Single-chain Diabody Research Articles

An IL-7 fusion protein targeting EDA fibronectin upregulates TCF1 on CD8+ T-cells, preferentially accumulates to neoplastic lesions, and boosts PD-1 blockade

BackgroundAnti-PD-1 antibodies have revolutionized cancer immunotherapy due to their ability to induce long-lasting complete remissions in a proportion of patients. Current research efforts are attempting to identify biomarkers and suitable...

Optimizing the Design and Geometry of T Cell-Engaging Bispecific Antibodies Targeting CEA in Colorectal Cancer.

Metastatic colorectal cancer remains a leading cause of cancer-related deaths, with a 5-year survival rate of only 15%. T cell-engaging bispecific antibodies (TCBs) represent a class of biopharmaceuticals that redirect cytotoxic T cells toward tumor cells, thereby turning immunologically "cold" tumors into "hot" ones. The carcinoembryonic antigen (CEA) is an attractive tumor-associated antigen that is overexpressed in more than 98% of patients with colorectal cancer. In this study, we report the comparison of four different TCB formats employing the antibodies F4 (targeting human CEA) and 2C11 (targeting mouse CD3ε). These formats include both antibody fragment-based and IgG-based constructs, with either one or two binding specificities of the respective antibodies. The 2 + 1 arrangement, using an anti-CEA single-chain diabody fused to an anti-CD3 single-chain variable fragment, emerged as the most potent design, showing tumor killing at subnanomolar concentrations across three different CEA+ cell lines. The in vitro activity was three times greater in C57BL/6 mouse colon adenocarcinoma cells (MC38) expressing high levels of CEA compared with those expressing low levels, highlighting the impact of CEA density in this assay. The optimal TCB candidate was tested in two different immunocompetent mouse models of colorectal cancer and showed tumor growth retardation. Ex vivo analysis of tumor infiltrates showed an increase in CD4+ and CD8+ T cells upon TCB treatment. This study suggests that bivalent tumor targeting, monovalent T-cell targeting, and a short spatial separation are promising characteristics for CEA-targeting TCBs.

Bispecific antibodies promote natural killer cell-mediated elimination of HIV-1 reservoir cells

The persistence of CD4+ T cells carrying latent human immunodeficiency virus-1 (HIV-1) proviruses is the main barrier to a cure. New therapeutics to enhance HIV-1-specific immune responses and clear infected cells will probably be necessary to achieve reduction of the latent reservoir. In the present study, we report two single-chain diabodies (scDbs) that target the HIV-1 envelope protein (Env) and the human type III Fcγ receptor (CD16). We show that the scDbs promoted robust and HIV-1-specific natural killer (NK) cell activation and NK cell-mediated lysis of infected cells. Cocultures of CD4+ T cells from people with HIV-1 on antiretroviral therapy (ART) with autologous NK cells and the scDbs resulted in marked elimination of reservoir cells that was dependent on latency reversal. Treatment of human interleukin-15 transgenic NSG mice with one of the scDbs after ART initiation enhanced NK cell activity and reduced reservoir size. Thus, HIV-1-specific scDbs merit further evaluation as potential therapeutics for clearance of the latent reservoir.

TCR-mimic bispecific antibodies to target the HIV-1 reservoir

HIV-1 infection is incurable due to the persistence of the virus in a latent reservoir of resting memory CD4+ T cells. “Shock-and-kill” approaches that seek to induce HIV-1 gene expression, protein production, and subsequent targeting by the host immune system have been unsuccessful due to a lack of effective latency-reversing agents (LRAs) and kill strategies. In an effort to develop reagents that could be used to promote killing of infected cells, we constructed T cell receptor (TCR)-mimic antibodies to HIV-1 peptide-major histocompatibility complexes (pMHC). Using phage display, we panned for phages expressing antibody-like variable sequences that bound HIV-1 pMHC generated using the common HLA-A*02:01 allele. We targeted three epitopes in Gag and reverse transcriptase identified and quantified via Poisson detection mass spectrometry from cells infected in vitro with a pseudotyped HIV-1 reporter virus (NL4.3 dEnv). Sequences isolated from phages that bound these pMHC were cloned into a single-chain diabody backbone (scDb) sequence, such that one fragment is specific for an HIV-1 pMHC and the other fragment binds to CD3ε, an essential signal transduction subunit of the TCR. Thus, these antibodies utilize the sensitivity of T cell signaling as readouts for antigen processing and as agents to promote killing of infected cells. Notably, these scDbs are exquisitely sensitive and specific for the peptide portion of the pMHC. Most importantly, one scDb caused killing of infected cells presenting a naturally processed target pMHC. This work lays the foundation for a novel therapeutic killing strategy toward elimination of the HIV-1 reservoir.

Design and Characterization of Novel Antibody-Cytokine Fusion Proteins Based on Interleukin-21.

Interleukin-21 (IL21) is a pleiotropic cytokine involved in the modulation of both innate and adaptive immunity. IL21 is mainly secreted by natural killer (NK) and activated CD4+ T-cells. The biology of this cytokine can be associated to proinflammatory responses reflecting its potent stimulatory activity of NK and CD8+ T-cells. Here we describe four formats of novel IL21-based antibody–cytokine fusion proteins, targeting the extra domain A (EDA) of fibronectin and explore their potential for cancer treatment. The fusion proteins were designed, expressed, and characterized. F8 in single-chain diabody (scDb) format fused to IL21 at its C-terminus exhibited a promising profile in size exclusion chromatography (SEC) and SDS-PAGE. The lead candidate was further characterized in vitro. A cell-based activity assay on murine cytotoxic T-cells showed that human IL21, compared to murine IL21 partially cross-reacted with the murine receptor. The prototype was able to recognize EDA as demonstrated by immunofluorescence analysis on tumor sections. In an in vivo quantitative biodistribution experiment, F8(scDb)-murine IL21 did not preferentially accumulate at the site of disease after intravenous injection, suggesting that additional protein engineering would be required to improve the tumor-homing properties of IL21-based product.

Structural arrangement of the VH and VL domains in the COBRA™ T-cell engaging single-chain diabody.

COBRA™ (COnditional Bispecific Redirected Activation) T-cell engagers are designed to target solid tumors as a single polypeptide chain prodrug that becomes activated by proteolysis in the tumor microenvironment. One COBRA molecule comprises seven Ig domains: three single-domain antibodies (sdAbs) recognizing a tumor target or human serum albumin (HSA), and CD3ε-binding variable fragment heavy chain (VH) and variable fragment light chain (VL) and their inactivated counterparts, VHi and VLi. Pairing of VH and VL, and VLi and VHi into single-chain variable fragments (Fv) is prevented by shortened inter-domain linkers. Instead, VH and VL are expected to interact with VLi and VHi, respectively, thus making a diabody whose binding to CD3ε on the T-cells is impaired. We analyzed the structure of an epidermal growth factor receptor (EGFR) COBRA in solution using negative stain electron microscopy (EM) and small-angle X-ray scattering (SAXS). We found that this EGFR COBRA forms stable monomers with a very dynamic interdomain arrangement. At most, only five domains at a time appeared ordered, and only one VH-VL pair was found in the Fv orientation. Nonenzymatic posttranslational modifications suggest that the CDR3 loops in the VL-VHi pair are exposed but are buried in the VH-VLi pair. The MMP9 cleavage rate of the prodrug when bound to recombinant EGFR or HSA is not affected, indicating positioning of the MMP9-cleavable linker away from the EGFR and HSA binding sites. Here, we propose a model for EGFR COBRA where VH and VLi form an Fv, and VL and VHi do not, possibly interacting with other Ig domains. SAXS and MMP9 cleavage analyses suggest that all COBRA molecules tested have a similar structural architecture.

Detection of salinomycin and lasalocid in chicken liver by icELISA based on functional bispecific single-chain antibody (scDb) and interpretation of molecular recognition mechanism

Salinomycin (SAL) and lasalocid (LAS) are widely used as ionophore antibiotics for coccidiosis control. However, their common use as feed additives has led to the occurrence of feed cross-contamination, which has toxic effects on non-target animals. There have been few reports on multiple-residue detection for SAL and LAS in recent years. In this study, two single-chain antibody fragments (scFvs) capable of specifically recognizing SAL and LAS were constructed. Using LAS-scFv and SAL-scFv as parent antibodies, a complete bispecific single-chain diabody (scDb) against both LAS and SAL was built using splicing by overlap extension polymerase chain reaction (SOE-PCR). In addition, the key amino acid sites and interaction energy of antibody variable regions for small-molecule recognition were preliminarily studied by homology modeling and molecular docking. Finally, IC50 values of 12.9 and 8.6ng/mL, with a linear range of 6.9-24.0 and 4.7-16.0ng/mL, were obtained for LAS-scFv and SAL-scFv, respectively. An indirect competitive enzyme-linked immunosorbent assay (icELISA) method was established using scDb to obtain an IC50 of 3.5ng/mL for LAS and 4.1ng/mL for SAL, which showed better sensitivity and specificity than those of the parent scFv antibodies. The recoveries of LAS and SAL in chicken liver were 89.2-92.7%(CV<4.7%) and 88.6-90.2% (CV<6.8%)), respectively.

Selected Articles from This Issue

In this First Disclosure, Smith and colleagues describe ASN004, a Dolaflexin-containing antibody drug conjugate targeting the 5T4 oncofetal antigen expressed on a wide range of tumors. ASN004 contains a scFv-Fc antibody and a Auristatin F Hydroxypropylamide payload with a drug-to-antibody ratio of 10-12. In a single dose study, 1 mg/kg achieved complete tumor regression in a cervical cancer model. ASN004 was also shown to have greater activity than the HER2-targeted trastuzumab-DM1 in a gastric model with high HER2 expression despite low 5T4 expression. Pharmacokinetics revealed low free payload drug and dose-dependent exposure. Their results support the continued clinical development of ASN004.In this First Disclosure, Duranti and colleagues outline a single-chain diabody targeting the hERG1 potassium channel and the β1 subunit of integrin receptors (scDb-hERG1-β1). The diabody bound with high affinity to the two proteins only when they were linked together and spared normal cells. The scDb-hERG1-β1 was antiproliferative against tumor spheroids through inhibition of AKT and HIF-1α signaling. The diabody also showed a favorable pharmacokinetic and toxicologic profile when injected into mice. Taken together, the disclosure provides evidence for the continued clinical development of scDb-hERG1-β1.VEGF inhibitors and immune therapies have continued to create favorable outcomes for renal cell carcinoma (RCC) patients. For those who don't respond to these therap. options, Yang and colleagues hypothesized mTOR inhibition will remain a salvage option either by itself or in combined therapy. A recent clinical trial demonstrated mTOR inhibition combined with the vascular agent BNC105P failed to show increased efficacy compared to mTOR inhibition alone. In this study, they demonstrate the transcriptomic gene signatures of patients from that clinical study that correlated with benefit with mTOR inhibition. Their findings were then validated using the phase III CheckMate 025 study.There remains an urgent need for new drug discovery in pediatric cancer with an added goal of reducing long-term toxicity. In this article, Vassal and colleagues underline the outcome of a pediatric cancer international multistakeholder meeting to develop a consensus set of requirements for preclinical drug development in pediatric cancers. The recommendations include the use of cell-based and mouse models as well as proof-of-concept data packages for clinical development. They are designed to represent the minimum guidelines necessary for the international development of drugs for pediatric cancer within the new regulatory environment set up by the RACE act.

A scDb-based trivalent bispecific antibody for T-cell-mediated killing of HER3-expressing cancer cells

HER3 is a member of the EGF receptor family and elevated expression is associated with cancer progression and therapy resistance. HER3-specific T-cell engagers might be a suitable treatment option to circumvent the limited efficacy observed for HER3-blocking antibodies in clinical trials. In this study, we developed bispecific antibodies for T-cell retargeting to HER3-expressing tumor cells, utilizing either a single-chain diabody format (scDb) with one binding site for HER3 and one for CD3 on T-cells or a trivalent bispecific scDb-scFv fusion protein exhibiting an additional binding site for HER3. The scDb-scFv showed increased binding to HER3-expressing cancer cell lines compared to the scDb and consequently more effective T-cell activation and T-cell proliferation. Furthermore, the bivalent binding mode of the scDb-scFv for HER3 translated into more potent T-cell mediated cancer cell killing, and allowed to discriminate between moderate and low HER3-expressing target cells. Thus, our study demonstrated the applicability of HER3 for T-cell retargeting with bispecific antibodies, even at moderate expression levels, and the increased potency of an avidity-mediated specificity gain, potentially resulting in a wider safety window of bispecific T-cell engaging antibodies targeting HER3.

Harnessing the hERG1/β1 Integrin Complex via a Novel Bispecific Single-chain Antibody: An Effective Strategy against Solid Cancers.

mAbs, either mono- or bispecific (bsAb), represent one of the most successful approaches to treat many types of malignancies. However, there are certain limitations to the use of full length mAbs for clinical applications, which can be overcome by engineered antibody fragments. The aim of this study was to develop a small bsAb, in the format of a single-chain diabody (scDb), to efficiently target two proteins, the hERG1 potassium channel and the β1 subunit of integrin receptors, which specifically form a macromolecular complex in cancer cells. We provide evidence that the scDb we produced binds to the hERG1/β1 complex in cancer cells and tissues, but does not bind to the hERG1 channel in nonpathologic tissues, in particular the heart. The scDb-hERG1-β1 (i) downregulates the formation of the hERG1/β1 complex, (ii) inhibits Akt phosphorylation and HIF-1α expression, and (iii) decreases cell survival, proliferation, and migration in vitro These effects only occur in cancer cells (either colon, pancreatic, or breast), but not in normal cells. In vivo, the scDb-hERG1-β1 shows a good pharmacokinetic profile, with a half-life of 13.5 hours and no general, cardiac, or renal toxicity when injected intravenously up to the dose of 8 mg/kg. The scDb-hERG1-β1 accumulates into subcutaneous xenografted tumors, arising from either colon or pancreatic human cancer cells, and induces a reduction of tumor growth and vascularization. Overall, the scDb-hERG1-β1 represents an innovative single-chain bispecific antibody for therapeutic applications in solid cancers that overexpress the hERG1/β1 integrin signaling complex.

Expression and purification of a novel single-chain diabody (scDb-hERG1/β1) from Pichia pastoris transformants

In the last decades, protein engineering has developed particularly in biotechnology and pharmaceutical field. In particular, the engineered antibody subclass has arisen. The single chain diabody format (scDb), conjugating small size with antigen specificity, offers versatility representing a gold standard for a variety of applications, spacing from research to diagnostics and therapy. Along with such advantages, comes the challenge of optimizing their production, improving expression systems, purification procedures and stability. All such parameters are detrimental for protein production in general and above all for recombinant antibody expression, which has to be fine-tuned, choosing a proper protein-expression host and adjusting expression protocols accordingly. In the present paper, we present data regarding the production and purification of a single chain diabody directed against the macromolecular complex hERG1/β1 integrin. We focus on the expression of clones deriving from the transformation of Pichia pastoris yeast cells. In particular, we compare two different clones arose from two separate transformation processes, demonstrating that both are suitable for proper protein expression. Moreover, we have set up an expression protocol and compared the yields obtained using two purification machines: Akta Pure and Akta Start, with a positive outcome.

Diabodies see the unseeable

Immunotherapy RAS oncogene mutations are common in various cancers, controlling their growth and survival. Targeting mutant RAS proteins with antibodies has been unsuccessful because of low surface expression, even when targeting mutant RAS peptides presented by human leukocyte antigen (HLA) on the surface of cancer cells. Douglass et al. used phage display to generate single-chain variable fragments (scFvs) specific for mutant RAS peptide-HLA complexes. The authors tested various bispecific, T cell–engaging antibody formulations, finding that single-chain diabodies (scDbs) combining the aforementioned scFv with an anti-CD3 scFv were able to induce T cell activation and subsequent killing of tumor cells expressing mutant RAS peptide-HLA complexes. This scDb approach opens the door for antibody-based therapies against mutant neoantigens expressed at very low levels on the surface of cancer cells. Sci. Immunol. 6 , eabd5515 (2021).

Bispecific antibodies targeting mutant RAS neoantigens.

Mutations in the RAS oncogenes occur in multiple cancers, and ways to target these mutations has been the subject of intense research for decades. Most of these efforts are focused on conventional small-molecule drugs rather than antibody-based therapies because the RAS proteins are intracellular. Peptides derived from recurrent RAS mutations, G12V and Q61H/L/R, are presented on cancer cells in the context of two common human leukocyte antigen (HLA) alleles, HLA-A3 and HLA-A1, respectively. Using phage display, we isolated single-chain variable fragments (scFvs) specific for each of these mutant peptide-HLA complexes. The scFvs did not recognize the peptides derived from the wild-type form of RAS proteins or other related peptides. We then sought to develop an immunotherapeutic agent that was capable of killing cells presenting very low levels of these RAS-derived peptide-HLA complexes. Among many variations of bispecific antibodies tested, one particular format, the single-chain diabody (scDb), exhibited superior reactivity to cells expressing low levels of neoantigens. We converted the scFvs to this scDb format and demonstrated that they were capable of inducing T cell activation and killing of target cancer cells expressing endogenous levels of the mutant RAS proteins and cognate HLA alleles. CRISPR-mediated alterations of the HLA and RAS genes provided strong genetic evidence for the specificity of the scDbs. Thus, this approach could be applied to other common oncogenic mutations that are difficult to target by conventional means, allowing for more specific anticancer therapeutics.

Targeting a neoantigen derived from a common TP53 mutation.

TP53 (tumor protein p53) is the most commonly mutated cancer driver gene, but drugs that target mutant tumor suppressor genes, such as TP53, are not yet available. Here, we describe the identification of an antibody highly specific to the most common TP53 mutation (R175H, in which arginine at position 175 is replaced with histidine) in complex with a common human leukocyte antigen-A (HLA-A) allele on the cell surface. We describe the structural basis of this specificity and its conversion into an immunotherapeutic agent: a bispecific single-chain diabody. Despite the extremely low p53 peptide-HLA complex density on the cancer cell surface, the bispecific antibody effectively activated T cells to lyse cancer cells that presented the neoantigen in vitro and in mice. This approach could in theory be used to target cancers containing mutations that are difficult to target in conventional ways.

A Novel Antibody-IL15 Fusion Protein Selectively Localizes to Tumors, Synergizes with TNF-based Immunocytokine, and Inhibits Metastasis.

IL15 is an immunostimulatory cytokine that holds promises for cancer therapy, but its performance (alone or as partner for fusion proteins) has often been limited by suboptimal accumulation in the tumor and very rapid clearance from circulation. Most recently, the Sushi Domain (SD, the shortest region of IL15 receptor α, capable of binding to IL15) has been fused to IL15-based anticancer products to increase its biological activity. Here, we describe two novel antibody fusion proteins (termed F8-F8-IL15 and F8-F8-SD-IL15), specific to the alternatively spliced EDA domain of fibronectin (a marker of tumor neoangiogenisis, expressed in the majority of solid and hematologic tumors, but absent in normal healthy tissues) and featuring the F8 antibody in single-chain diabody format (with a short linker between VH and VL, thus allowing the domains to pair with the complementary ones of another chain). Unlike previously described fusions of the F8 antibody with human IL15, F8-F8-IL15 and F8-F8-SD-IL15 exhibited a preferential uptake in solid tumors, as evidenced by quantitative biodistribution analysis with radioiodinated protein preparations. Both products were potently active in vivo against mouse metastatic colon carcinomas and in sarcoma lesion in combination with targeted TNF. The results may be of clinical significance, as F8-F8-IL15 and F8-F8-SD-IL15 are fully human proteins, which recognize the cognate tumor-associated antigen with identical affinity in mouse and man.

Development of bispecific anti-c-Met/PD-1 diabodies for the treatment of solid tumors and the effect of c-Met binding affinity on efficacy

ABSTRACT Although the blockade of the programmed cell death protein 1/programmed cell death ligand 1 (PD-1/PD-L1) pathway has become a promising treatment strategy for several types of cancers, the constitutive activation of c-Met in tumors may cause a low overall response rate to PD-1 inhibitors. Increasing evidence indicates that the dual inhibition of c-Met and PD-1 could improve the efficacy of anti-PD-1/PD-L1 monoclonal antibodies for tumor immunotherapy. In this study, we developed two bispecific single-chain diabodies targeting c-Met and PD-1 for the treatment of solid tumors based on protein homology modeling, and we identified that the binding affinity of diabody-mp to c-Met was 50-folds higher than that of diabody-pm. The results of in vitro studies revealed that both diabodies suppressed HGF-induced proliferation, migration, and invasion of tumor cells, inhibiting the activation of c-Met signaling by antagonizing HGF binding to c-Met. Moreover, they promoted T cell activation by blocking the PD-1 pathway, mediating tumor cellular cytotoxicity through T cell engagement. In vivo studies with mice models demonstrated that diabody-mp exhibited higher therapeutic efficacy than other structural antibodies, greatly enhancing the survival of c-Met-positive tumor-bearing mice compared to single or combined c-Met and PD-1 blockade therapy. Furthermore, diabody-mp, which had a higher c-Met binding affinity, showed better anti-tumoral activity than diabody-pm, which had a lower c-Met binding affinity. In conclusion, bispecific anti-PD-1/c-Met diabody-mp, with high c-Met-associated affinity, inhibited tumor growth by activating T cells, suggesting its therapeutic potential for c-Met-positive solid tumors.

A novel format for recombinant antibody-interleukin-2 fusion proteins exhibits superior tumor-targeting properties in vivo

The targeted delivery of interleukin-2 to the tumor is gaining attention as an avenue to potentiate the action of T and NK cells at the site of disease. We have previously described the fusion of the L19 antibody, specific to the EDB domain of fibronectin, with human interleukin-2, using a non-covalent homodimeric diabody format. Here, we describe four novel formats for the L19-IL2 fusion, featuring different arrangements of antibody and IL2. A comparative quantitative biodistribution analysis in tumor-bearing mice using radioiodinated proteins revealed that the novel format (L19L19-IL2, with the antibody in single-chain diabody format) exhibited the best biodistribution results. In vitro assays on peripheral blood mononuclear cells showed a decrease activation of regulatory T cells when single IL2 domain was used. In vivo, both L19-IL2 and L19L19-IL2 inhibited tumor growth in immunocompetent mouse models of cancer. T-cell analysis revealed similar levels of CD4+ and FoxP3+ cells, with an expansion of the CD8+ T cell in mice treated with L19-IL2 and L19L19-IL2. The percentage of CD4+ regulatory T cells was markedly decreased with L19L19-IL2 combined with a mouse-specific PD-1 blocker. Collectively, these data indicate that the new L19L19-IL2 format exhibits favorable tumor-homing properties and mediates a potent anti-cancer activity in vivo.

Abstract 534: Discovery of an anti-claudin18.2/anti-PD-L1 bispecific antibody SPX-301 in the SMARTOPTM format

Abstract This research aimed to produce anti-claudin18.2 (CLDN 18.2)/anti-PD-L1 bispecific antibodies to enhance the therapeutic index and expand the responsive population of the parent anti-CLDN 18.2 and anti-PD-L1 antibodies through the mechanisms of tumor-specific distribution and synergistic tumor killing. CLDN 18.2 is highly expressed in several tumors but is minimally expressed in normal tissues and a chimeric IgG1 CLDN 18.2 antibody zolbetuximab significantly improved PFS and OS in gastric cancer patients in a CLDN 18.2-dependent manner on the background of EOX. However, the response rate was moderate (~10%), leaving room for further improvement. Anti-PD-L1 antibodies showed remarkable efficacy in lung cancer and melanoma but in various settings of gastric cancer they showed weaker benefits, which warrant optimization. We designed anti-claudin18.2/anti-PD-L1 bispecific antibodies in the SMARTOPTM format, where a single-chain diabody containing optimized CLDN 18.2 and PD-L1 binding variable domains is connected to the Fc domain by linkers that can be optimized by phage display libraries. The bispecific antibodies were selected by affinity at low pH values and slow off-rate to improve the therapeutic window by enhancing tumor retention and preserving or improving binding affinity in the low pH tumor microenvironment. Immunogenicity was optimized by mutating the surface residues and disulfide bonds were inserted to improve stability. A representative bispecific antibody SPX-301 showed comparable binding affinity to the CLDN 18.2 and PD-L1 antigens to the parent antibodies (SPX-101 and durvalumab, respectively). SPX-301 represents a novel anti-claudin18.2/anti-PD-L1 bispecific antibody in the SMARTOPTM format. The format demonstrated excellence retention of antigen binding affinity, high expression levels, minimal aggregation, and straightforward purification. The preclinical characterizations of SPX-301 will also be presented. Citation Format: Guidong Zhu, Jingdong Ye, Jichun Ma, Jingdong Qin, Yi Cai. Discovery of an anti-claudin18.2/anti-PD-L1 bispecific antibody SPX-301 in the SMARTOPTM format [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 534.

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.

Abstract 1216: Novel strategies in cancer immunotherapy: Harness the hERG1 β1 macromolecular complex via a new bispecific antibody and its bifunctional TRAIL

Abstract Introduction Among hindrances in cancer treatment, the lack of appropriate markers to be exploited for targeted therapy, and the need of new potential drugs are two big challenges.hERG1 potassium channels are a novel class of oncological targets and, in cancer, they are known to interact with integrins. It has been recently demonstrated that macromolecular complexes formed between hERG1 and β1 integrins selectively occurs in many types of cancer (Becchetti A et al., 2017). In this scenario, hERG1 could be exploited as a therapeutic target providing non cardiotoxic strategies aimed at blocking hERG1. Materials and Methods A scDb, a bifunctional single-chain diabody, directed against hERG1/β1 complex, was developed via SOE-PCR methodology. Such antibody was tested on HCT116 cells in lateral motility and western blotting experiments. Moreover immunohistochemistry (IHC) was performed on metastatic colorectal cancer (mCRC) paraffin embedded samples using the scDb, an anti-hERG1 and an anti-β1 integrin. Moreover, we improved the pro-apoptotic effects of the nude hERG1/β1-scDb, developing a trifunctional recombinant protein, in which the scDb-hERG1-β1 was linked to the tumour necrosis factor-related apoptosis-inducing ligand (TRAIL). Results and Discussion Performing IHC on sequential sections of mCRC confirmed the specificity of the scDb for both hERG1 and β1 integrin. In vitro data provide evidences that the administering of the bispecific antibody has an impact on lateral motility. Moreover, signaling pathways are also affected by the antibody treatment, as AKT phosphorylation and HIF1α levels are decreased when the molecule is administered, suggesting a possible effect of the bispecific antibody on the VEGF-A signaling pathway, which are consistent with our previous hypothesis (Becchetti A et al., 2017) of a possible cross-talk leading to a deep impact on VEGF expression and, thus, on neoangiogenesis. Encouraging results were obtained when testing the scDb-hERG1-β1 -TRAIL fusion protein, which was capable to produce a strong induction of apoptosis in ALL cells (32.6 vs 11% at 24h; 62.4% vs 10% at 48h), with no effects on TRAIL- resistant (Panc1) tumour cells. Conclusions scDb-hERG1/β1 could be used as a potential new therapeutic tool for cancer patients’ treatment as well as for early molecular diagnosis. In fact, the selective expression of hERG1/β1 complex in cancer cells and its role in angiogenesis and cancer progression suggests that a molecule selectively targeting the complex will be an invaluable tool for cancer treatment. In this view, we have recently licensed a patent which will be exploited with the final aim to undergo clinical trials. Becchetti A, et al. (2017) Science Signaling 10(473). Patent. Inventors: Arcangeli A, Duranti C. et al. Patent Ref: 102017000083637 Citation Format: Claudia Duranti, Jessica Iorio, Stefano Coppola, Giulia Petroni, Tiziano Lottini, Lara Magni, Elena Lastraioli, Annarosa Arcangeli. Novel strategies in cancer immunotherapy: Harness the hERG1 β1 macromolecular complex via a new bispecific antibody and its bifunctional TRAIL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1216.