Single-chain Antibody Fragment Research Articles

Brolucizumab: an evolution in treatment for neovascular age-related macular degeneration

ABSTRACTIntroduction: The treatment paradigm for neovascular age-related macular degeneration (nAMD) has had a profound shift over the past 15 years with the advent of anti-vascular endothelial growth factor (VEGF) agents. However, the ability to sustain optimal visual outcomes is challenging with disease chronicity and treatment burden. Brolucizumab, a single-chain antibody fragment targeting all isoforms of VEGF, is a new molecule with properties that allow for less frequent doses of anti-VEGF to be administered with low systemic exposure.Areas covered: The authors will provide a comprehensive review of the current literature and clinical trial data surrounding brolucizumab in the treatment of nAMD, while comparing it with other emerging therapies.Expert opinion: Brolucizumab is a novel anti-VEGF inhibitor, with Phase I, II, and III trial data indicating safety, tolerability, and efficacy for the treatment of nAMD when given 12-weekly. Precision dosing with brolicizumab may best achieve the opportunity for vision improvement and maintenance of vision gains, while potentially maintaining patients on manageable dosing intervals with disease stability, improved treatment adherence, and reduced burden.

Nanobody-Based high-performance immunosorbent for selective beta 2-microglobulin purification from blood

Removing β2-microglobulin (β2M) from blood circulation is considered to be the most effective method to delay the occurrence of dialysis-related amyloidosis (DRA). The ideal extracorporeal β2M removal system should be cost-effective, highly specific and having a high capacity. However, the traditional technologies based on size exclusion do not have an adequate specificity, and alternative immunosorbents have limited applications due to low capacity and their high cost. Nanobodies (Nbs), the smallest functional recombinant antibody fragments, offer several advantages to overcome these obstacles. In this study, an anti-β2M Nb with a C-terminal thiol-tag was successfully prepared from E. coli for site-directed and oriented immobilization and usage as capture ligand in a β2M-selective immunosorbent. The prepared immunosorbent showed a high binding capacity of up to 7 mg β2M per mL resin, which is 17 times higher than that of previous studies using single-chain variable antibody fragments (scFv). Furthermore, an exceptional high specificity has been demonstrated as other human serum proteins were not adsorbed during dynamic adsorption experiments. About 80% of the original binding capacity of the immunosorbent was restored after four consecutive easy regenerations, whereas 90% of the original capacity was retained after 1-month storage of the resin. Moreover, the mathematical model fitted very well the in vitro perfusion. The results with this pioneering immunosorbent confirm its possible clinical application and is expected to reach the required clinical effect of immunoadsorption therapy. Statement of SignificanceDialysis-related amyloidosis (DRA), associated with the accumulation of β2-microglobulin (β2M), is a serious complication of end-stage kidney disease. Removing β2M from blood circulation by extracorporeal blood purification is considered to be the most effective method to delay the occurrence of DRA. However, the existing methods are incapable to eliminate sufficient quantities of β2M from circulation, either because of lack of specificity, high cost or for low capacity. In this manuscript, we provide a practical and economic immunosorbent based on anti-β2M nanobody for DRA. The prepared immunosorbent was reusable and storable, and demonstrated high specificity and realized a high binding capacity of up to 7 mg β2M per mL resin, which is 17 times higher than that of the previous studies.

Bacterial expression and characterization of an anti-CD22 single-chain antibody fragment

Single-chain variable fragment (scFv) antibodies are fusion proteins of the variable regions of the heavy and light chains of immunoglobulins connected with a short linker peptide. They possess unique and superior features compared to whole antibodies for immunotherapy of various carcinomas, including hematologic B-cell malignancies.In the presented study we obtained efficient production of the recombinant anti-CD22 scFv in Escherichia coli expression system. The active recombinant protein was successfully recovered from inclusion bodies. Assays were performed to assess the in vitro targeting properties and specificity of the obtained anti-CD22 scFv antibody in the CD22 positive and negative lymphoma cell lines.

Cryo-EM Structures of Chromatosomes Containing Human Linker Histone Variants

Linker and core histones together condense genomic DNA by forming the chromatosome, the structural unit of metazoan chromatin. There are 11 linker histone variants in human but no high-resolution structures of chromatosomes are available. Aided by a single-chain antibody fragment, we determined the cryo-EM structures of three chromatosomes containing different linker histone variants at near atomic resolution. In these structures, the globular domains of the linker histones bind to the nucleosome dyad, but the linker DNAs displayed different conformations. We observed no well-defined densities for linker histones tails but NMR studies showed that many residues in their C-terminal tails were not observable, suggesting that the C-terminal tails likely interact with the linker DNA dynamically. Our study provides insights into the structures and dynamics of chromatosomes, which have implications for understanding the roles of different linker histone variants in chromatin structure and function.

Understanding the Spatial Topology of Artificial Immunological Synapses Assembled in T Cell-Redirecting Strategies: A Major Issue in Cancer Immunotherapy.

T cell-redirection strategies aim to selectively eliminate cancer cells by physically linking T lymphocytes with cancer cells using tumor-targeted cell-cell bridging (CCB) molecules, such as membrane-anchored chimeric antigen receptors (CARs) or soluble bispecific antibodies (bsAbs) that specifically recognize a cell-surface tumor-associated antigen (TAA) (Blanco et al., 2019). In the CAR approach, a TAA-specific antibody is genetically fused to intracellular T cell signaling domains. CARs have evolved greatly since their initial description, as single-chain antibody fragment (scFv)-based receptors containing the signaling domain of the CD3ζ chain (CD247) of the T cell receptor (TCR) (Eshhar et al., 1993). Subsequently, constructs incorporating signaling domains of costimulatory molecules (e.g., CD28 or 4-1BB) in tandem with the CD3ζ signaling domain were generated (Finney et al., 1998). Engrafting T-cells with such receptors, termed second-generation CARs, enables sustained proliferation and increased cytokine secretion. Third-generation CARs contain two costimulatory domains, in addition to the CD3ζ signaling domain (Carpenito et al., 2009; Milone et al., 2009). Current CAR-T cell therapy involves the isolation of autologous T cells using leukapheresis, followed by in vitro stimulation, genetic modification to express the TAA-specific CAR, and expansion to infuse back into the patient (Blanco et al., 2019). The bsAbs are designed to simultaneously bind to the TAA in the surface of tumor cells and the CD3e chain of the TCR/CD3 complex in the surface of T cells (Blanco et al., 2019). More than a 100 different bsAb formats have been reported, including small bsAbs composed only by two antigen-binding sites, IgG-like bsAbs and larger and non-IgG bsAbs formed by different antigen-binding moieties, often combined with oligomerization modules (Nunez-Prado et al., 2015; Brinkmann and Kontermann, 2017). By connecting CD3 signaling molecules with a recognition process independent of the TCR variable domains, T cells can be hot-wired to recognize a user-defined cell-surface TAA that is not associated with the major histocompatibility complex (MHC) to activate effector cell responses and kill cancer cells (Blanco et al., 2019). Nonetheless, the precise molecular mechanisms by which T cells are activated through these CCB molecules are poorly understood. The administration of bsAbs and CAR-T cells has achieved remarkable clinical outcomes in hematological tumors, and several products have been approved by regulatory agencies for clinical use. Blinatumomab, an anti-CD19xanti-CD3 bsAb designed in the BiTE (bispecific T cell-engager) format, was approved by the US Food and Drug Administration (FDA) for the treatment of relapsed or refractory B cell acute lymphoblastic leukemia (B-ALL) (Przepiorka et al., 2015). Two CD19-specific second generation CAR-T cell products, tisagenlecleucel and axicabtagene ciloleucel (axi-cel), have been approved by the US FDA for the treatment of pediatric and young adult patients with relapsed or refractory B-ALL (Maude et al., 2018) and adult patients with relapsed or refractory diffuse large B cell lymphomas (Neelapu et al., 2017), respectively. However, the utility of these approaches in the treatment of solid tumors targeting TAAs has been limited by organ toxicities related to activation of T cell effector functions by non-tumor cells expressing low levels of the TAA, as well as systemic cytokine-associated toxicities (Alonso-Camino et al., 2016).

Transferrin-binding peptide functionalized polymersomes mediate targeted doxorubicin delivery to colorectal cancer in vivo

Transferrin receptor (TfR) is a promising target validated in the clinical trials for managing various malignancies. Transferrin (Tf) and single chain antibody fragment can target TfR and are typically conjugated to nanomedicines via post-modification, which poses significant production challenges. Here, we report that the polymersomes functionalized with a Tf-binding peptide CGGGHKYLRW (TBP-Ps) can selectively and stably bind Tf and subsequently mediate targeted doxorubicin (Dox) delivery to TfR over-expressing HCT-116 colorectal cancer cells in vitro and in vivo. The Tf surface density of the polymersomes could be controlled by the surface content of TBP. Interestingly, modifying Dox-loaded TBP-Ps with Tf led to greatly increased cellular uptake and inhibitory effect of HCT-116 cells. Tf-bound TBP-Ps demonstrated rapid accumulation in the tumor xenografts in nude mice following i.v. injection. More importantly, Dox-loaded Ps with Tf binding significantly enhanced the antitumor efficacy in mice bearing HCT-116 tumors compared to polymersomes without Tf binding. Surface functionalization of the nanoparticles with Tf-binding peptide provides an appealing strategy in formulating Tf-targeted nanomedicines.

Brolucizumab as treatment of wet age-related maculopathy.

Given the success in stabilizing vision with current anti-vascular endothelial growth factor (VEGF) options, one main target for future anti-VEGF drug development includes creating medications with longer durations of action. Achieving this goal will decrease the number of overall injections and follow-up visits required to ensure better patient compliance. The smallest anti-VEGF created so far is brolucizumab (Beovu; Novartis). It is a 26-kDa IgG single-chain antibody fragment that delivers 11 times more anti-VEGF per injection than aflibercept. Brolucizumab was approved by the U.S. Food and Drug Administration (FDA) in late 2019 for the treatment of wet age-related macular degeneration, and has been also approved for the same indication in Japan and the European Union in 2020. In this article, we compare brolucizumab to current FDA-approved anti-VEGF treatments, address the studies associated with brolucizumab, discuss brolucizumab's side effects, and conclude with recommendations.

Tandem CAR-T cells targeting CD70 and B7-H3 exhibit potent preclinical activity against multiple solid tumors.

Purpose: Given that heterogeneous expression and variants of antigens on solid tumors are responsible for relapse after chimeric antigen receptor (CAR)-T cell therapy, we hypothesized that combinatorial targeting two tumor-associated antigens would lessen this problem and enhance the antitumor activity of T cells.Methods: The co-expression level of CD70 and B7-H3 was analyzed in multiple tumor tissue samples. Further, two putative antigens were identified in The Cancer Genome Atlas and Gene Expression Profiling Interactive Analysis database. Two CD70 targeted CARs with different antigen binding domain, truncated CD27 and CD70 specific single-chain antibody fragment (scFv), were designed to screen a more suitable target-antigen binding moiety. Accordingly, we designed a bivalent tandem CAR (TanCAR) and further assessed the anti-tumor efficacy of TanCAR-T cells in vitro and in vivo.Results: Our results indicated that co-expression of CD70 and B7-H3 was observed on multiple tumor types including kidney, breast, esophageal, liver, colon cancer, glioma as well as melanoma. The CD70 targeted CAR-T cells with binding moiety of CD70 specific scFv exhibit a higher affinity and antitumor effect against CD70+ tumor cells. TanCAR-T cells induced enhanced ability of cytolysis and cytokine release over unispecific CAR-T cells when encountering tumor cells expressing two target-antigens. Further, low doses of TanCAR-T cells could also effectively control the lung cancer and melanoma xenografts and improved overall survival of the treated animals.Conclusion: TanCAR-T cells targeting CD70 and B7-H3 exhibit enhanced antitumor functionality and improve the problem of antigenic heterogeneity and variant in the treatment against solid tumor and melanoma.

Selection and characterization of scFv antibody against nucleocapsid protein of Porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome virus (PRRSV) is a widespread infectious agent in pigs. Nucleocapsid (N) protein of PRRSV has been identified as the most immunodominant viral protein. The main goal of the work was the selection and characterization of a single-chain antibody fragments (scFv) antibody specific to the N protein. Specific scFv antibody clone D5 was selected from the Tomlinson phagemid library and purified by immobilized metal affinity chromatography from the periplasmatic space of E. coli cells. The antibody was then characterized by sequencing and the ability to recognize the native virus N protein by Western blot and competitive ELISA. Pepscan analysis identified the position of the binding epitope between amino acids 62–84 of the N protein. Our study could help to improve the diagnostics and prevention of PRRSV in Central Europe.

Brolucizumab: First Approval.

Brolucizumab (Beovu®) is a low molecular weight, single-chain antibody fragment vascular endothelial growth factor (VEGF) inhibitor being developed by Novartis for the treatment of exudative (wet) age-related macular degeneration (AMD), diabetic macular oedema and macular oedema secondary to retinal vein occlusion. Based primarily on the results of the phase III HAWK and HARRIER trials brolucizumab was recently approved in the US for the treatment of wet AMD. This article summarizes the milestones in the development of brolucizumab leading to this first approval.

Simple strategy for single-chain fragment antibody-conjugated probe construction

AimsA combination of biomarker and instrument technology diagnosis methods, especially antigen-targeted imaging methods, is required to increase the accuracy of the diagnosis of cancer. Currently, the targeting efficiency is limited by the conjugation methods used for the conjugation of antibodies and imaging materials. Here, a simple strategy for the conjugation of a probe and a single-chain fragment antibody (scFv) that does not change the characteristics of the antibody was shown. Main methodsAn ScFv was conjugated with superparamagnetic iron oxide (SPIO) or indocyanine green (ICG) via a linker by utilizing the reaction between cysteine and maleimide. The characterization of the probe was performed by flow cytometry, confocal imaging, optical imaging and magnetic resonance imaging (MRI). Key findingsAfter conjugation, the scFv retained high affinity, antigen specificity, and strong internalization ability. The application of the conjugated probe was also confirmed by optical imaging and MRI. SignificanceThe proposed strategy provides a simple method for the production of high efficiency antigen-targeted imaging probes for tumor diagnosis.

A microbial expression system for high-level production of scFv HIV-neutralizing antibody fragments in Escherichia coli

Monoclonal antibodies (mABs) are of great biopharmaceutical importance for the diagnosis and treatment of diseases. However, their production in mammalian expression hosts usually requires extensive production times and is expensive. Escherichia coli has become a new platform for production of functional small antibody fragment variants. In this study, we have used a rhamnose-inducible expression system that allows precise control of protein expression levels. The system was first evaluated for the cytoplasmic production of super folder green fluorescence protein (sfGFP) in various production platforms and then for the periplasmic production of the anti-HIV single-chain variable antibody fragment (scFv) of PGT135. Anti-HIV broadly neutralizing antibodies, like PGT135, have potential for clinical use to prevent HIV transmission, to promote immune responses and to eradicate infected cells. Different concentrations of L-rhamnose resulted in the controlled production of both sfGFP and scFv PGT135 antibody. In addition, by optimizing the culture conditions, the amount of scFv PGT135 antibody that was expressed soluble or as inclusions bodies could be modulated. The proteins were produced in batch bioreactors, with yields of 4.9 g/L for sfGFP and 0.8 g/L for scFv. The functionality of the purified antibodies was demonstrated by their ability to neutralize a panel of different HIV variants in vitro. We expect that this expression system will prove very useful for the development of a more cost-effective production process for proteins and antibody fragments in microbial cells.

One-step site-specific antibody fragment auto-conjugation using SNAP-tag technology.

Antibody-based diagnostic and therapeutic agents play a substantial role in medicine, especially in cancer management. A variety of chemical, genetic and enzymatic site-specific conjugation methods have been developed for equipping antibodies with effector molecules to generate homogeneous antibody conjugates with tailored properties. However, most of these methods are relatively complicated and expensive and require several reaction steps. Self-labeling proteins such as the SNAP-tag are an innovative solution for addressing these challenges. The SNAP-tag is a modified version of the human DNA repair enzyme alkylguanine-DNA alkyltransferase (AGT), which reacts specifically with O(6)-benzylguanine (BG)-modified molecules via irreversible transfer of an alkyl group to a cysteine residue. It provides a simple, controlled and robust site-specific method for labeling antibodies with different synthetic small effector molecules. Fusing a SNAP-tag to recombinant antibodies allows efficient conjugation of BG-containing substrates by autocatalytic, irreversible transfer of the alkyl group to a cysteine residue in the enzyme's active site under physiological conditions and with a 1:1 stoichiometry. This protocol describes how to generate site-specific SNAP-tag single-chain antibody fragment (scFv) conjugates with different types of BG-modified effector molecules. A specific example is included for the design and production of an scFv-photosensitizer conjugate and its characterization as an immuno-theranostic agent. This protocol includes DNA sequences encoding scFV-SNAP-tag fusion proteins and outlines strategies for expression, purification and testing of the resulting scFv-SNAP-tag-based immuno-conjugates. All experiments can be performed by a graduate-level researcher with basic molecular biology skills within an 8-week time frame.

A VEGFR2-MICA bispecific antibody activates tumor-infiltrating lymphocytes and exhibits potent anti-tumor efficacy in mice.

MHC class I-related chain A (MICA) is one of the major ligands for natural killer group 2 member D (NKG2D), which is an activating NK receptor. MICA is expressed on the surface of human epithelial tumor cells, and its shedding from tumor cells leads to immunosuppression. To activate immune response in the tumor microenvironment, we designed an anti-VEGFR2-MICA bispecific antibody (JZC01), consisting of MICA and an anti-VEGFR2 single chain antibody fragment (JZC00) and explored its potential anti-tumor activity. JZC01 targeted vascular endothelial growth factor receptor 2 (VEGFR2) and inhibited tumorigenesis by blocking the VEGFR2 signaling pathway. Additionally, JZC01 promoted NK and CD8+ T cells to release IFN-γ and engaged activated lymphocytes to lysis of VEGFR2-expressing tumor cells. The in vivo anti-tumor activity of JZC01 was investigated by establishing a Lewis lung cancer cell-transplanted mouse model. It effectively reduced the tumor vascular density and increased the infiltration and activation of NK and CD8+ T cells in the tumor microenvironment. Thus, JZC01 functions in anti-tumor angiogenesis and anti-tumor immune activation, and showed improved anti-tumor efficacy combined with docetaxel, which provides a new insight into anti-tumor therapy.

Magnetic nanocarriers for the specific delivery of siRNA: Contribution of breast cancer cells active targeting for down-regulation efficiency.

The association between superparamagnetic iron oxide nanoparticles (SPION), carrying small interfering RNA (siRNA) as therapeutic agents and humanized anti- human epidermal growth factor receptor-2 (HER2) single-chain antibody fragments (scFv) for the active delivery into HER2-overexpressing cells appears as an interesting approach for patients with HER2-overexpressing advanced breast cancer. The obtained Targeted Stealth Magnetic siRNA Nanovectors (TS-MSN) are formulated by combining: (i) the synthesis protocol of Targeted Stealth Fluorescent Particles (T-SFP) which form the core of TS-MSN and (ii) the formulation protocol allowing the loading of T-SFP with polyplexes (siRNA and cationic polymers). TS-MSN have suitable physico-chemical characteristics for intravenous administration and protect siRNA against enzymatic degradation up to 24 h. The presence of HER2-targeting scFv on TS-MSN allowed an improved internalization (3-4 times more compared to untargeted S-MSN) in HER2-overexpressing breast cancer cells (BT-474). Furthermore, anti-survivin siRNA delivered by TS-MSN in HER2-negative breast-cancer control cells (MDA-MB-231) allowed significant down-regulation of the targeted anti-apoptotic protein of about 70%. This protein down-regulation increased in HER2+ cells to about 90% (compared to 70% with S-MSN in both cell lines) indicating the contribution of the HER2-active targeting. In conclusion, TS-MSN are promising nanocarriers for the specific and efficient delivery of siRNA to HER2-overexpressing breast cancer cells.

Enhancing Recombinant Protein Yields in the E. coli Periplasm by Combining Signal Peptide and Production Rate Screening.

Proteins that contain disulfide bonds mainly mature in the oxidative environment of the eukaryotic endoplasmic reticulum or the periplasm of Gram-negative bacteria. In E. coli, disulfide bond containing recombinant proteins are often targeted to the periplasm by an N-terminal signal peptide that is removed once it passes through the Sec-translocon in the cytoplasmic membrane. Despite their conserved targeting function, signal peptides can impact recombinant protein production yields in the periplasm, as can the production rate. Here, we present a combined screen involving different signal peptides and varying production rates that enabled the identification of more optimal conditions for periplasmic production of recombinant proteins with disulfide bonds. The data was generated from two targets, a single chain antibody fragment (BL1) and human growth hormone (hGH), with four different signal peptides and a titratable rhamnose promoter-based system that enables the tuning of protein production rates. Across the screen conditions, the yields for both targets significantly varied, and the optimal signal peptide and rhamnose concentration differed for each protein. Under the optimal conditions, the periplasmic BL1 and hGH were properly folded and active. Our study underpins the importance of combinatorial screening approaches for addressing the requirements associated with the production of a recombinant protein in the periplasm.

B7-H3 as a Novel CAR-T Therapeutic Target for Glioblastoma

Glioblastoma (GBM) remains one of the most malignant primary tumors in adults, with a 5-year survival rate less than 10% because of lacking effective treatment. Here, we aimed to explore whether B7-H3 could serve as a novel therapeutic target for GBM in chimeric antigen receptor (CAR) T cell therapy. In this study, a CAR targeting B7-H3 was constructed and transduced into T cells by lentivirus. Antitumor effects of B7-H3-specific CAR-T cells were assessed with primary and GBM cell lines both in vitro and in vivo. Our results indicated that B7-H3 was positively stained in most of the clinical glioma samples, and its expression levels were correlated to the malignancy grade and poor survival in both low-grade glioma (LGG) and GBM patients. Specific antitumor functions of CAR-T cells were confirmed by cytotoxic and ELISA assay both in primary glioblastoma cells and GBM cell lines. In the orthotropic GBM models, the median survival of the CAR-T-cell-treated group was significantly longer than that of the control group. In conclusion, B7-H3 is frequently overexpressed in GBM patients and may serve as a therapeutic target in CAR-T therapy.

Design, Expression, Purification and Characterization of the Recombinant Immunotoxin 4D5 scFv-TRAIL

Immunotoxins are chimeric proteins that combine antibodies and toxins in targeted cancer therapy. Despite the promising application prospects, immunogenicity and nonspecific cytotoxicity of immunotoxins with toxins from bacterial or plant origin have hindered their clinical application. In this study, an anti-HER2 single-chain antibody fragment 4D5, was fused to a human-origin antitumor drug, TRAIL to produce a humanized immunotoxin in Escherichia coli BL21 (DE3). An appropriate linker between the domains was selected using the software Antheprot 5.0. The 4D5 scFv-TRAIL was obtained using a SUMO tag for solubility-enhancing and purified by Ni–NTA affinity chromatography after tag removing. The purity and yield of 4D5 scFv-TRAIL reached 92% and 11.6 mg/L in shake flasks, respectively. Additionally, MTT assays showed 4D5 scFv-TRAIL exhibited selective in vitro cytotoxicity on the breast cancer cell line MDA-MB-231 without cytotoxic effect on the normal cell FBHE. Therefore, 4D5 scFv-TRAIL is a potential humanized immunotoxin for treatment of HER2-overexpressing tumor cells.

Enhancing the detection of Toxoplasma gondii via an anti-SAG1 scFv-alkaline phosphatase immunoconjugate

The purpose of this study was to implement a fluorometric method for enhancing the detection sensitivity of Toxoplasma gondii in biological fluids. To address this challenge, we designed and produced a recombinant immunoconjugate tool based on a single-chain antibody fragment anti-T. gondii SAG1 antigen (scFvSG15) genetically fused to the bacterial alkaline phosphatase (AP) using 4-methyl-umbelliferyl-phosphate as fluorogenic substrate. The anti-SAG1 scFv-AP conjugate was fully bifunctional and was used successfully in different assays including immunoblot, fluorometric ELISA and direct immunofluorescence. The fluorometric immunoassay afforded an extremely low detection limit (1 tachyzoite/well), which was in agreement with the real-time PCR control test. The immunofluorescence imaging has provided captivating visual evidence of T. gondii detection. These results strongly suggest that the recombinant anti-SAG1-AP conjugate generated here might serve as useful and highly sensitive immunoassay probe to direct detect T. gondii in a one-step procedure, opening up new perspectives for diagnosis of toxoplasmosis.

Pharmacokinetic Analysis of a Novel Human EGFRvIII:CD3 Bispecific Antibody in Plasma and Whole Blood Using a High-Resolution Targeted Mass Spectrometry Approach.

Bispecific single chain antibody fragments (bi-scFv) represent an emerging class of biotherapeutics. We recently developed a fully human bi-scFv (EGFRvIII:CD3 bi-scFv) with the goal of redirecting CD3-expressing T cells to recognize and destroy malignant, EGFRvIII-expressing glioma. In mice, we showed that EGFRvIII:CD3 bi-scFv effectively treats orthotopic patient-derived malignant glioma and syngeneic glioblastoma. Here, we developed a targeted assay for pharmacokinetic (PK) analysis of EGFRvIII:CD3 bi-scFv, a necessary step in the drug development process. Using microflow liquid chromatography coupled to a high resolution parallel reaction monitoring mass spectrometry, and data analysis in Skyline, we developed a bottom-up proteomic assay for quantification of EGFRvIII:CD3 bi-scFv in both plasma and whole blood. Importantly, a protein calibrator, along with stable isotope-labeled EGFRvIII:CD3 bi-scFv protein, were used for absolute quantification. A PK analysis in a CD3 humanized mouse revealed that EGFRvIII:CD3 bi-scFv in plasma and whole blood has an initial half-life of ∼8 min and a terminal half-life of ∼2.5 h. Our results establish a sensitive, high-throughput assay for direct quantification of EGFRvIII:CD3 bi-scFv without the need for immunoaffinity enrichment. Moreover, these pharmacokinetic parameters will guide drug optimization and dosing regimens in future IND-enabling and phase I studies of EGFRvIII:CD3 bi-scFv.