the binding capacity of antibodies derived from these sources to autologous cancer and autologous noncancer breast tissue. Antibody clonality of B cells derived from tumor and lymph node was determined by analyzing heavy and light chain immunoglobulin sequences. The number of shared clonal groups observed between tumor and lymph node antibodies was significant for both heavy (p = 0.004) and light chain (p = 0.012) populations. Panning with phage-displayed single-chain variable fragment libraries derived from the tumor and lymph node B cells resulted in multiple antibodies that bound autologous tumor. Sequence analysis of enriched antibodies recovered after the third round of panning the tumor and TDLN libraries against autologous tumor lysates had a genetic relationship. These results indicate that B cells infiltrating a patient's breast cancer and B cells present in the tumor-draining lymph node are clonally and functionally related.

Effects of signal sequence on phage-displayed disulfide-stabilized single chain antibody variable fragment (sc-dsFv) libraries

Phage-displayed single chain variable fragment (scFv) libraries are powerful tools in antibody engineering. Disulfide-stabilized scFv (sc-dsFv) with an interface disulfide bond is structure-wise more stable than the corresponding scFv. A set of recently discovered signal sequences replacing the wild type (pelB) signal peptidase cleavage site in the c-region has been shown to be effective in rescuing the expression of sc-dsFv libraries on the phage surface. However, the effects of the other regions of the signal sequence on the expression of the sc-dsFv libraries and on the formation of the interface disulfide bond in the phage-displayed sc-dsFv have not been clear. In this work, selected novel signal sequence variants in the h-region were shown to be equally effective in promoting sc-dsFv library expression on the phage surface; the expression level and complexity of the sc-dsFv libraries were comparable to the corresponding scFv libraries produced with the wild-type (pelB) signal sequence. The interface disulfide bond in the phage-displayed sc-dsFv was proven to form to a large extent in the library variant ensemble generated with signal sequence variants in both the h-region and the c-region. The sc-dsFv engineering platform established in this work can be applied to many of the known scFv molecules which are in need of a more stable version for the applications under harsh conditions or for longer shelf-life.

Screening and Identification of Recombinant Anti-Idiotype Antibodies against Gastric Cancer and Colon Cancer Monoclonal Antibodies by a Phage-Displayed Single-Chain Variable Fragment Library

Several monoclonal antibodies (McAbs) have been developed that show high sensitivity and specificity to gastric cancer and colorectal cancer. However, few of the antigens recognized by these antibodies have been identified. The authors now report the selection of anti-idiotype (anti-id) antibodies of MGb1 McAb against gastric cancer and MC5 McAb against colorectal cancer using phage-displayed single-chain variable fragment (ScFv) libraries. After purification, the anti-id antibodies were approximately 30 kd and could be recognized by MGb1/MC5 McAb. Anti-id antibodies significantly blocked the binding of MGb1 and MC5 to gastric cancer/colorectal cancer cells, respectively, suggesting that the antibodies were specific to MGb1 and MC5. Antibodies against gastric and colorectal cancer could be detected in mice at 6 weeks after immunization with the anti-id antibodies. At week 8, antibody titers reached 1:400. The anti-id antibodies may be useful as novel reagents for developing vaccines against gastric cancer and colorectal cancer.

Signal sequence as a determinant in expressing disulfide-stabilized single chain antibody variable fragments (sc-dsFv) against human VEGF

Phage-displayed single chain variable fragment (scFv) libraries have been powerful tools in antibody engineering. But the scFv structures are frequently unstable due to the dissociation of the dimeric interface between the two variable domains. One solution is the sc-dsFv construct, where the single chain variable domain fragment is stabilized with an additional interface disulfide bond, leading to stable and homogeneous dimeric interface for the sc-dsFv structure. However, the phagemid system that is capable of effective expression for both sc-dsFv-pIII fusion proteins on phage surface and secreted non-fusion sc-dsFv in bacterial culture medium has not been demonstrated. In this work, a biological combinatorial approach was applied to optimize the signal sequence N-terminal to the sc-dsFv-pIII fusion protein encoded in a phagemid. The optimized sc-dsFv phage display systems were compatible with both the phage-based directed evolution procedure and the high throughput screening of the soluble sc-dsFv. The utility of the phagemid systems was demonstrated in generating anti-VEGF sc-dsFv with VEGF-binding affinity one order of magnitude higher than the corresponding scFv, due only to the interface disulfide bond in the sc-dsFv. Moreover, the protein stability of the sc-dsFv construct was unmatched by the corresponding scFv. These advantages of the sc-dsFv were gained through the interface disulfide bond of the sc-dsFv and the novel signal sequence in the phagemid.

A phage-displayed single chain variable fragment that interacts with hepatitis B core antigen: Library construction, selection and diagnosis

Background Phage display is an alternative method for constructing and selecting antibodies with desired specificity towards an antigen. Objectives To construct a library of single chain variable fragment (ScFv) towards hepatitis B core antigen (HBcAg). To isolate a ScFv phage clone that interacts with HBcAg and to develop a phage-ELISA for detecting the antigen. Study design Mice were inoculated with HBcAg and RNA was extracted from their spleen cells. The genes encoding heavy (V H) and light (V L) chains were amplified, linked via PCR and cloned into a phagemid vector. Phage particles displaying ScFv were panned against HBcAg and a selected clone was characterized and employed as a diagnostic reagent for detecting HBcAg in serum samples. Results A phage clone that interacts with HBcAg was selected from the antibody library. The binding of the phage to HBcAg was inhibited by a cyclic peptide bearing the WSFFSNI sequence. A phage-ELISA was established using the recombinant phage and as low as 10 ng of HBcAg can be detected by the assay. Conclusion The ScFv displayed on the surface of filamentous phage is an alternative choice for diagnosis of HBcAg in serum samples.

Chicken recombinant antibodies specific for very virulent infectious bursal disease virus

A phage-displayed single chain variable fragment (scFv) antibody library was constructed from the immune spleen cells of chickens immunized with very virulent infectious bursal disease virus (vvIBDV) strain CS89. A library consisting of around 9.2 x 10(7) clones was subjected to 3 rounds of panning against captured CS89 virus. Analysis of individual clones by nucleotide sequencing revealed at least 22 unique scFv antibodies binding to vvIBDV in ELISA. Testing of the scFv antibody panel in ELISA against classical, variant or vaccine strains and a wide variety of vvIBDV isolates from the UK, China, France, Belgium, Africa, Brazil, Indonesia and the Netherlands identified one antibody, termed chicken recombinant antibody 88 (CRAb 88) that was specific for vvIBDV. CRAb 88 was capable of recognizing all vvIBDV strains tested regardless of their country of origin and showed no reactivity with classical, variant or vaccine strains, lending support to the use of this scFv as a powerful diagnostic tool for the differentiation of vvIBDV strains. Immunoprecipitation studies revealed that CRAb 88 was directed towards a highly conformational epitope located within the major neutralizing protein VP2. Sequence analysis of the hypervariable region of VP2 of the IBDV strains tested indicate that Ile(256) and Ile(294) may play roles in binding of CRAb 88. This is the first reagent of its type capable of positively distinguishing vvIBDV from other IBDV strains.

Construction and characterization of recombinant single-chain variable fragment antibodies against Toxoplasma gondii MIC2 protein

The protozoan parasite Toxoplasma gondii produces a family of microneme proteins that are thought to play diverse roles in aiding the parasite's intracellular existence. Among these, TgMIC2 has a putative function in parasite adhesion to the host cell to initiate the invasion process. The invasion process may be localized and inhibited by monoclonal antibodies against the protein(s) involved. Here we report on the construction of a phage-displayed single-chain variable fragment (scFv) library from mice immunized with whole T. gondii parasites. The library was subsequently panned against recombinant TgMIC2 (rpTgMIC2) and 2 different groups of antibody clones were obtained, based on fingerprinting and sequencing data. The expressed recombinant scFv antibody was able to recognize rpTgMIC2 in a Western blot detection experiment. These results show that the phage display technology allows quick and effective production of monoclonal antibodies against parasite antigens. By panning the scFv-displayed library, we should be able to obtain a plethora of multi-functional scFv antibodies towards T. gondii proteins.

Isolation and characterization of rabbit single chain antibodies to human Reg Iα protein

To investigate the role of Reg Iα in human inflammatory bowel disease (IBD), we made two phage-displayed single chain variable fragment (scFv) libraries from rabbits immunized with recombinant or native human Reg Iα. After one to three rounds of panning, we were able to isolate phage-displaying scFvs, which bound to human Reg Iα. Anti-Reg Iα scFvs from both libraries showed similar immunoreactivity to different processed forms of the protein. Despite several DNA fingerprint patterns among these clones, their deduced amino acid sequences are highly homologous with 100% identity in the complementarity-determining regions (CDRs) of the variable segment of heavy chain (VH) region and a small variation in the CDR1 of the variable segment of light chain (VL) region. We also expressed and purified soluble myc-tagged or glutathione S-transferase (GST) fusion scFv proteins from bacteria. Immunohistochemical studies using one of our anti-Reg Iα scFv antibodies showed prominent staining in the metaplastic Paneth cell population and light staining in the lamina propria. This scFv antibody is now being used for studies of the role of Reg Iα in human IBD.