Tandem Single-chain Variable Fragment Research Articles

A trispecific antibody induces potent tumor-directed T-cell activation and antitumor activity by CD3/CD28 co-engagement.

Aim: A novel CD19xCD3xCD28 trispecific antibody with a tandem single-chain variable fragments (scFv) structure was developed for the treatment of B-cell malignancies. Methods: The trispecific antibody in inducing tumor-directed T-cell activation and cytotoxicity was evaluated in vitro and in vivo and compared with its bispecific counterpartBiTE-CD19xCD3lacking aCD28-targeting domain. Results: The trispecific antibody with aco-stimulatory domain exhibited augmented T-cell activation and memory T-cell differentiation capability and itinduced faster tumor cell lysis than the bispecific antibody. RNAseq analysis revealed that the trispecific antibody modulates CD3/TCR complex-derived signal and upregulates antiapoptotic factors to influence thesurvival of Tcells. Conclusion: By CD3/CD28 co-engagement, the trispecific antibody demonstrated its advantages in T-cell immunity and potential use as a more powerful and long-lasting T-cell engager.

Bispecific T-Cell Engaging Antibodies Against MUC16 Demonstrate Efficacy Against Ovarian Cancer in Monotherapy and in Combination With PD-1 and VEGF Inhibition.

Immunotherapy for ovarian cancer is an area of intense investigation since the majority of women with relapsed disease develop resistance to conventional cytotoxic therapy. The paucity of safe and validated target antigens has limited the development of clinically relevant antibody-based immunotherapeutics for this disease. Although MUC16 expression is almost universal in High Grade Serous Ovarian Cancers, engagement of the shed circulating MUC16 antigen (CA-125) presents a theoretical risk of systemic activation and toxicity. We designed and evaluated a series of bispecific tandem single-chain variable fragments specific to the retained portion of human MUC16 ectodomain (MUC16ecto) and human CD3. These MUC16ecto- BiTEDs retain binding in the presence of soluble MUC16 (CA-125) and show cytotoxicity against a panel of ovarian cancer cells in vitro. MUC16ecto- BiTEDs delay tumor progression in vivo and significantly prolong survival in a xenograft model of ovarian peritoneal carcinomatosis. This effect was significantly enhanced by antiangiogenic (anti-VEGF) therapy and immune checkpoint inhibition (anti-PD1). However, the combination of BiTEDs with anti-VEGF was superior to combination with anti-PD1, based on findings of decreased peritoneal tumor burden and ascites with the former. This study shows the feasibility and efficacy of MUC16ecto- specific BiTEDs and provides a basis for the combination with anti-VEGF therapy for ovarian cancer.

A modular and controllable T cell therapy platform for acute myeloid leukemia

Targeted T cell therapy is highly effective in disease settings where tumor antigens are uniformly expressed on malignant cells and where off-tumor on-target-associated toxicity is manageable. Although acute myeloid leukemia (AML) has in principle been shown to be a T cell-sensitive disease by the graft-versus-leukemia activity of allogeneic stem cell transplantation, T cell therapy has so far failed in this setting. This is largely due to the lack of target structures both sufficiently selective and uniformly expressed on AML, causing unacceptable myeloid cell toxicity. To address this, we developed a modular and controllable MHC-unrestricted adoptive T cell therapy platform tailored to AML. This platform combines synthetic agonistic receptor (SAR) -transduced T cells with AML-targeting tandem single chain variable fragment (scFv) constructs. Construct exchange allows SAR T cells to be redirected toward alternative targets, a process enabled by the short half-life and controllability of these antibody fragments. Combining SAR-transduced T cells with the scFv constructs resulted in selective killing of CD33+ and CD123+ AML cell lines, as well as of patient-derived AML blasts. Durable responses and persistence of SAR-transduced T cells could also be demonstrated in AML xenograft models. Together these results warrant further translation of this novel platform for AML treatment.

Designing a new bispecific tandem single-chain variable fragment antibody against tumor necrosis factor-α and interleukin-23 using in silico studies for the treatment of rheumatoid arthritis.

Rheumatoid arthritis disease is a chronic auto-immune inflammatory disease that mainly causes synovial joint inflammation and cartilage destruction. The tumor necrosis factor-α (TNF-α) is a pivotal cytokine that plays an important role in rheumatoid arthritis. The treatments focusing on a single cytokine inhibition are clinically able to produce meaningful responses in only about half of the treated patients due to multiple cytokines involved in this disease. In the present study, a bispecific tandem single-chain variable fragment was designed in order to suppress both human tumor necrosis factor-α and interleukin-23 (IL23) as a potential therapeutic drug candidate for this disease. To do so, at first, eight bispecific tandem single-chain variable fragment models were built against tumor necrosis factor-α and interleukin-23 cytokines with different domain orders by the homology modeling, and then 50ns molecular dynamics simulation was performed for each one and then structural properties were exploited. The MD simulation results indicate the fact that the domains' order strongly affects tandem single-chain variable fragment properties, and in overall, the fragment VLAIL23+Linker+VHAIL23+linker+VLATNF+Linker +VHATNF +His6 (VL and VH are light and heavy chain variable fragments and AIL23 and ATNF are anti-interleukin 23 and anti-tumor necrosis factor-α, respectively, and His6 is the six histidine) not only separated antibody domains accurately but also had better stability and solvation free energy. Therefore, this structure can be considered as an effective potential drug for rheumatoid arthritis. It is expected that the findings of this research could shed a light on the treatment approaches of the rheumatoid arthritis disease.

Abstract PR12: Assessing in vivo mechanisms regulating the therapeutic activity of bi-specific T-cell engager (BiTE®) molecules in immunocompetent mice expressing a chimeric human/mouse CD3ϵ receptor

Abstract The development and success of Blincyto®, a Bi-Specific T cell Engager (BiTE®), to treat acute lymphoblastic leukemia has expanded the class of anticancer immunotherapy agents. BiTE® molecules consist of a single-chain Fc antibody containing tandem single-chain variable fragments (scFv) recognizing the CD3 receptor on T lymphocytes and a tumor-associated antigen and induce redirected T-cell cytotoxicity and tumor cell lysis. Despite the clinically validated efficacy of BiTE® molecules, there has been little elucidation of the parameters governing their in vivo activity. This is due, in part, to a lack of immunocompetent murine tumor models that allow for engagement of endogenous T cells in the context of an intact immune system, which more accurately mimic the human therapeutic setting. We have developed a genetically engineered mouse model (huCD3 mouse) in which a human/mouse chimeric CD3ϵ receptor recognized by BiTE® molecules was engineered into the mouse CD3ϵ genomic locus. Here, we characterize the immune cell distribution of the huCD3 mouse and utilize BiTE® molecules and syngeneic tumor models to elucidate pharmacokinetic, pharmacodynamic, and efficacy relationships in vivo. The peripheral B and T cell distribution profile of huCD3 mice and WT littermates are comparable, but T cells isolated from huCD3 mice express lower levels of. In T cell-dependent cytotoxic (TDCC) assays, an anti-huEpCAM BiTE® molecule exhibited similar potency against MC38 mouse cells expressing human EpCAM co-cultured with T cells from the huCD3 mice or a human donor, if mouse T-cells were preactivated in vitro. In vivo, MC38-huEpCAM tumors grew robustly and anti-huEpCAM BiTEÂ treatment resulted in dose-dependent tumor growth inhibition, with several tumor-free animals at higher doses. Flow cytometry analysis of disaggregated tumors and serum cytokine analysis indicated that BiTE® treatment resulted in T-cell activation and a cytokine response at doses that correlate with tumor regression. In vivo studies were also performed using B16F10-huEPCAM cells, which generate tumors with reduced T-cell infiltration compared to MC38-huEpCAM. Additionally, we used WT mice reconstituted with different ratios of huCD3e and WT bone marrow to investigate the T-cell number requirement for optimal BiTE®-mediated efficacy. We also evaluated the efficacy of an anti-mouse CD19 BiTE® molecule against MC38-CD19 tumor cells to compare the requirements for antitumor efficacy versus normal B-cell depletion in the blood and in lymphoid organs. Finally, we performed a rational combination study using BiTE® molecules and a PD1 inhibitor, demonstrating that this combination can lead to the eradication of poorly T cell-infiltrated solid tumors. These data begin to elucidate the in vivo mechanisms regulating the efficacy of BiTE® molecules under physiologically relevant conditions. This abstract is also being presented as Poster A29. Citation Format: Brian Belmontes, Hong Tan, Wendy Zhong, Danny Chui, Kevin Cook, Sarah O'Brien, Melissa Martin, Brandy Alexander, Jason Eng, John Harrold, Famke Aeffner, Matthew Chun, Alice Bakker, Mercedesz Balazs, Jason DeVoss, Angela Coxon, Jude Canon, Jackson Egen, Olivier Nolan-Stevaux. Assessing in vivo mechanisms regulating the therapeutic activity of bi-specific T-cell engager (BiTE®) molecules in immunocompetent mice expressing a chimeric human/mouse CD3ϵ receptor [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr PR12.

Selection for Anti-transferrin Receptor Bispecific T-cell Engager in Different Molecular Formats.

Selecting an ideal molecular format from diverse structures is a major challenge in developing a bispecific antibody (BsAb). To choose an ideal format of anti-CD3 × anti-transferrin receptor (TfR) bispecific antibodies for clinical application, we constructed TfR bispecific T-cell engager (BiTE) in two extensively applied formats, including single-chain tandem single-chain variable fragments (scFvs) and double-chain diabodies, and evaluated their functional characterizations in vitro. Results demonstrated that TfR-BiTE in both formats directed potent killing of TfR+ HepG2 cells. However, compared to two-chain diabodies, scFvs were more efficient in antigen binding and TfR+ target killing. Furthermore, different domain orders in scFvs would also be evaluated because single-TfR-CD3-His was preferable to single-CD3-TfR-His in immunotherapeutic strategies. Thus, the single-chain tandem TfR-CD3 format was favored for further investigation in cancer therapy.

Preclinical evaluation of AMG 160, a next-generation bispecific T cell engager (BiTE) targeting the prostate-specific membrane antigen PSMA for metastatic castration-resistant prostate cancer (mCRPC).

301 Background: mCRPC is a disease of high unmet medical need, especially for patients who fail novel hormonal therapies and chemotherapy. BiTE molecules provide an off the shelf therapy that activates a patient’s own immune system and redirects T cells to kill tumor cells. The BiTE mechanism of action is distinct from other immunotherapies and may unlock immune response in mCRPC. PSMA is a compelling BiTE target that is highly expressed on PCa compared to normal tissue and has increased expression in mCRPC. Methods: AMG 160 is a fully human, half-life extended (HLE) BiTE that targets PSMA on tumor cells and CD3 on T cells. AMG 160 comprises two tandem single chain variable fragments fused to an Fc domain. Results: AMG 160 binds human and non-human primate (NHP) PSMA and CD3, leading to T cell activation and proliferation and cytokine production. AMG 160 redirects T cells to kill PSMA-positive cancer cell lines in vitro, including those with low PSMA levels or androgen-independent signaling. Weekly dosing of AMG 160 induces significant antitumor activity in established PCa xenograft model. The pharmacokinetics (PK) and pharmacodynamics of AMG 160 were tested in NHP. AMG 160 treatment led to BiTE target engagement in vivo, including transient T cell activation and cytokine release in blood, and mixed cellular infiltrates in multiple organs known to express PSMA. AMG 160 treatment was well tolerated. Cytokine release associated with the first dose could be attenuated using a step dose regimen. The half-life of AMG 160 in NHP was about one week. Based on allometric scaling, the PK profile of AMG 160 may be projected to enable dosing every other week in humans. Conclusions: AMG 160 is a potent HLE BiTE with specificity for PSMA-positive tumor cells. A Phase 1 study is planned to evaluate the safety and efficacy of AMG 160 in patients with mCRPC.

Rational design of a trispecific antibody targeting the HIV-1 Env with elevated anti-viral activity

HIV-1 broadly neutralizing antibodies (bNAbs) are being explored as passively administered therapeutic and preventative agents. However, the extensively diversified HIV-1 envelope glycoproteins (Env) rapidly acquire mutations to evade individual bNAbs in monotherapy regimens. The use of a “single” agent to simultaneously target distinct Env epitopes is desirable to overcome viral diversity. Here, we report the use of tandem single-chain variable fragment (ScFv) domains of two bNAbs, specific for the CD4-binding site and V3 glycan patch, to form anti-HIV-1 bispecific ScFvs (Bi-ScFvs). The optimal Bi-ScFv crosslinks adjacent protomers within one HIV-1 Env spike and has greater neutralization breadth than its parental bNAbs. Furthermore, the combination of this Bi-ScFv with a third bNAb recognizing the Env membrane proximal external region (MPER) results in a trispecific bNAb, which has nearly pan-isolate neutralization breadth and high potency. Thus, multispecific antibodies combining functional moieties of bNAbs could achieve outstanding neutralization capacity with augmented avidity.

Large-Scale Purification of r28M: A Bispecific scFv Antibody Targeting Human Melanoma Produced in Transgenic Cattle.

Background30 years ago, the potential of bispecific antibodies to engage cytotoxic T cells for the lysis of cancer cells was discovered. Today a variety of bispecific antibodies against diverse cell surface structures have been developed, the majority of them produced in mammalian cell culture systems. Beside the r28M, described here, no such bispecific antibody is known to be expressed by transgenic livestock, although various biologicals for medical needs are already harvested—mostly from the milk—of these transgenics. In this study we investigated the large-scale purification and biological activity of the bispecific antibody r28M, expressed in the blood of transgenic cattle. This tandem single-chain variable fragment antibody is designed to target human CD28 and the melanoma/glioblastoma-associated cell surface chondroitin sulfate proteoglycan 4 (CSPG4).ResultsWith the described optimized purification protocol an average yield of 30 mg enriched r28M fraction out of 2 liters bovine plasma could be obtained. Separation of this enriched fraction by size exclusion chromatography into monomers, dimers and aggregates and further testing regarding the biological activity revealed the monomer fraction as being the most appropriate one to continue working with. The detailed characterization of the antibody’s activity confirmed its high specificity to induce the killing of CSPG4 positive cells. In addition, first insights into tumor cell death pathways mediated by r28M-activated peripheral blood mononuclear cells were gained. In consideration of possible applications in vivo we also tested the effect of the addition of different excipients to r28M.ConclusionSumming up, we managed to purify monomeric r28M from bovine plasma in a large-scale preparation and could prove that its biological activity is unaffected and still highly specific and thus, might be applicable for the treatment of melanoma.

Bispecific Her2 × cotinine antibody in combination with cotinine–(histidine)2–iodine for the pre-targeting of Her2-positive breast cancer xenografts

Cotinine has optimal characteristics as a hapten for pre-targeted radioimmunotherapy (PRIT). This study was performed to evaluate the applicability of cotinine/anti-cotinine antibody to PRIT. We developed and prepared a tandem, single-chain, variable fragment Fc fusion protein [tandem single-chain variable fragment (scFv) Fc fusion protein] that is reactive to both human epidermal growth factor receptor 2 (Her2) and cotinine. Its simultaneous reactivity to Her2 and cotinine was tested in an enzyme-linked immunosorbent assay (ELISA) and two radioimmunoassays (RIA) employing Her2-coated RIA tubes and a Her2-overexpressing cell line. For in vivo imaging, mice bearing Her2-positive tumors were injected with a mixture of tandem scFv Fc fusion and (125)I-cotinine-conjugated histidine dipeptide ((125)I-cotinine peptide). After a delay, (125)I-cotinine peptide was injected again. ELISA and RIA results showed that tandem scFv Fc fusion protein successfully bound to both Her2 and cotinine. In single-photon emission computed tomography (SPECT), the complex of tandem scFv Fc fusion protein and (125)I-cotinine peptide was localized to Her2-positive tumor xenografts in mice 4 h after the first injection. Enhanced radioactivity at the site of the Her2-positive tumor lesion was monitored 1 h after the second injection. With these findings, we conclude that the tandem scFv Fc fusion protein and cotinine hapten system have the potential to be applied in PRIT.

Abstract C239: Development of EphA10/CD3 bispecific tandem scFv as a drug candidate against breast cancer.

Abstract Background: Monoclonal antibodies (mAbs) are clinically used to treat several types of cancer due to their high specificity and potential to activate immune systems. The therapeutic effects of mAbs are unsatisfactory, however, in cases of aggressive cancer or low expression level of target protein. Recently, various antibody formats has been developed through genetic engineering technologies to improve therapeutic effect. Most notably bispecific antibodies (BsAb) are a highly promising form of target therapy. A BsAb that simultaneously targets the antigens on cancer cells and activated T-cells reportedly has the potential to show cytotoxicity more effectively than conventional mAbs. In our efforts to develop a new bispecific monoclonal antibody drug against breast cancer, we attempted to generate a tandem single-chain variable fragment (taFv) targeting both the T-cell CD3 antigen and Ephrin receptor A10 (EphA10), which were previously identified as breast cancer novel biomarker proteins. Methods: We constructed the taFv (EphA10/CD3) expression vector that contain two single chain Fv (scFv) derived from the anti-EphA10 antibody and anti-CD3 antibody joined by a flexible five amino acid linker (G4S). The taFv was expressed transiently in CHO-S cells and verified for identity by Western blot analysis, was purified via its C-terminal His tag by metal ion affinity chromatography (IMAC). Flow cytometry was used to confirm dual specificity of taFv against cells expressing the appropriate targets. The ability of cytotoxic for taFv from PBMC was analyzed by lactate dehydrogenase (LDH) release assay. Results: The taFv monomer was purified from CHO-S cells together with the taFv dimer. By flow cytometry analysis, each monomer and dimer bound both antigens EphA10 and CD3 while binding to CD3 was lower than full-body IgG. And each taFvs showed highly cytotoxicity specifically against EphA10 over-expression cancer cell line. Most notably, the cytotoxicity of taFv dimer was higher than taFv monomer at low Effector/Target ratio and low concentration. Conclusion: In this research, it's proved that this taFv (EphA10/CD3) could induce EphA10 specific cytotoxicity. So, we are now ongoing to evaluate the therapeutic effects of each taFvs (monomer and dimer) in vivo. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C239. Citation Format: Shintaro Taki. Development of EphA10/CD3 bispecific tandem scFv as a drug candidate against breast cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C239.

Application of bispecific antibody against antigen and hapten for immunodetection and immunopurification

We present a bispecific antibody that recognizes an antigen and a hapten and can be applied to various biological assays, including immunoblotting and immunoprecipitation. In immunoblot analysis of serum, an anti-C5 × anti-cotinine bispecific tandem single-chain variable fragment (scFv)-Fc fusion protein and cotinine-conjugated horseradish peroxidase (HRP) generated a clean signal without the high background that was observed in a parallel experiment using HRP-conjugated goat anti-rabbit immunoglobulin G (Fc-specific) antibody. In immunoprecipitation analysis of serum, use of the bispecific tandem scFv-Fc fusion protein and cotinine-crosslinked magnetic beads significantly reduced the amount of protein contaminants compared with a parallel experiment done with protein A agarose beads. In subsequent immunoblot analysis, use of cotinine–HRP as the secondary probe instead of HRP-conjugated goat anti-rabbit IgG (Fc-specific) antibody successfully eliminated the band corresponding to the bispecific tandem scFv-Fc fusion protein.

Cancer T Cell Immunotherapy with Bispecific Antibodies and Chimeric Antigen Receptors

Solid tumors contain several different types of malignant cells. This cellular heterogeneity complicates therapy for at least two reasons. First, each subpopulation may respond differently to a given treatment. Second, cancer cells are plastic, and thus may convert from a therapy-sensitive to a therapy-resistant cell type represented by another subpopulation. Therefore, successful therapies will have to target numerous malignant cell types, not just the rapidly proliferating cells as most standard treatments do. Immunotherapies with T cells engineered to recognize cancer cells via bispecific antibodies (bsAbs) or chimeric antigen receptors (CARs) are particularly promising approaches with potential to ablate both dividing and non/slow-dividing subpopulations of cancer cells. Here, we discuss several patents associated with exceptionally effective bsAbs of the tandem single-chain variable fragment (taFv) class and untangle a part of the complex network of patents directly or indirectly related to CARs. Furthermore, we speculate on the future of bsAbs and CARs for both treatment and prevention of solid tumors such as prostate cancer.

Domain order of a bispecific diabody dramatically enhances its antitumor activity beyond structural format conversion: the case of the hEx3 diabody

The domains of bispecific diabodies (BsDbs) can be ordered in four different ways; however, the influence of domain order on the cytotoxicity of BsDbs that retarget immune cells against tumor cells had not been addressed. We previously reported the marked antitumor effects of a humanized BsDb that targets epidermal growth factor receptor and CD3 (hEx3-Db). Here, we rearranged the domains of hEx3-Db to examine the influence of domain order on the function of BsDbs. We successfully prepared homogenous dimers of hEx3-Db in all four domain configurations. Interestingly, all three rearranged hEx3s inhibited cancer growth more effectively than did the original hEx3-Db, in which both components were in variable heavy domain (VH)-variable light domain (VL) order (redesignated as hEx3-HL), and the highest effects were observed with hEx3-LH (hEx3-Db with both components in VL-VH order). In addition, hEx3-LH had comparable in vitro growth inhibitory effects to those of the tandem single-chain variable fragment (scFv) format of hEx3-Db (hEx3-tandem scFv (taFv)), which we previously showed to have greater cytotoxicity than does hEx3-HL. Flow cytometry suggested that the enhanced cytotoxicity of hEx3-LH is attributable to structural superiority for cross-linking, similar to that of hEx3-taFv. Furthermore, hEx3-LH inhibited cancer growth in mice more effectively than did hEx3-taFv; this difference may be due to differences in antibody stability. Our results show that merely rearranging the domain order of BsDbs can enhance their effects beyond those with structural format conversion.

Development of a Diphtheria Toxin Based Antiporcine CD3 Recombinant Immunotoxin

Anti-CD3 immunotoxins, which induce profound but transient T-cell depletion in vivo by inhibiting eukaryotic protein synthesis in CD3+ cells, are effective reagents in large animal models of transplantation tolerance and autoimmune disease therapy. A diphtheria toxin based antiporcine CD3 recombinant immunotoxin was constructed by fusing the truncated diphtheria toxin DT390 with two identical tandem single chain variable fragments (scFv) derived from the antiporcine CD3 monoclonal antibody 898H2-6-15. The recombinant immunotoxin was expressed in a diphtheria-toxin resistant yeast Pichia pastoris strain under the control of the alcohol oxidase promoter. The secreted recombinant immunotoxin was purified sequentially with hydrophobic interaction chromatography (Butyl 650 M) followed by strong anion exchange (Poros 50 HQ). The purified antiporcine CD3 immunotoxin was tested in vivo in four animals; peripheral blood CD3+ T-cell numbers were reduced by 80% and lymph node T-cells decreased from 74% CD3+ cells pretreatment to 24% CD3+ cells remaining in the lymph node following 4 days of immunotoxin treatment. No clinical toxicity was observed in any of the experimental swine. We anticipate that this conjugate will provide an important tool for in vivo depletion of T-cells in swine transplantation models.

Cytotoxic Enhancement of a Bispecific Diabody by Format Conversion to Tandem Single-chain Variable Fragment (taFv): THE CASE OF THE hEx3 DIABODY

Diabodies (Dbs) and tandem single-chain variable fragments (taFv) are the most widely used recombinant formats for constructing small bispecific antibodies. However, only a few studies have compared these formats, and none have discussed their binding kinetics and cross-linking ability. We previously reported the usefulness for cancer immunotherapy of a humanized bispecific Db (hEx3-Db) and its single-chain format (hEx3-scDb) that target epidermal growth factor receptor and CD3. Here, we converted hEx3-Db into a taFv format to investigate how format affects the function of a small bispecific antibody; our investigation included a cytotoxicity assay, surface plasmon resonance spectroscopy, thermodynamic analysis, and flow cytometry. The prepared taFv (hEx3-taFv) showed an enhanced cytotoxicity, which may be attributable to a structural superiority to the diabody format in cross-linking target cells but not to differences in the binding affinities of the formats. Comparable cross-linking ability for soluble antigens was observed among hEx3-Db, hEx3-scDb, and hEx3-taFv with surface plasmon resonance spectroscopy. Furthermore, drastic increases in cytotoxicity were found in the dimeric form of hEx3-taFv, especially when the two hEx3-taFv were covalently linked. Our results show that converting the format of small bispecific antibodies can improve their function. In particular, for small bispecific antibodies that target tumor and immune cells, a functional orientation that avoids steric hindrance in cross-linking two target cells may be important in enhancing the growth inhibition effect.

Conversion of CTLA-4 from inhibitor to activator of T cells with a bispecific tandem single-chain Fv ligand.

Abs or their recombinant fragments against surface receptors of the Ig superfamily can induce or block the receptors' native function depending on whether they induce or prevent the assembly of signalosomes on their cytoplasmic tails. In this study, we introduce a novel paradigm based on the observation that a bispecific tandem single-chain variable region fragment ligand of CTLA-4 by itself converts this inhibitory receptor into an activating receptor for primary human T lymphocytes. This reversal of function results from increased recruitment of the serine/threonine phosphatase 2A to the cytoplasmic tail of CTLA-4, consistent with a role of this phosphatase in the regulation of CTLA-4 function, and assembly of a distinct signalosome that activates an lck-dependent signaling cascade and induces IL-2 production. Our data demonstrate that the cytoplasmic domain of CTLA-4 has an inherent plasticity for signaling that can be exploited therapeutically with recombinant ligands for this receptor.