scFv Fusion Research Articles

A novel LAG3 neutralizing antibody improves cancer immunotherapy by dual inhibition of MHC-II and FGL1 ligand binding

LAG3 is an inhibitory immune checkpoint expressed on activated T and NK cells. Blocking the interaction of LAG3 with its ligands MHC-II and FGL1 renders T cells improved cytotoxicity to cancer cells. Current study generated a panel of LAG3 monoclonal antibodies (mAbs) through immunization of mice followed by phage display. Some of them bound to the D1-D2 domain of LAG3, which is known for the engagement of its ligands FGL1 and MHC-II. Three outperformers, M208, M226, and M234, showed stronger blocking activity than Relatlimab in the FGL1 binding. Furthermore, M234 showed dual inhibition of FGL1 (IC50 of 20.6 nM) and MHC-II binding (IC50 of 6.2 nM) to LAG3. In vitro functional tests showed that M234 significantly stimulated IFN-γ secretion from activated PBMC cells. In vivo studies in a mouse model of hepatocellular carcinoma xenografts demonstrated that combining M234 IgG with GPC3-targeted bispecific antibodies significantly improved efficacy. In addition, GPC3-targeted CAR-T cells secreting IL-21-M234 scFv fusion protein exhibited enhanced activity in inhibiting tumor growth and greatly increased the survival rate of mice. Taken together, M234 has potential in cancer immunotherapy and warrants further clinical trial.

A bifunctional anti-PCSK9 scFv/Exendin-4 fusion protein exhibits enhanced lipid-lowering effects via targeting multiple signaling pathways in HFD-fed mice

Fusion protein which encompasses more than one functional component, has become one of the most important representatives of macromolecular drugs for disease treatment since that monotherapy itself might not be effective enough to eradicate the disease. In this study, we sought to construct a bifunctional antibody fusion protein by fusing anti-PCSK9 scFv with Exendin-4 for simultaneously lowering both LDL-C and TG. Firstly, three Ex4-anti-PCSK9 scFv fusion proteins were constructed by genetically connecting the C-terminal of Exendin-4 to the N-terminal of anti-PCSK9 scFv through various flexible linker peptides (G4S)n (n = 2, 3, 4). After soluble expression in E. coli, the most potent Ex4-(G4S)4-anti-PCSK9 scFv fusion protein was selected based on in vitro activity assays. Then, we investigated the in vivo therapeutic effects of Ex4-(G4S)4-anti-PCSK9 scFv on the serum lipid profile and bodyweight changes as well as underlying molecular mechanism in HFD-fed C57BL/6 mice. The data showed that Ex4-(G4S)4-anti-PCSK9 scFv exhibits enhanced effects of lowering both LDL-C and TG in serum, reducing food intake and body weight via blocking PCSK9/LDLR, activating AMPK/SREBP-1 pathways, and up-regulating sirt6. Conclusively, Ex4-(G4S)4-anti-PCSK9 has the potential to serve as a promising therapeutic agent for effectively treating dyslipidemia with high levels of both LDL-C and TG.

Therapeutic potential of a novel IP-10-(anti-HER2 scFv) fusion protein for the treatment of HER2-positive breast cancer.

Interferon-γ-inducible protein 10 (IP-10) is a potent antitumor agent and acts by its angiostatic and immunomodulatory properties. IP-10 can target to tumor site by linking with single chain variable fragment (scFv) that recognized specific tumor antigen. In this study, we evaluated biological activity of the fusion protein including IP-10 and anti-HER2 scFv (IP-10-(anti-HER2 scFv)). The HER2- and cell-based ELISA as well as the flow cytometry analysis demonstrated that the fusion protein specifically binds to HER2 antigen. In addition, competitive ELISA demonstrated that the fusion protein recognized the same epitope of HER2 antigen as trastuzumab. The results of MTT assay demonstrated that the growth of HER2-enriched SK-BR3 cells was inhibited in the presence of the fusion protein. Moreover, the cytotoxic effect of the fusion protein was not significantly different from that of trastuzumab. However, no significant cytotoxic effect compared to trastuzumab and anti-HER2 scFv was observed in HER2-low-expressing MDA-MB-231 cells. The obtained findings demonstrated that IP-10-(anti-HER2 scFv) can selectively reduce the cell viability in HER2+ cells. Moreover, similar inhibitory effect on growth of both SK-BR-3 and MDA-MB-231 cell lines was observed in the presence of anti-HER2 scFv protein even at high concentration after 72h. The chemotaxis properties of the fusion protein were also analyzed by a chemotaxis assay. It was demonstrated that the fusion protein induced migration of activated T cell similar to recombinant IP-10 protein. Our findings suggested that IP-10-(anti-HER2 scFv) fusion protein can specifically direct IP-10 to the HER2-expressing tumor cells and may act as an adjuvant along with HER2-based vaccine to gather the elicited immune response at the site of HER2-overexpressimg tumors.

Recombinant cell penetrating peptides and intrabodies targeting membrane-bound mutated KRAS antigens

One of the drivers for early carcinogenesis involves specific somatic point substitution mutations in the KRAS gene which damages its ability to conduct signal transduction. Although antibodies can be used for the targeting of KRAS antigen, their localization along the cell’s inner membrane serves as a barrier against the accessibility of the antibodies. This study describes the evaluation of two internalization strategies, namely the endocytosis-based cell penetrating peptide (CPP) approach and the adenoviral-based intrabody (IB) approach, for the delivery of an anti-mutant KRAS single-chain variable fragment (scFv) into the cell. Splicing by overhang extension polymerase chain reaction (SOE-PCR) was used for the fusion of scFv with an enhanced green fluorescence protein (eGFP) and Antennapedia-PTD (Antp), a cell penetrating signal peptide. The fused construct (Antp-scFv-eGFP) at a concentration of 0.085 mg/ml was expressed in E. coli (BL21), while recombinant adenoviral particles containing the scFv-eGFP gene were harvested from HEK 293 cells. Both SW480 and HeLa cells were treated with Antp-scFv-eGFP and recombinant adenoviral particles, and their eGFP localization and intensity were compared to determine their scFv binding efficiencies. The IB approach was shown to exhibit a 3-fold higher fluorescence signal intensity compared to the CPP approach. This proof-of-concept study demonstrated that both antigens for either screening, diagnostic approaches can be potentially adopted when targeting various intracellular or therapeutic purposes.

Abstract 2811: CT120, a novel fully human anti-CD19 x anti-CD22 dual targeted chimeric antigen receptor T cell product for the treatment of B-NHL and B-ALL

Abstract Background: Anti-CD19 chimeric antigen receptor T cell therapies (CAR-T) have shown impressive clinical outcomes in relapsed/refractory (R/R) CD19+ B-cell non-Hodgkin lymphomas (B-NHL) and acute lymphoblastic leukemias (B-ALL). However, 30%-60% of patients with B-ALL who initially responded to anti-CD19 CAR-T therapies relapsed either due to poor CAR-T persistence or CD19 antigen escape, and preliminary data also indicated CD19 antigen loss in patients with B-NHL relapsed after anti-CD19 CAR-T therapy. In addition, the immunogenicity of murine origin CAR construct is another cause of limited in vivo CAR-T persistence. Design: To overcome these problems, a novel fully human anti-CD19×anti-CD22 dual targeted CAR-T cell product - CT120, has been developed. The CT120 CAR molecule contains tandem fusion of anti-CD19 and anti-CD22 scFv, which can effectively recognize CD19 and CD22 antigen independently, to avoid antigen escape. The intracellular CAR domain contains CD3ζ and 4-1BB co-stimulatory domain, which can improve in vivo persistence. In addition, both anti-CD19 and anti-CD22 antibody were generated from proprietary fully human libraries to minimize possible immunogenicity. The CAR molecule was transferred to autologous T cells through a lentiviral backbone. Results: The anti-CD19 and anti-CD22 scFv binds to its antigen in nanomolar range, and CT120 CAR-T cells can bind to both CD19 and CD22 antigens in a noncompetitive manner. CT120 CAR-T cells showed potent in vitro cytotoxicity not only to target B-cell lymphoma/leukemia cell lines (Raji, Daudi, and NALM-6), but also to the CD19 knockout (CD19-CD22+) and CD22 knockout (CD19+CD22-) Raji cell lines. CT120 can secret multiple cytokines, such as IL-2, TNF-α and IFN-γ, and proliferate efficiently after engagement of target cells. In vivo efficacy study demonstrated that CT120 CAR-T cells expanded effectively in mice and completely inhibit Raji and NALM-6 tumor cell growth. Furthermore, CT120 showed a favorable safety profile and promising efficacy in patients with r/r B-NHL and B-ALL in an investigator-initiated trial (IIT). Certain patients who relapsed after previous murine origin CAR-T treatment also benefited from CT120 therapy. Conclusion: Collectively, CT120 is a potent and safe anti-CD19×anti-CD22 dual target CAR-T product for the treatment of B-NHL and B-ALL, and now it is in registered phase 1/2 clinical trial. Citation Format: Panpan Niu, Qianli Hu, Jialu Mo, Guangrong Meng, Xiangyin Jia, Wei Cheng, Qiaoe Wei, Zhenyu Dai, Xuefeng Wu, Guang Hu, Taochao Tan, Jianfeng Zhou, Yongkun Yang, gang Hu. CT120, a novel fully human anti-CD19 x anti-CD22 dual targeted chimeric antigen receptor T cell product for the treatment of B-NHL and B-ALL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2811.

BR2 cell penetrating peptide effectively delivers anti-p21Ras scFv to tumor cells with ganglioside expression for therapy of ras-driven tumor

PurposeCell membrane penetrating peptide BR2 can bind with ganglioside and introduce foreign drugs into tumor cells. In this study, we employed BR2 to carry the broad-spectrum anti-p21Ras scFv prepared in our laboratory into ganglioside expressing tumor cells for therapy of ras-driven tumors.MethodsBR2-p21Ras scFv gene was cloned to prokaryotic expression vector and expressed in E. coli BL21, then the fusion protein was purified with HisPur Ni-NTA. The immunoreactivity of the fusion protein with p21Ras was detected by ELISA and western blotting. The membrane-penetrating and immune co-localization with p21Ras of the fusion protein were determined by immunofluorescence. The antitumor activity was investigated using MTT, wound healing, colone formation, and apoptosis assays in vitro.ResultsBR2-p21Ras scFv fusion protein was successfully expressed and purified. We found that the fusion protein could specifically penetrate into human tumor cell lines which express ganglioside including human neuroblastoma cell line SK-N-SH, human colon cancer cell line HCT116 and human glioma cell line U251. After entering tumor cells the fusion protein bonded specifically with p21Ras. In vitro experiments revealed that it could significantly inhibit the proliferation, migration, and colone formation of HCT116, SK-N-SH, and U251 cells and promote the apoptosis of these tumor cells.ConclusionsBR2-p21Ras scFv can penetrate ganglioside expressing tumor cells and inhibit the growth of ras-driven tumor by binding with p21Ras, and producing an inhibitory effect. It is suggested that BR2-p21Ras scFv is a potential ras-driven tumor therapeutic antibody.

GlycoTAIL and FlexiTAIL as Half-Life Extension Modules for Recombinant Antibody Fragments.

Many therapeutic proteins are small in size and are rapidly cleared from circulation. Consequently, half-life extension strategies have emerged to improve pharmacokinetic properties, including fusion or binding to long-lasting serum proteins, chemical modifications with hydrophilic polymers such as PEGylation, or, more recently, fusion to PEG mimetic polypeptides. In the present study, two different PEG mimetic approaches, the GlycoTAIL and the FlexiTAIL, were applied to increase the hydrodynamic radius of antibody fragments of different sizes and valencies, including scFv, diabody, and scFv-EHD2 fusion proteins. The GlycoTAIL and FlexiTAIL sequences of varying lengths are composed of aliphatic and hydrophilic residues, with the GlycoTAIL furthermore comprising N-glycosylation sites. All modified proteins could be produced in a mammalian expression system without reducing stability and antigen binding, and all modified proteins exhibited a prolonged half-life and increased drug disposition in mice. The strongest effects were observed for proteins comprising a FlexiTAIL of 248 residues. Thus, the GlycoTAIL and FlexiTAIL sequences represent a flexible and modular system to improve the pharmacokinetic properties of proteins.

Inhibition of human lung cancer cells by anti-p21Ras scFv mediated by the activatable cell-penetrating peptide.

Activatable cell-penetrating peptide (ACPP) is a tumour-targeting cell-penetrating peptide. Here, we used ACPP to carry anti-p21Ras scFv for Ras-driven cancer therapy. The ACPP-p21Ras scFv fusion protein was prepared by a prokaryotic expression system and Ni-NTA column purification. The human tumour cell lines A549, SW480, U251 and Huh7 and the normal cell line BEAS 2B were used to study the tumor-targeting and membrane-penetrating ability of ACPP-p21Ras scFv. The antitumour activity of ACPP-p21Ras scFv on A549 cells and H1299 cells in vitro was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, scratch wound healing, plate cloning and apoptosis assays. The penetration pathway of ACPP was determined by enhanced green fluorescent protein. The ACPP-p21Ras scFv fusion protein was successfully obtained at a concentration of 1.8 mg/ml. We found that ACPP-p21Ras scFv could penetrate tumour cell membranes with high expression of matrix metalloproteinase-2 (MMP-2), effectively inhibit the migration and proliferation of A549 cells and H1299 cells, and promote the apoptosis of A549 cells and H1299 cells. The membrane penetration experiment demonstrated that ACPP could enter A549 cells by direct penetration. The ability of ACPP to penetrate the membrane was affected by the addition of a membrane affinity inhibitor and a change in the potential difference across the cell membrane but not by the addition of endocytosis inhibitors and a change in temperature. The ACPP-p21Ras scFv fusion protein can penetrate tumour cells with MMP-2 expression and has antitumour activity against A549 cells and H1299 cells in vitro. This molecule is expected to become a potential antitumour drug for Ras gene-driven lung cancer.

A Novel Method for Highly Sensitive Detection and Isolation of Chimeric Antigen Receptor and Cancer Associated Antigen-Expressing Cells

Chimeric antigen receptor (CAR)-based cellular therapy is a revolutionary approach to treat cancer as witnessed by recent success in clinical trials for various hematological malignancies. Currently, flow cytometry based detection of fluorochrome-tagged antibodies or proteinL that binds to the Extra-Cellular Domain (ECD) of CAR molecule are the widely used methods for the detection of CARexpression. Here, we have developed a novel luciferase based assay for detecting the expression of CAR. Our assay is accurate, highly sensitive (10-5), and has a broad linearity by taking advantage of the extreme brightness of recently discovered marine luciferases (Gluc/Nluc/Tluc16/Mluc/Loluc/Paluc/Htluc). The assay is based on recombinant fusion protein technology by fusing the ECD of a CAR target in frame with one of the marine luciferases (for detection) along with several small peptide tags -Flag/ Strep -tag II/AcV5/His (for isolation). Initially, a fusion construct was made by cloning the ECD of CD19 fused in frame with Nluc. The fusion protein was produced using 293FT cells, and tested by a simple binding assay that involved 45 minutes incubation at 4oC followed by washing and detection of luminescence. More than 103 fold increases in luminescence was observed between FMC63-CARtransduced-T/NK cells and uninfected cells or a non-specific CAR transduced cells. Essentially, identical results were obtained by replacing Nluc with other marine luciferases or by using cells transduced with five distinct CARs targeting CD19. Similar strategy was successfully applied for the specific detection of CARs targeting CD20, CD30, CD33, CD123, CD138, BCMA, and SLAM7. We also show that the small peptide tags in the fusion protein can be used for the specific isolation of CAR+ve cells using anti-tag antibodies by FACS. Additionally, we purified ECD-fusion proteins for CD19 and CD33 using Strep -Tactin protein purification columns. Purified fusion proteins were fully active, as observed by successful and specific binding to respective CAR-T/NK cells. Furthermore, direct conjugation of purified ECD-fusion proteins with fluorochromes resulted in a single-step detection and/or isolation of CAR+vecells.In another embodiment, an antigen positive cell can be detected by fusing the single-chain fragment variable (scFv) of its specific monoclonal antibody with one of the marine luciferases. A fusion construct was made by cloning scFv of a CD19 antibody clone (FMC63) fused in frame with Nluc, followed by the fusion protein production and binding assay. Again, a 103 fold increase in luminescence was observed between CD19+ve cell lines (Raji, Nalm6, and BV173) and CD19-ve cell lines (HL60 or Raji-CD19-/- by Crispr-Cas9). Similar results were obtained using other marine luciferases in place of Nluc or by replacing FMC63 with five distinct scFv targeting CD19. Next, we made fusion proteins using scFv of antibody clones targeting CD20, CD30, CD33, CD123, CD138, BCMA, and SLAM7 and tested by binding assay using a panel of cell lines comprising 8 hematological malignancies. Strikingly, all the scFv fusion proteins bound strictly to their respective antigen+ve cell lines.Until now, only a limited number of targets have been pursued using CAR technology, which can be attributed to the lack of a High Throughput Screening (HTS)-based approach to find novel targets. To check the compatibility of our scFv-luciferase fusion protein technology as a HTS approach to discover novel CAR targets, a library of 78 distinct scFv-Nluc constructs were made, followed by the production of fusion proteins. Binding assays were performed in 96-well plates using Raji, BV173, MM1S, and U266 cell lines. Fusion proteins which are supposed to bind their respective targets were readily identified by the assay - Raji (CD19 and CD20 targeting scFv); BV173 (CD19 and CD33 targeting scFv); MM1S and U266 (CD138, BCMA, and SLAM7 targeting scFv). Most importantly, few novel CAR targets were identified thereby opening up the possibility of applying this screening approach to patient tumor cells, that may result in precision medicine model in cellular-therapy space by tailoring CAR therapy for individual patients. Finally, we demonstrate the utility of scFv-luciferase fusion protein technology in accurately detecting the serial engraftment of B-ALL and AML-patient derived xenografts in NSG mice using scFv of antibodies targeting CD19 and CD33, respectively. DisclosuresChaudhary:University of Southern California: Patents & Royalties: P.M.C. have filed patent application related to the technology described in this manuscript.

Novel Bispecific Antibody Targeting CD45 and 90Y-DOTA Is Effective Therapy for Acute Myeloid Leukemia in Preclinical Murine Models

Background : Acute myeloid leukemia (AML) is a heterogeneous disease with a variety of chromosomal and molecular aberrations, rendering AML a challenging disease to cure with targeted agents. Despite the genetic variability, AML uniformly responds to radiation therapy. We have previously shown therapeutic efficacy targeting CD45+ AML using the beta-emitter 90Y and a streptavidin-biotin (SA) pretargeted radioimmunotherapy (PRIT) system. Theoretical concerns, such as endogenous biotin in humans, may limit translation of this approach to the clinic. To circumvent such hurdles, we developed bispecific antibody constructs targeting both CD45 and 90Y-labeled-DOTA-biotin, and evaluated this approach in murine models of AML.Methods : We constructed a pair of plasmids using the pfuse system that code for heavy and light chains for antibody bispecific to murine CD45 (30F11) and Y-DOTA (C825). Plasmid pairs were co-transfected into HEK 293T cells for production and purificaiton of the bispecific antibody. Binding to CD45 and 90Y-labeled-DOTA-biotin was confirmed, after which biodistribution studies with 30F11-Fc-C825 were performed in a disseminated murine leukemia model. B6SJLF1 mice (n=5 per group) were injected intravenously with 105 SJL murine leukemia cells, followed 2 days later with 0.67 nmol 30F11-Fc-C825, then 5 μg DOTA-Y-dextran clearing agent 23 hr later to clear any unbound bispecific construct from circulation. Mice were then injected with 50 µCi 90Y-DOTA-biotin IP 24 hr after the bispecific antibody infusion. Tissues were harvested at 6 and 24 hr after radiolabeled DOTA-biotin, and then analyzed for radioactivity to calculate the percent injected dose per gram of tissue (% ID/g).Results : Target organs (spleen and bone marrow) from leukemia bearing mice treated with 30F11-Fc-C825 had 9.0 ± 1.5 and 8.1 ± 1.2% ID/g, respectively, 24 hr post injection. Non-target organs such as kidneys and lungs each had <0.5% ID/g at this time point. Targeting translated into a therapeutic benefit, as mice bearing SJL leukemia treated with 30F11-Fc-C825 and 800 µCi of 90Y-DOTA-biotin had median overall survival of 43 days, compared to leukemic control mice (30 days, p<0.0001).To investigate this approach in a clinically relevant human leukemia model, an anti-human CD45 (BC8) scFv fusion protein was similarly engineered onto a high-affinity anti-Y-DOTA (C825) scFv fusion protein. This bispecific construct (BC8-Fc-C825) was expressed and purified from CHO cells. Binding to human CD45 and 90Y-labeled-DOTA-biotin was confirmed by flow cytometry. In biodistribution studies, female athymic mice (n=5 per group) were injected subcutaneously with 107 human AML cells (HEL). Mice were then injected intravenously with 1.4 nmol BC8-Fc-C825, BC8-SA, or non-targeting CC49-Fc-C825 as the first step before receiving clearing agent. Radiolabeled 90Y-DOTA-biotin (50 µCi) was injected IP 24 hr after bispecific delivery. HEL tumors from mice treated with BC8-Fc-C825 showed peak uptake at 24 hr post injection with 7.75 ± 0.02% ID/g, with minimal uptake in non-target organs [e.g., 0.5 ± 0.3% ID/g in kidneys]. This compared favorably with HEL tumors from mice treated with BC8-SA as the first step, which had peak uptake of 6.7 ± 0.01% ID/g 24 hr post-injection. Therapy studies took athymic mice (n=10 per group) that had been given subcutaneous HEL xenografts, before receiving BC8-Fc-C825, BC8-SA, or non-binding control CC49-Fc-C825. Treated mice were given clearing agent, followed by 800 to 1500 µCi of 90Y-labeled DOTA-biotin. Mice bearing HEL tumors treated with BC8-Fc-C825 followed by 1400 µCi of 90Y-DOTA-biotin, resulted in 6 of 10 mice surviving 170 days post-injection, whereas untreated controls and HEL-bearing mice treated with non-targeting negative control CC49-Fc-C825 and 1400 µCi of 90Y-DOTA-biotin required euthanasia due to tumor size by day 26 and 32, respectively.Conclusion : Our bispecific constructs both showed selectivity for CD45+ targets and 90Y-DOTA. In two leukemia models, targeting of 90Y-DOTA to target hematopoietic tissues translated into survival benefits. Given the therapeutic efficacy, bispecific constructs with the capacity to pretarget CD45 and capture 90Y-DOTA should be added to the armamentarium of PRIT approaches. [Display omitted] DisclosuresOrozco:Actinium Pharmaceuticals: Other: Research Funding to Institution for sponsored Clinical Trials. Till:Mustang Bio: Patents & Royalties, Research Funding; Genentech: Research Funding. Gopal:Seattle Genetics: Consultancy, Research Funding. Orcutt:inviCRO: Employment, Equity Ownership. Pagel:Actinium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Press:Roche: Honoraria, Research Funding; BMS: Honoraria; Bayer: Consultancy.

Development of an Antigen-Antibody Co-Display System for Detecting Interaction of G-Protein-Coupled Receptors and Single-Chain Variable Fragments.

G-protein-coupled receptors (GPCRs), especially chemokine receptors, are ideal targets for monoclonal antibody drugs. Considering the special multi-pass transmembrane structure of GPCR, it is often a laborious job to obtain antibody information about off-targets and epitopes on antigens. To accelerate the process, a rapid and simple method needs to be developed. The split-ubiquitin-based yeast two hybrid system (YTH) was used as a blue script for a new method. By fusing with transmembrane peptides, scFv antibodies were designed to be anchored on the cytomembrane, where the GPCR was co-displayed as well. The coupled split-ubiquitin system transformed the scFv-GPCR interaction signal into the expression of reporter genes. By optimizing the topological structure of scFv fusion protein and key elements, including signal peptides, transmembrane peptides, and flexible linkers, a system named Antigen-Antibody Co-Display (AACD) was established, which rapidly detected the interactions between antibodies and their target GPCRs, CXCR4 and CXCR5, while also determining the off-target antibodies and antibody-associated epitopes. The AACD system can rapidly determine the association between GPCRs and their candidate antibodies and shorten the research period for off-target detection and epitope identification. This system should improve the process of GPCR antibody development and provide a new strategy for GPCRs antibody screening.

APDL1-CART cells exhibit strong PD-L1-specific activity against leukemia cells.

Chimeric antigen receptor (CAR) T cells target specific tumor antigens and lyse tumor cells in an MHC-independent manner. However, the efficacy of CAR-T cell and other cancer immunotherapies is limited by the expression of immune-checkpoint molecules such as programmed death-ligand 1 (PD-L1) on tumor cells, which binds to PD-1 receptors on T cells leading to T cell inactivation and immune escape. Here, we incorporated a PD-L1-targeted single-chain variable fragment (scFv) fusion protein sequence into a CAR vector to generate human anti-PD-L1-CAR-T cells (aPDL1-CART cells) targeting the PD-L1 antigen. Unlike control T cells, aPDL1-CART cells significantly halted the expansion and reduced the viability of co-cultured leukemia cells (Raji, CD46, and K562) overexpressing PD-L1, and this effect was paralleled by increased secretion of IL-2 and IFN-γ. The antitumor efficacy of aPDL1-CART cells was also evaluated in vivo by co-injecting control T cells or aPDL1-CART cells along with PDL1-CA46 cells to generate subcutaneous xenografts in NCG mice. Whereas large tumors developed in mice inoculated with PDL1-CA46 cells alone or together with control T cells, no tumor formation was detected in xenografts containing aPDL1-CART cells. Our data suggest that immune checkpoint-targeted CAR-T cells may be useful for controlling and eradicating immune-refractory hematological malignancies.

Evaluation of Hydra HALT-1 as a toxin moiety for recombinant immunotoxin

BackgroundImmunotoxin is a hybrid protein consisting of a toxin moiety that is linked to a targeting moiety for the purpose of specific elimination of target cells. Toxins used in traditional immunotoxins are practically difficult to be produced in large amount, have poor tissue penetration and a complex internalization process. We hypothesized that the smaller HALT-1, a cytolysin derived from Hydra magnipapillata, can be used as the toxin moiety in construction of a recombinant immunotoxin.ResultsIn this study, pro-inflammatory macrophage was selected as the target cell due to its major roles in numerous inflammatory and autoimmune disorders. We aimed to construct macrophage-targeted recombinant immunotoxins by combining HALT-1 with anti-CD64-scFv in two orientations, and to assess whether their cytotoxic activity and binding capability could be preserved upon molecular fusion. The recombinant immunotoxins, HALT-1-scFv and scFv-HALT-1, were successfully constructed and expressed in Escherichia coli (E. coli). Our data showed that HALT-1 still exhibited significant cytotoxicity against CD64+ and CD64− cell lines upon fusion with anti-CD64 scFv, although it had half cytotoxic activity as compared to HALT-1 alone. As positioning HALT-1 at N- or C-terminus did not affect its potency, the two constructs demonstrated comparable cytotoxic activities with IC50 lower in CD64+ cell line than in CD64− cell line. In contrast, the location of targeting moieties anti-CD64 scFv at C-terminal end was crucial in maintaining the scFv binding capability.ConclusionsHALT-1 could be fused with anti-CD64-scFv via a fsexible polypeptide linker. Upon the successful production of this recombinant HALT-1 scFv fusion protein, HALT-1 was proven effective for killing two human cell lines. Hence, this preliminary study strongly suggested that HALT-1 holds potential as the toxin moiety in therapeutic cell targeting.

Anthrax Protective Antigen Retargeted with Single-Chain Variable Fragments Delivers Enzymes to Pancreatic Cancer Cells.

The nontoxic, anthrax protective antigen/lethal factor N-terminal domain (PA/LFN ) complex is an effective platform for translocating proteins into the cytosol of cells. Mutant PA (mPA) was recently fused to epidermal growth factor (EGF) to retarget delivery of LFN to cells bearing EGF receptors (EGFR), but the requirement for a known cognate ligand limits the applicability of this approach. Here, we render practical protective antigen retargeting to a variety of receptors with mPA single-chain variable fragment (scFv) fusion constructs. Our design enables the targeting of two pancreatic cancer-relevant receptors, EGFR and carcinoembryonic antigen. We demonstrate that fusion to scFvs does not disturb the basic functions of mPA. Moreover, mPA-scFv fusions enable cell-specific delivery of diphtheria toxin catalytic domain and Ras/Rap1-specific endopeptidase to pancreatic cancer cells. Importantly, mPA-scFv fusion-based treatments display potent cell-specific toxicity in vitro, opening fundamentally new routes toward engineered immunotoxins and providing a potential solution to the challenge of targeted protein delivery to the cytosol of cancer cells.

The Use of a Shear Device to Monitor the Stability of a Single-Chain Variable Fragment (scFv) Fusion Protein MFECP1

Antibody based drugs are increasingly being used to treat a vast array of diseases because of their unique affinity to target specific antigen proteins on the surfaces of target cancer cells. Fusions of antibodies and conjugated biopharmaceuticals are progressively being used as this gives the opportunity to target other cytotoxic molecules to unwanted cells. It is critical to ensure these types of drug products are not fragile or uneconomical to produce at a large scale. A very small amount of precious protein solution can be characterised in an Ultra scale-down (USD) shear device to uncover if fusion proteins are prone to shear stress. This article presents how the purified and deglycosylated form of the MFECP1 fusion protein was quantified with an ELISA from 700-50 ng/ml, with a +/- 10% deviation in the standard curve. It also describes how the same MFECP1 fusion protein was analysed to establish the optimum experimental control conditions that were required to observe changes due to hydrodynamic-associated degradation in a shear device. Lastly, it looks at how a first order kinetic relationship can be used to model the rate of MFECP1 fusion protein degradation and how this was used to quantify the rate of protein loss during different shear environments with and without air/liquid interfaces.

Recombinant fusion proteins for targeting dendritic cell subsets in therapeutic cancer vaccine.

Dendritic cells (DCs) are professional antigen-presenting cells, which are optimal for the priming of a T cell response against pathogens and tumors. Therefore, many efforts are made to develop therapeutic cancer vaccines which preferentially target the antigen to DC subsets. To this aim, we developed two types of recombinant fusion proteins, which favor antigen delivery to pro-inflammatory DCs as well as the crosstalk between specialized subpopulations of DCs. The first approach combines peptide/CpG vaccination with the recruitment of iNKT cells to the tumor site via CD1d-antitumor scFv fusion proteins. The second approach is targeting the tumor antigen to cross-presenting Xcr1+ DCs via a fusion protein made of Xcl1 fused to a synthetic long peptide followed by an IgG1 Fc fragment. Both strategies allow a potent tumor-specific CD8 T cell response associated with tumor regression or tumor growth delay depending on the model. In the case of iNKT cell activation, the strategy relies on a strong IL-12 release by splenic DCs, while in the second case, the T cell response is strictly dependent on the presence of Xcr1+ cross-presenting DCs.

Efficient development and expression of scFv recombinant proteins against PD-L1 surface domain and potency in cancer therapy.

PD-L1 is a 40kDa trans-membrane protein of B7 family and an important T cell regulator. Binding of PD-L1 and PD-1 inhibits proliferation and activation of T cell results cell exhaustion. This phenomenon can be reversed by blocking PD-L1/PD-1 interactions with single chain variables fragment (scFv) fusion proteins and by direct inhibition of tumor cells with drug conjugates. The human phage-displayed scFv library was utilized to generate scFv against the PD-L1 antigen by affinity bio-panning. The positive clones were selected by continuous transfection of bacterial cells and sequence analysis. The binding affinity and specificity of the scFv and antibody fragments were determined by using surface plasmon resonance biosensor, western blot analysis, and immunofluorescence assay. After three rounds of panning selection, about 30% of clones have a binding affinity with targeted PD-L1 antigen. Eight positive clones with accurate sequences were isolated and analyzed for binding affinity with PD-L1 antigen. Three of those with accurate sequences and binding affinity were selected for the recombinant formation and soluble expression by Escherichia coli host machinery. The highly positive recombinant clones with the exact orientation of FR and CDR domains were developed and can be used as a drug carrier tools in ADC formation or direct inhibition of immune checkpoint in cancer immunotherapy. The conjugate achieved its initial potency and need efficient improvement to enhance direct tumor suppression and bio-therapeutics strategies enrichment.

Bispecific anti-mPDGFRβ x cotinine scFv-Cκ-scFv fusion protein and cotinine-duocarmycin can form antibody-drug conjugate-like complexes that exert cytotoxicity against mPDGFRβ expressing cells

Antibody selection for antibody-drug conjugates (ADCs) has traditionally depended on its internalization into the target cell, although ADC efficacy also relies on recycling of the receptor-ADC complex, endo-lysosomal trafficking, and subsequent linker/antibody proteolysis. In this study, we observed that a bispecific anti-murine platelet-derived growth factor receptor beta (mPDGFRβ) x cotinine single-chain variable fragment (scFv)-kappa constant region (Cκ)-scFv fusion protein and cotinine-duocarmycin can form an ADC-like complex to induce cytotoxicity against mPDGFRβ expressing cells. Multiple anti-mPDGFRβ antibody candidates can be produced in this bispecific scFv-Cκ-scFv fusion protein format and tested for their ability to deliver cotinine-conjugated cytotoxic drugs, thus providing an improved approach for antibody selection in ADC development.

A targeted immunotherapy approach for HER2/neu transformed tumors by coupling an engineered effector domain with interferon-γ

ABSTRACT Despite substantial clinical progress with targeted therapies, current antibody-based approaches have limited efficacy at controlling HER2/neu-positive breast cancers, especially in the absence of chemotherapies. Previously, we showed that the combination of IFNγ and anti-HER2/neu antibody synergistically reduces tumor growth in an in vivo implanted mammary tumor model. Here, we report a recombinant approach to produce an anti-HER2/neu scFv and IFNγ fusion protein using an engineered effector domain (EED) scaffold. The new molecule induces in vitro apoptosis in an IFNγ receptor-dependent manner. At a very low dose in the in vivo xenografted tumor models, the new EED-IFNγ fusion protein demonstrates superior activity over the anti-HER2/neu antibody and is even active on tumors that are resistant to anti-HER2/neu antibody therapy. Examination of tumor infiltrated macrophages and lymphocytes reveals that the fusion protein can induce changes in tumor microenvironment to support immune reactivity against tumors. Our studies have defined a targeted immunotherapy approach for the treatment of cancers.

Tumour endothelial marker 1/endosialin-mediated targeting of human sarcoma

BackgroundTumour endothelial marker 1 (TEM1/endosialin/CD248) is a tumour-restricted cell-surface protein expressed by human sarcomas. We previously developed a high-affinity human single-chain variable fragment (scFv)-Fc fusion protein (78Fc) against TEM1 and demonstrated its specific binding to human and mouse TEM1. Patient and methodsClinical sarcoma specimens were collected between 2000 and 2015 at the Hospital of the University of Pennsylvania, as approved by the institutional review board and processed by standard formalin-fixed paraffin embedded techniques. We analysed TEM1 expression in 19 human sarcoma subtypes (n = 203 specimens) and eight human sarcoma–cell lines. Near-infrared (NIR) imaging of tumour-bearing mice was used to validate 78Fc binding to TEM1+ sarcoma in vivo. Finally, we tested an immunotoxin conjugate of anti-TEM1 78Fc with saporin (78Fc-Sap) for its therapeutic efficacy against human sarcoma in vitro and in vivo. ResultsTEM1 expression was identified by immunohistochemistry in 96% of human sarcomas, of which 81% expressed TEM1 both on tumour cells and the tumour vasculature. NIR imaging revealed specific in vivo targeting of labelled 78Fc to TEM1+ sarcoma xenografts. Importantly, 78Fc-Sap was effective in killing in vitro TEM1+ sarcoma cells and eliminated human sarcoma xenografts without apparent toxicity in vivo. ConclusionTEM1 is an important therapeutic target for human sarcoma, and the high-affinity TEM1-specific scFv fusion protein 78Fc is suitable for further clinical development for therapeutic applications in sarcoma.