Single-chain Fv Fragment Research Articles

Clonal array profiling of scFv-displaying phages for high-throughput discovery of affinity-matured antibody mutants

"Antibody-breeding" approach potentially generates therapeutic/diagnostic antibody mutants with greater performance than native antibodies. Therein, antibody fragments (e.g., single-chain Fv fragments; scFvs) with a variety of mutations are displayed on bacteriophage to generate diverse phage-antibody libraries. Rare clones with improved functions are then selected via panning against immobilized or tagged target antigens. However, this selection process often ended unsuccessful, mainly due to the biased propagation of phage-antibody clones and the competition with a large excess of undesirable clones with weaker affinities. To break radically from such panning-inherent problems, we developed a novel method, clonal array profiling of scFv-displaying phages (CAP), in which colonies of the initial bacterial libraries are examined one-by-one in microwells. Progenies of scFv-displaying phages generated are, if show sufficient affinity to target antigen, captured in the microwell via pre-coated antigen and detected using a luciferase-fused anti-phage scFv. The advantage of CAP was evidenced by its application with a small error-prone-PCR-based library (~ 105 colonies) of anti-cortisol scFvs. Only two operations, each surveying only ~ 3% of the library (9,400 colonies), provided five mutants showing 32–63-fold improved Ka values (> 1010 M−1), compared with the wild-type scFv (Ka = 3.8 × 108 M−1), none of which could be recovered via conventional panning procedures operated for the entire library.

Abstract 2167: A protease-mediated regulatory chimeric antigen receptor (CAR): “Scissors” CAR improves target-specificity through regulation of T-cell activity

Abstract [Introduction] CAR-T cell therapy is an attractive methodology in the field of cancer immunotherapy. The anti-CD19 CAR-T cell therapy shows excellent therapeutic effects against refractory B-cell malignancies. However, the CAR-T cells attack not only target-tumor-cells, but also normal tissues expressing the target molecule, for example, normal B-cells with CD19. For the CAR-T cell therapy, such “on-target / off-tumor” effect is one of major obstacles to be overcome. Here, we show the protease-mediated regulatory CAR-T cell system, which improved the target-specificity by recognizing two distinct antigens. We designed two types of CAR: "effector CAR" and "scissors CAR". The “effector CAR” is constituted of a single chain Fv fragment (scFv) targeting an antigen (protein X) on tumor cells, Human Immunodeficiency Virus protease (HIVPR) recognition site, and a functional domain of CD3-ζ. The “scissors CAR” is constituted of a scFv targeting another antigen (protein Y) and HIVPR. The “scissors CAR” induces cleavage of the “effector CAR” leading to inactivation when the CAR-T cells contact with cells expressing both proteins X and Y. [Material and Methods] For proof of principle, we first constructed the anti-CD19 “mCherry CAR” harboring mCherry fluorescence protein in the cytoplasmic region under the HIVPR recognition site. Also, we constructed the anti-HER2 “scissors CAR”. To analyze the target-cell dependent cleavage of “mCherry CAR”, 293T cells expressing the CARs were co-cultured with target-cells, including Raji (CD19+, HER2-) and engineered SK-BR-3 (CD19+, HER2+). Locations of mCherry were analyzed under the microscopy and by Western blotting. Next, to assess T-cell activation, we established Jurkat cells expressing both the anti-CD19 “effector CAR” and the anti-HER2 “scissors CAR”. These cells were co-cultured with the target-cells described above. T-cell activation was analyzed by both flowcytometric analysis and measurement of IL-2 mRNA expression. [Results] Transduced anti-CD19 “mCherry CAR” was detected as a membranous protein in 293T cell. Co-cultivation of 293T cell expressing both the anti-CD19 “mCherry CAR” and the anti-HER2 “scissors CAR” with Raji (CD19+, HER2-) did not change the localization of mCherry. However, engineered SK-BR-3 (CD19+, HER2+) induced cleavage of the recognition site and translocation of the mCherry from the membrane to cytoplasm. Furthermore, the anti-HER2 “scissors CAR” attenuated T-cell activation driven by the anti-CD19 “effector CAR” when Jurkat cells expressing both the CARs contacted with the target-cells expressing both CD19 and HER2. [Discussion] This novel protease-mediated CAR-T system recognized expression pattern of target molecules and regulated T-cell activity. Our CAR-T system would attenuate the adverse effects and contribute to expansion in application of CAR-T cell therapy. Citation Format: Satoru Aoyama, Shunichiro Yasuda, Daisuke Watanabe, Hiroki Akiyama, Keigo Okada, Yoshihiro Umezawa, Ayako Nogami, Osamu Miura, Norihiko Kawamata. A protease-mediated regulatory chimeric antigen receptor (CAR): “Scissors” CAR improves target-specificity through regulation of T-cell activity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2167.

Seeking high-priority mutations enabling successful antibody-breeding: systematic analysis of a mutant that gained over 100-fold enhanced affinity

“Antibody-breeding” has provided therapeutic/diagnostic antibody mutants with greater performance than native antibodies. Typically, random point mutations are introduced into the VH and VL domains of parent antibodies to generate diverse libraries of single-chain Fv fragments (scFvs), from which evolved mutants are selected. We produced an scFv against estradiol-17β with 11 amino acid substitutions and a >100-fold improved affinity constant (Ka = 1.19 × 1010 M−1) over the parent scFv, enabling immunoassays with >30-fold higher sensitivity. We systematically analyzed contributions of these substitutions to the affinity enhancement. Comparing various partial scFv revertants based on their Kas indicated that a revertant with four substitutions (VH-L100gQ, VL-I29V, -L36M, -S77G) exhibited somewhat higher affinity (Ka = 1.46 × 1010 M−1). Finally, the VH-L100gQ substitution, occurring in VH complementarity-determining region (CDR) 3, was found to be the highest-priority for improving the affinity, and VL-I29V and/or VL-L36M cooperated significantly. These findings encouraged us to reconsider the potential of VH-CDR3-targeting mutagenesis, which has been frequently attempted. The substitution(s) wherein might enable a “high rate of return” in terms of selecting mutants with dramatically enhanced affinities. The “high risk” of generating a tremendous excess of “junk mutants” can be overcome with the efficient selection systems that we developed.

Impact of enzymatic reduction on bivalent bispecific antibody fragmentation and loss of product purity upon reoxidation.

Antibody disulfide bond (DSB) reduction during manufacturing processes is a widely observed phenomenon attributed to host cell reductases present in harvest cell culture fluid. Enzyme-induced antibody reduction leads to product fragments and aggregates that increase the impurity burden on the purification process. The impact of reduction on bivalent bispecific antibodies (BisAbs), which are increasingly entering the clinic, has yet to be investigated. We focused on the reduction and reoxidation properties of a homologous library of bivalent BisAb formats that possess additional single-chain Fv (scFv) fragments with engineered DSBs. Despite all BisAbs having similar susceptibilities to enzymatic reduction, fragmentation pathways were dependent on the scFv-fusion site. Reduced molecules were allowed to reoxidize with and without low pH viral inactivation treatment. Both reoxidation studies demonstrated that multiple, complex BisAb species formed as a result of DSB mispairing. Furthermore, aggregate levels increased for all molecules when no low pH treatment was applied. Combined, our results show that complex DSB mispairing occurs during downstream processes while aggregate formation is dependent on sample treatment. These results are applicable to other novel monoclonal antibody-like formats containing engineered DSBs, thus highlighting the need to prevent reduction of novel protein therapeutics to avoid diminished product quality during manufacturing.

A Novel Protease-Mediated Chimeric Antigen Receptor (CAR): “Double-Arm” CAR-T Cell System Improves Target Specificity of CAR-T Cell Therapy

【 Introduction 】 CAR-T cell therapy is an attractive methodology in the field of cancer immunotherapy. Anti-CD19 CAR is used for treating refractory B cell malignancies and shows excellent therapeutic effects. However, there are several disadvantages to be overcome in this therapy. "On-target / off-tumor" effect is one of such adverse effects. Current CAR-T cell therapy targets single cell-surface molecule, which leads to damage of normal cells expressing the target protein. Improvement of target-cell-specificity is one of important issues in CAR-T cell therapy to avoid this serious adverse effect. Here, we show the protease-mediated "Double-Arm" CAR-T cell system (Figure), which improved the specificity of CAR-T cell therapy by recognizing two distinct cell-surface proteins. We designed two types of CAR: "Effector CAR" and "Scissors CAR" (Figure, left panel). The "effector CAR" is constituted of a single chain Fv fragment (scFv) targeting a cell-surface protein (protein X) on tumor cells, Human Immunodeficiency Virus protease (HIVPR) recognition polypeptide sequence, and a functional domain of CD3-zeta. The "scissors CAR" is constituted of a recognition portion targeting another protein (protein Y) and HIVPR. The HIVPR induces cleavage of the recognition polypeptide sequence in the effector CAR leading to inactivation of the effector CAR when the CAR-T cells contact with cells expressing both protein X and Y (Figure, right panel). 【 Material and Methods 】 For proof of principle, we first constructed "anti-CD19 mCherry CAR" harboring mCherry fluorescence protein in the cytoplasmic region under the HIVPR recognition polypeptide sequence. Also, we constructed "anti-CD19 scissors CAR" and "anti-HER2 scissors CAR". To analyze the target-cell-dependent cleavage of mCherry CAR, 293T cells expressing these CARs were co-cultured with target cells, including K562 (CD19-, HER2-), Raji (CD19+, HER2+), or SK-BR-3 (CD19-, HER2+). To obtain target cells expressing both CD19 and HER2, Raji and SK-BR-3 cells were molecularly manipulated. (1) To evaluate efficiency of this system, after co-cultivation of CAR-transduced 293T cell and target cells (K562, Raji, SK-BR-3), the localization of mCherry was examined under the microscopy and Western blotting. (2) To assess the T cell activation, we constructed "anti-CD19 effector CAR" and established Jurkat cells expressing both the "effector CAR" and the "anti-HER2 scissors CAR". These cells were co-cultured with wild type or engineered Raji or SK-BR-3 cells. T cell activation was analyzed with flowcytometric analysis and IL-2 mRNA expression measured with qRT-PCR. 【 Results 】 (1) Transduced "anti-CD19 mCherry CAR" was detected as a membrane-bound protein in 293T cell. Co-cultivation of 293T cell expressing both the "anti-CD19 mCherry CAR" and "anti-HER2 scissors CAR" with engineered Raji cells expressing both CD19 and HER2 induced cleavage of the recognition site and translocation of the mCherry from the membrane to cytoplasm. In addition, the cleavage was inhibited by a HIVPR inhibitor, Saquinavir. These results suggested that this novel system would regulate CAR-T cell activities through HIVPR-mediated cleavage of the "effector CAR" in vitro. (2) Jurkat cells expressing "anti-CD19 effector CAR" were activated through the target-cell-dependent manner. In addition, "anti-HER2 scissors CAR" attenuated T cell activation driven by "anti-CD19 effector CAR" when Jurkat cells expressing both the "anti-CD19 effector CAR" and the "anti-HER2 scissors CAR" contacted with the target cells expressing both CD19 and HER2. 【Discussion】 This is a novel protease-mediated controllable CAR system. The "scissors CAR" regulated activity of CAR-T cells depending on expression pattern of target molecules on the target cells. Our "Double-Arm" CAR-T cell system (Figure) would improve target specificity. It would attenuate the adverse effects and contribute to expansion in application of CAR-T cell therapy other than B cell malignancies. Figure Disclosures No relevant conflicts of interest to declare.

Biophysical characterization and single-chain Fv construction of a neutralizing antibody to measles virus.

The measles virus (MV) is a major cause of childhood morbidity and mortality worldwide. We previously established a mouse monoclonal antibody, 2F4, which shows high neutralizing titers against eight different genotypes of MV. However, the molecular basis for the neutralizing activity of the 2F4 antibody remains incompletely understood. Here, we have evaluated the binding characteristics of a Fab fragment of the 2F4 antibody. Using the MV infectious assay, we demonstrated that 2F4 Fab inhibits viral entry via either of two cellular receptors, SLAM and Nectin4. Surface plasmon resonance (SPR) analysis of recombinant proteins indicated that 2F4 Fab interacts with MV hemagglutinin (MV-H) with a KD value at the nm level. Furthermore, we designed a single-chain Fv fragment of 2F4 antibody as another potential biopharmaceutical to target measles. The stable 2F4 scFv was successfully prepared by the refolding method and shown to interact with MV-H at the μm level. Like 2F4 Fab, scFv inhibited receptor binding and viral entry. This indicates that 2F4 mAb uses the receptor-binding site and/or a neighboring region as an epitope with high affinity. These results provide insight into the neutralizing activity and potential therapeutic use of antibody fragments for MV infection.

PEGylated immunoliposome-loaded endoglin single-chain antibody enhances anti-tumor capacity of porcine α1,3GT gene

Tumor could not be completely removed due to the absence of immune storm against tumor. The porcine α1,3 galactosyltransferase (α1,3 GT) induce the hyperacute rejection by synthesizing Galα1-3Galβ1-(3)4GlcNAc-R (αGal) on the surface of graft endothelial cells (ECs) during xeno-transplantation. This study aimed to develop anti-endoglin single-chain Fv fragments (ENG-scFv) conjugated PEGylated immunoliposomes (iLPs) to induce immune storm against tumor. Immune fluorescence was performed to detect the binding of ENG-scFv to human ENG, the endosomal/lysosomal escape of ENG-scFv-iLPs/α1,3 GT, and αGal expression in hENG-HEK293 cells. In vitro MTT assay was performed to measure ENG-scFv-iLPs/α1,3 GT cytotoxicity. NOD/SCID mouse born A549 tumor model was used to evaluate the therapeutic potency of ENG-scFv-iLPs/α1,3 GT. ENG-scFv-iLPs enabled efficient targeting delivery of α1,3 GT plasmid to ENG + tumors neovascular endothelial cells (TnECs), promoted endosomal/lysosomal escape due to the pH-sensitive ability, then synthesized carbohydrate epitope αGal on the surface of these cells to achieve the purpose of destroying the tumor. The mechanism of uptake for nanoparticles was energy driven, the clathrin-mediated endocytosis was the main endocytic pathway of the ENG-mAb-iLPs/α1,3 GT and lipid-raft-mediated of the ENG-scFv-iLPs/α1,3 GT, and macropinocytosis was also involved in intracellular entry. The inhibition of tumor angiogenesis and proliferation by ENG-scFv-iLPs/α1,3 GT was closely related to down-regulation of VEGF. Our findings establish an alternative therapeutic paradigm for scFv-conjugated nanoparticles to induce tumor cell apoptosis and inhibit tumor growth early. Such iLPs nanocarrier could efficiently release α1,3 GT to their distinct sites of action, where the endoglin + tumor neovascular endothelial cells (ENG + TnECs) exist, in a site-specific manner. Therefore, we believe that these scFv-targeted core-shell immunocomplexes are an important potential α1,3 GT delivery system for various solid tumor-targeted therapy.

An Antibody Fab Fragment-based Chimeric Antigen Receptor Could Efficiently Eliminate Human Thyroid Cancer Cells.

Thyroid cancer remains a significant health problem worldwide. Traditional chemotherapy does generate long-term benefit but are usually accompanied by severe side effects. Immunotherapy by adoptive infusion of T cells is now an attractive alternative to chemotherapy. Chimeric antigen receptor engineered lymphocytes have produced tremendous clinical outcomes in treating leukemia or lymphoma, but not in solid tumors, which is in part due to the low affinity of single chain Fv fragment or the rapid loss of transfused T cells. In present research, we designed a novel Fab based chimeric antigen receptor, which inherits the advantages of Fab fragment as well as the natural TCR receptor. The novel Fab CAR could recognize the tumor antigens independent of MHC/peptide complex, and mimic the natural activation process of endogenous TCR, therefore extend the life span of CAR-engineered T cells and generate durable clinical effects.

Induced Pluripotent Stem Cell-Derived NK Cells Genetically Modified to Express NKG2C/DAP12 Mediate Potent Function When Targeted through an NKG2C/IL-15/CD33 Tri-Specific Killer Engager (TriKE)

Adoptive transfer of haplo-donor NK cells can achieve remission in patients with relapsed or refractory acute myeloid leukemia (AML). This strategy is limited by NK cell heterogeneity, lack of specificity, and single dosed strategies. To overcome these barriers, we genetically modified induced pluripotent stem cells (iPSCs) to stably over-express NKG2C alone or along with DAP12. CD94/NKG2C is a heterodimeric receptor that binds to HLA-E and associates with DAP12, a protein containing an immunoreceptor tyrosine-based activating motif. Efficient expression of CD94/NKG2C on the cell surface requires the presence of DAP12, and charged amino acids in the transmembrane domains of DAP12 and NKG2C mediate this interaction. Physiologic NKG2C expression is limited to subsets of NK and CD8+ T cells that expand in response to cytomegalovirus (CMV) infection.Engineered iPSCs were differentiated into CD34+ hematopoietic progenitor cells and subsequently differentiated into CD56+ NK cells (iNK cells) using a stepwise differentiation and expansion strategy. Surface NKG2C expression was low on unmodified iNK cells (10.7%) with higher levels of NKG2C on the surface of iNK cells derived from the NKG2C only line (65.1%). These results demonstrate that transgenic NKG2C can pair with endogenous CD94 and DAP12 to form stable structures on the cell surface. Importantly, surface NKG2C expression was highest on iNK cells derived from the NKG2C/DAP12 line (84.1%) with higher MFI, demonstrating the importance of engineering a line with both components (Figure 1A). To validate function via engagement of NKG2C on iNK cells, we used a redirected antibody dependent cellular cytotoxicity (ADCC) assay where P815 cells (which bind the Fc portion of antibodies) were coated with a monoclonal anti-NKG2C agonist antibody at a range of concentrations (0.01-10 mg/ml) and co-cultured with unmodified iNK cells, NKG2C iNK cells or NKG2C/DAP12 iNK cells. We found that at all concentrations tested, NKG2C/DAP12 iNK cells demonstrated the strongest functional response as measured by the frequencies of surface CD107a expression, interferon (IFN)-g production and tumor necrosis factor (TNF) production. Intermediate functional responses were observed for NKG2C iNK cells (Figure 1B).Having demonstrated stable expression and intact signaling of NKG2C/DAP12 in iNK cells, we next created a Tri-specific Killer Engager (TriKE) molecule with single chain Fv fragments specific for NKG2C (on iNK cells) and CD33 (on AML cells), as well as IL-15 (to support iNK cell survival and proliferation). This NKG2C/CD33/IL-15 TriKE was tested in functional assays where NKG2C iNK cells or NKG2C/DAP12 iNK cells were used as effectors against CD33+ THP1 cells (an AML cell line shown to be refractory to NK cell cytotoxicity). Without the addition of NKG2C/CD33/IL-15 TriKE, the frequencies of both NKG2C iNK cells and NKG2C/DAP12 iNK cells producing IFN-g was relatively low (< 10%). Addition of NKG2C/IL-15/CD33 TriKE in the assay markedly increased the frequency of IFN-g production by both NKG2C iNK cells (19.1%) and NKG2C/DAP12 iNK cells (25.8%) (Figure 1C). Finally, we directly tested the ability of the NKG2C/IL-15/CD33 TriKE molecule to trigger target killing by engineered iNK cells monitored over a 24-hour period. THP1 target cells were dye-labeled and plated in wells with either NKG2C iNK cells or NKG2C/DAP12 iNK cells at a 5:1 effector-to-target ratio with or without NKG2C/CD33/IL-15 TriKE. We found that in the absence of TriKE, there was no killing of THP1 targets. In contrast, both NKG2C iNK cells and NKG2C/DAP12 iNK cells mediated robust THP1 killing with the addition of NKG2C/CD33/IL-15 TriKE, with NKG2C/DAP12 iNK cells exhibiting the strongest response (Figure 1D). Our results provide a proof-of-concept that iNK cells engineered to express NKG2C/DAP12 can be used in combination with a novel NKG2C/IL-15/CD33 TriKE molecule to effectively target cancer cells. Engaging NK cells through NKG2C, restricted to adaptive NK cells or genetically modified iPSC will be more specific than CD16, which will bind to CD16A on NK cells but also have off-target binding to CD16B on neutrophils. Because of the enormous expansion and engineering capacity of the iNK cell platform, we are in a unique position to create an “off-the-shelf” NK cellular therapy that is targeted, specific and efficacious. DisclosuresCichocki:Fate Therapeutics Inc.: Consultancy, Research Funding. Felices:GT Biopharma: Research Funding. Chu:Fate Therapeutics Inc.: Employment. Ge:Fate Therapeutics Inc.: Employment. Bjordahl:Fate Therapeutics Inc.: Employment. Kaufman:Fate Therapeutics: Consultancy, Research Funding. Malmberg:Fate Therapeutics Inc.: Consultancy, Research Funding. Valamehr:Fate Therapeutics Inc.: Employment.

Comparison of rabies virus protection by single chain and leucine zipper Fv fragments cocktail derived from a monoclonal antibody cocktail

Monoclonal antibodies (MAbs) are a unique and attractive class of biologics and are potential substitutes for post-exposure rabies prophylaxis. The safety, tolerance, and broad neutralization efficiency of a MAb cocktail called CL184, composed of the antibodies CR4098 and CR57, was confirmed in a phase I clinical trial. We have prepared a series of single-chain Fv fragments (scFvs) and leucine zipper Fv fragments (zipFvs) from CR57 and CR4098. In this study, we selected and formed scFv and zipFv cocktails and compared their protective effects against the rabies virus. Mice and hamster challenge models demonstrated the improved protection of the zipFv cocktail compared with scFv cocktail, because of its stronger affinity. The results indicate that zipFv production is a promising novel method for the genetic engineering of antibody fragments and improving affinity through systematic screening may be important when designing small molecule antibodies against RV.

Antibodies and Engineered Antibody Fragments against M13 Filamentous Phage to Facilitate Phage-Display-Based Molecular Breeding.

Antibodies are essential for characterizing various analytes. "Molecular-breeding" approaches enable rapid generation of antibody mutants with desirable antigen-binding abilities. Typically, prototype antibodies are converted to single-chain Fv fragments (scFvs), and random mutations are genetically introduced to construct molecular libraries with a vast diversity. Improved species therein are then isolated via phage display genotype-phenotype-connecting systems to separate them from a large excess of nonspecific scFvs. During these experiments, counting of phage particles is routinely performed. However, current methods depend on the time-consuming overnight cultivation of phage-infected bacteria on agar plates to estimate phage numbers as plaque-forming units (pfu) or colony-forming units, the results of which fluctuate considerably. Immunochemical systems capturing phage particles should be a more convenient and robust alternative. We therefore generated monoclonal antibodies against M13 filamentous phage, which is commonly used for phage display, by employing hybridoma technology. Combinatorial use of two such antibodies (Ab-M13#53 and #71; both specific to the major coat protein pVIII) enabled development of a sandwich enzyme-linked immunosorbent assay (ELISA) that could measure ca. 107-1010 phage pfu/mL. To construct a more convenient system, Ab-M13#71 was converted to the scFv form and further fused with an alkaline phosphatase variant. Using this fusion protein, the sandwich ELISA enabled rapid (within 90 min) and reliable phage counting without reducing the sensitivity, and the results were reasonably consistent with those of infection-based methods. The present anti-phage antibodies and scFvs might also enable visualization of individual phage particles by combining them with sensitive fluorescent staining.

A fusion-protein approach enabling mammalian cell production of tumor targeting protein domains for therapeutic development.

A single chain Fv fragment (scFv) is a fusion of the variable regions of heavy (VH ) and light (VL ) chains of immunoglobulins. They are important elements of chimeric antigen receptors for cancer therapy. We sought to produce a panel of 16 extracellular protein domains of tumor markers for use in scFv yeast library screenings. A series of vectors comprising various combinations of expression elements was made, but expression was unpredictable and more than half of the protein domains could not be produced using any of the constructs. Here we describe a novel fusion expression system based on mouse TEM7 (tumor endothelial marker 7), which could facilitate protein expression. With this approach we could produce all but one of the tumor marker domains that could not otherwise be expressed. In addition, we demonstrated that the tumor associated antigen hFZD10 produced as a fusion protein with mTEM7 could be used to enrich scFv antibodies from a yeast display library. Collectively our study demonstrates the potential of specific fusion proteins based on mTEM7 in enabling mammalian cell production of tumor targeting protein domains for therapeutic development.

Selective CD28 Inhibition Modulates Alloimmunity and Cardiac Allograft Vasculopathy in Anti-CD154-Treated Monkeys.

Selective CD28 inhibition is actively pursued as an alternative to B7 blockade using cytotoxic T lymphocyte antigen 4 Ig based on the hypothesis that the checkpoint immune regulators cytotoxic T lymphocyte antigen 4 and programmed death ligand 1 will induce tolerogenic immune signals. We previously showed that blocking CD28 using a monovalent nonactivating reagent (single-chain anti-CD28 Fv fragment linked to alpha-1 antitrypsin [sc28AT]) synergizes with calcineurin inhibitors in nonhuman primate (NHP) kidney and heart transplantation. Here, we explored the efficacy of combining a 3-week "induction" sc28AT treatment with prolonged CD154 blockade. Cynomolgus monkey heterotopic cardiac allograft recipients received sc28AT (10 mg/kg, d0-20, n = 3), hu5C8 (10-30 mg/kg, d0-84, n = 4), or combination (n = 6). Graft survival was monitored by telemetry. Protocol biopsies and graft explants were analyzed for International Society of Heart and Lung Transplantation acute rejection grade and cardiac allograft vasculopathy score. Alloantibody, T-cell phenotype and regulatory T cells were analyzed by flow cytometry. Immunochemistry and gene expression (NanoString) characterized intra-graft cellular infiltration. Relative to modest prolongation of median graft survival time with sc28AT alone (34 days), hu5C8 (133 days), and sc28AT + hu5C8 (141 days) prolonged survival to a similar extent. CD28 blockade at induction, added to hu5C8, significantly attenuated the severity of acute rejection and cardiac allograft vasculopathy during the first 3 months after transplantation relative to hu5C8 alone. These findings were associated with decreased proportions of circulating CD8 and CD3CD28 T cells, and modulation of inflammatory gene expression within allografts. Induction with sc28AT promotes early cardiac allograft protection in hu5C8-treated NHPs. These results support further investigation of prolonged selective CD28 inhibition with CD40/CD154 blockade in NHP transplants.

Rapid Selection of High-Affinity Antibody scFv Fragments Using Ribosome Display.

Ribosome display has proven to be a powerful in vitro selection and evolution method for generating high-affinity binders from libraries of folded proteins. It works entirely in vitro, and this has two important consequences. First, since no transformation of any cells is required, libraries with much greater diversity can be handled than with most other techniques. Second, since a library does not have to be cloned and transformed, it is very convenient to introduce random errors in the library by PCR-based methods and select improved binders. Thus, a true directed evolution, an iteration between randomization and selection over several generations, can be conveniently carried out, e.g., for affinity maturation, either on a given clone or on the whole library. Ribosome display has been successfully applied to antibody single-chain Fv fragments (scFv), which can be selected not only for specificity but also for stability and catalytic activity. High-affinity binders with new target specificity can be obtained from highly diverse libraries in only a few selection rounds. In this protocol, the selection from the library and the process of affinity maturation and off-rate selection are explained in detail.

Application of chimeric antigen receptor-engineered T cells in ovarian cancer therapy.

Due to the critical role of T cells in the immune surveillance of ovarian cancer, adoptive T-cell therapies are receiving increased attention as an immunotherapeutic approach for ovarian cancer. Chimeric antigen receptors (CARs), constructed by incorporating the single-chain Fv fragment to a T-cell signaling domain such as CD3 ζ or Fc receptor γ chain, endow T cell with nonmajor histocompatibility complex-restricted specificity. Dual specificity, trans-signaling CARs and affinity-tuned single-chain Fv fragment have broadened the applicability of CAR-engineered T-cell therapy and may be considered preferential to T cell receptor T-cell therapy in clinical care. As new insights into the CAR-engineered T cells have emerged over the last decade, we review the development of CAR T-cell therapy and discuss the progress and safety concerns regarding its translation from basic research into clinical care of ovarian cancer.

Selection of novel affinity-matured human chondroitin sulfate proteoglycan 4 antibody fragments by yeast display.

Chondroitin sulfate proteoglycan 4 (CSPG4) is a promising target for cancer immunotherapy due to its high level of expression in a number of malignant tumors, and its essential role in tumor growth and progression. Clinical application of CSPG4-targeting immunotherapies is hampered by the lack of fully human high-affinity CSPG4 antibodies or antibody fragments. To overcome this limitation, we performed affinity maturation on a novel human CSPG4 single-chain Fv fragment (scFv) using the random mutagenesis approach and screened for improved variants from a yeast display library using a modified whole-cell panning method followed by fluorescence-activated cell sorting. After six rounds of panning and sorting, the top seven mutant scFvs were isolated and their binding affinities were characterized by flow cytometry and surface plasmon resonance. These highly specific, affinity-matured variants displayed nanomolar to picomolar binding affinities to the CSPG4 antigen. While each of the mutants harbored only two to six amino acid substitutions, they represented ~270-3000-fold improvement in affinity compared to the parental clone. Our study has generated affinity-matured scFvs for the development of antibody-based clinical therapeutics targeting CSPG4-expressing tumors.

Single-step colony assay for screening antibody libraries

We describe a method, single-step colony assay, for simple and rapid screening of single-chain Fv fragment (scFv) libraries. Colonies of Escherichia coli expressing the scFv library are formed on a hydrophilic filter that is positioned in contact with a membrane coated with an antigen. scFv expression is triggered upon treatment of colonies with an induction reagent, following which scFvs are secreted from the cells and diffused to the antigen-coated membrane. scFvs that exhibit binding affinity for the antigen are captured by the membrane-immobilized antigen. Lastly, detection of scFv binding of the antigen on the membrane allows identification of the clones on the filter that express antigen-specific scFvs. We tested this methodology by using an anti-rabbit IgG scFv, scFv(A10B), and a rat immune scFv library. Experiments conducted using scFv(A10B) revealed that this method improves scFv expression during the colony assay. By using our method to screen an immune library of 3×103 scFv clones, we established several clones exhibiting affinity for the antigen. Moreover, we tested 7 other antigens, including peptides, and successfully identified positive clones. We believe that this simple procedure and controlled scFv expression of the single-step colony assay could make the antibody screening both rapid and reliable and lead to successful isolation of positive clones from antibody libraries.

Engineering of a novel zipFv using leucine zipper motif against rabies virus glycoprotein G with improved protection potency in vivo

Rabies is an acute zoonotic infectious disease with a high fatality rate but is preventable with vaccination and rabies immunoglobulin (RIG). The single-chain Fv fragment (scFv), a small engineered antigen-binding protein derived from antibody variable heavy (VH) and light (VL) chains connected by a peptide linker, can potentially be used to replace RIG. Here, we produced two peptides VH-JUN-HIS and VL-FOS-HA separately in Escherichia coli and assembled them to form zipFv successfully in vitro. The new zipFv utilizes FOS and JUN leucine zippers to form an antibody structure similar to the IgG counterpart with two free N-terminal ends of VH and VL. The zipFv protein showed notable improvement in binding ability and affinity over its corresponding scFv. The zipFv also demonstrated greater stability in serum and the same protective rate as RIG against challenge with a standard rabies virus (CVS-24) in mice. Our results indicated zipFv as a novel and efficient antibody form with enhanced neutralizing potency.

Enhanced humoral and CD8 + T cell immunity in mice vaccinated by DNA vaccine against human respiratory syncytial virus through targeting the encoded F protein to dendritic cells

Human respiratory syncytial virus (RSV) is the most important cause of serious lower respiratory tract infection in infants, the elderly, and the immunocompromised population. There is no licensed vaccine against RSV until now. It has been reported that targeting antigen to DEC205, a phagocytosis receptor on dendritic cells (DCs), could induce enhanced CD4+ and CD8+ T cell responses in mice. To develop RSV DNA vaccine and target the encoded antigen protein to DCs, the ectodomain of fusion glycoprotein (sF, amino acids: 23–524) of RSV was fused with anti-DEC205 single-chain Fv fragment (scDEC) and designated scDECF. Following successful expression from the recombinant plasmid of pVAX1/scDECF, the recombinant protein of scDECF was found capable of specifically binding to DEC205 receptor on CHOmDEC205 cells, and facilitating uptake of RSV F by DC2.4 cells in vitro. Furthermore, the higher levels of RSV-specific IgG antibody responses and neutralization antibody titers, as well as RSV F-specific CD8+ T cell responses were induced in mice immunized intramuscularly by pVAX1/scDECF than by the control plasmid of pVAX1/scISOF encoding sF protein fused with isotype matched control single-chain Fv fragment (scISO). Compared with pVAX1/scISOF, both the ratio of IgG2a/IgG1, >1, and the enhanced IFN-γ cytokine were induced in mice following pVAX1/scDECF immunization, which exhibited a Th1 dominant response in pVAX1/scDECF vaccinated mice. Notably, the elevated efficiency of RSV F protein bound by DCs in vivo could also be observed in mice inoculated by pVAX1/scDECF. Collectively, these results demonstrate the enhanced IgG and CD8+ T cell immune responses have been induced successfully by DNA vaccine against RSV by targeting F antigen to DCs via the DEC205 receptor, and this DC-targeting vaccine strategy merits further investigation.

Immunoprotein-Mediated siRNA Delivery.

RNA interference strategies offer an alternative to small molecular drug targeting. Small interfering RNA (siRNA) constitutes a class of molecules that allows the effective and specific inhibition of the biosynthesis of any protein. Indeed, siRNA have emerged as a major tool in molecular biology techniques and an important approach to identify suitable therapy targets in cancer. However, siRNA therapy approaches in vivo are scarce. Two major problems hinder siRNA as a therapeutic tool: (1) delivery through the bloodstream leads to degradation or rapid renal clearance (stabilization) and (2) specific uptake by the desired cell type (specificity). This review summarizes the ongoing attempts to use RNAi against disease-causing factors. We compare methods to stabilize siRNA in different conjugates and that decorate these complexes with targeting molecules such as antibodies, single-chain Fv or Fab fragments, to enable specific uptake of the carriers by the respective cells. We propose the development of antibody-coupled siRNA complexes, which have shown to allow stabilization as well as targeted uptake of siRNA to cancer cells.