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Schematic diagram and biochemical analysis of EI-04. (A) Schematic diagram of EI-04, composed of an anti-EGFR Fab linked to an effectorless human Fc with a stability-engineered anti-IGF-1R scFv attached at the C-terminus. (B) SDS-PAGE analysis of EI-04 under non-reducing (lane 2) and reducing (lane 3) conditions. (C) Analytical size exclusion chromatogram of EI-04. Static light scattering measurements indicate that the material is monomeric with MW ~200 kDa. (D) Differential scanning calorimetry (DSC) analysis of EI-04. Raw data are shown as a solid black line, while the deconvoluted peaks, each representing an EI-04 domain (Fab, CH2, CH3, scFv), are shown as dotted lines. The deduced domain assignments are indicated above each peak.
Source publication
The epidermal growth factor receptor (EGFR) and the type I insulin-like growth factor receptor (IGF-1R) are two cell surface receptor tyrosine kinases known to cooperate to promote tumor progression and drug resistance. Combined blockade of EGFR and IGF-1R has shown improved anti-tumor activity in preclinical models. Here, we report the characteriz...
Contexts in source publication
Context 1
... V H was attached to a chime- ric aglycosylated IgG4.P/IgG1 constant domain (agly IgG4.P/ IgG1), with the stability engineered anti-IGF-1R scFv appended to the carboxyl-terminus of the C H 3 domain through a flexible (GGGGS) 3 linker. The resulting bispecific antibody targeting EGFR and IGF-1R (EI-BsAb), denoted EI-04, is illustrated in Figure 1A. ...
Context 2
... similar to that of EI-04 and M60-A02, with binding affinities measured at 0.58 nM and 0.27 nM, respectively (data not shown). In addition, the BsAb showed the expected target specificity in vitro-EI-04 bound only to EGFR, and not to recombinant human ErbB fam- ily members HER2, HER3 or HER4 as determined in a biolayer interferometry assay (Sup. Fig. ...
Context 3
... following a two-step purification schema consisting of pro- tein A and ion exchange chromatography consistently resulted in greater than 1 g of highly purified bispecific antibody from 10-20 L of culture media. The product quality of the final purified bispecific antibody was generally very high with purity >97 % as assessed by SDS-PAGE and SEC (Fig. 1B and 1C). The thermal stability profile of purified EI-04 as assessed by differen- tial scanning calorimetry (DSC), shown in Figure 1D, confirms the T m of the stability-engineered BIIB5 scFv domain (~68°C) within the context of the final bispecific antibody format. 44 Stability studies of purified EI-04 BsAb showed the material to remain ...
Context 4
... product quality of the final purified bispecific antibody was generally very high with purity >97 % as assessed by SDS-PAGE and SEC (Fig. 1B and 1C). The thermal stability profile of purified EI-04 as assessed by differen- tial scanning calorimetry (DSC), shown in Figure 1D, confirms the T m of the stability-engineered BIIB5 scFv domain (~68°C) within the context of the final bispecific antibody format. 44 Stability studies of purified EI-04 BsAb showed the material to remain monomeric, homogeneous and stable at 4°C over at least 2 months at 25 and 50 mg/ml in a citrate-based pre-formulation buffer (data not shown). ...
Citations
... BsAbs can be engineered to selectively target, modulate, and interconnect biologic activities of otherwise separately acting surface receptors and ligands in a predesigned manner [39]. Moreover, tetravalent bsAbs are known to have significantly enhanced avidity towards cells that simultaneously express both targets antigens of interest, as they have up to four binding sites available for enhancement of functional interactions [40]. Importantly, an increasing number of bsAb-based immunotherapeutics are currently being evaluated in preclinical and clinical studies. ...
Simple Summary
Blockade of the immunosuppressive CD73/ADO immune checkpoint has been suggested as a promising alternate immunotherapeutic approach for refractory ovarian cancer (OC). Despite promising preclinical results, midterm clinical trial reports indicate that the efficacy of the CD73-blocking antibody oleclumab is modest. The limited efficacy of oleclumab may be related to the fact that it indiscriminately binds to and blocks CD73 that is present in a massive surplus of normal cells. The aim of our study was to achieve a tumor-directed inhibition of the CD73 immune checkpoint on OC cells. To this end, we constructed a novel bispecific antibody, bsAb CD73xEpCAM, that blocks the CD73 immune checkpoint in an EpCAM-directed manner. Moreover, treatment of OC cells with bsAb CD73xEpCAM inhibited various pro-oncogenic features. Taken together, bsAb CD73xEpCAM may be useful as an alternate and more tumor-directed immunotherapeutic approach to overcome the CD73-mediated immunosuppression in OC patients.
Abstract
PD-1/PD-L1-inhibiting antibodies have shown disappointing efficacy in patients with refractory ovarian cancer (OC). Apparently, OC cells exploit nonoverlapping immunosuppressive mechanisms to evade the immune system. In this respect, the CD73-adenosine inhibitory immune checkpoint is of particular interest, as it rapidly converts pro-inflammatory ATP released from cancer cells to immunosuppressive adenosine (ADO). Moreover, cancer-cell-produced ADO is known to form a highly immunosuppressive extra-tumoral ‘halo’ that chronically inhibits the anticancer activity of various immune effector cells. Thus far, conventional CD73-blocking antibodies such as oleclumab show limited clinical efficacy, probably due to the fact that it indiscriminately binds to and blocks CD73 on a massive surplus of normal cells. To address this issue, we constructed a novel bispecific antibody (bsAb) CD73xEpCAM that inhibits CD73 expressed on the OC cell surface in an EpCAM-directed manner. Importantly, bsAb CD73xEpCAM showed potent capacity to inhibit the CD73 enzyme activity in an EpCAM-directed manner and restore the cytotoxic activity of ADO-suppressed anticancer T cells. Additionally, treatment with bsAb CD73xEpCAM potently inhibited the proliferative capacity of OC cells and enhanced their sensitivity to cisplatin, doxorubicin, 5FU, and ionizing radiation. BsAb CD73xEpCAM may be useful in the development of tumor-directed immunotherapeutic approaches to overcome the CD73-mediated immunosuppression in patients with refractory OC.
... In addition, such systems can result in higher avidity and affinity than the corresponding monospecific systems [26]. The underlying idea is that the simultaneous binding of the bispecific nanoplatform to both antigens on the surface of the same cell can limit escape mechanisms and improve target selectivity through a strong avidity effect [16,[26][27][28][29]. ...
Dual-receptor targeted (DRT) nanoparticles which contain two distinct targeting agents may exhibit higher cell selectivity, cellular uptake, and cytotoxicity toward cancer cells than single-ligand targeted nanoparticle systems without additional functionality. The purpose of this study is to prepare DRT poly(lactic-co-glycolic acid) (PLGA) nanoparticles for targeting the delivery of docetaxel (DTX) to the EGFR and PD-L1 receptor positive cancer cells such as human glioblastoma multiform (U87-MG) and human non-small cell lung cancer (A549) cell lines. Anti-EGFR and anti-PD-L1 antibody were decorated on DTX loaded PLGA nanoparticles to prepare DRT-DTX-PLGA via. single emulsion solvent evaporation method. Physicochemical characterizations of DRT-DTX-PLGA, such as particle size, zeta-potential, morphology, and in vitro DTX release were also evaluated. The average particle size of DRT-DTX-PLGA was 124.2 ± 1.1 nm with spherical and smooth morphology. In the cellular uptake study, the DRT-DTX-PLGA endocytosed by the U87-MG and A549 cells was single ligand targeting nanoparticle. From the in vitro cell cytotoxicity, and apoptosis studies, we reported that DRT-DTX-PLGA exhibited high cytotoxicity and enhanced the apoptotic cell compared to the single ligand-targeted nanoparticle. The dual receptor mediated endocytosis of DRT-DTX-PLGA showed a high binding affinity effect that leads to high intracellular DTX concentration and exhibited high cytotoxic properties. Thus, DRT nanoparticles have the potential to improve cancer therapy by providing selectivity over single-ligand-targeted nanoparticles.
... 1,2 A common building block for the construction of multispecific biologics is the single-chain variable fragment (scFv), consisting of the target-engaging variable heavy chain (VH) linked to the variable light chain (VL) via a flexible linker. Multispecific format platforms such as the BiTE, 3 IgG-scFv, 4 and XmAb 5 incorporate scFv modules. Although scFvs are prevalent in multispecific biologic candidates, they may display sub-optimal physical properties relative to conventional mAbs and generally require sequence modifications to produce a developable asset. ...
Over the last three decades, the appeal for monoclonal antibodies (mAbs) as therapeutics has been steadily increasing as evident with FDA’s recent landmark approval of the 100th mAb. Unlike mAbs that bind to single targets, multispecific biologics (msAbs) have garnered particular interest owing to the advantage of engaging distinct targets. One important modular component of msAbs is the single-chain variable fragment (scFv). Despite the exquisite specificity and affinity of these scFv modules, their relatively poor thermostability often hampers their development as a potential therapeutic drug. In recent years, engineering antibody sequences to enhance their stability by mutations has gained considerable momentum. As experimental methods for antibody engineering are time-intensive, laborious and expensive, computational methods serve as a fast and inexpensive alternative to conventional routes. In this work, we show two machine learning approaches – one with pre-trained language models (PTLM) capturing functional effects of sequence variation, and second, a supervised convolutional neural network (CNN) trained with Rosetta energetic features – to better classify thermostable scFv variants from sequence. Both of these models are trained over temperature-specific data (TS50 measurements) derived from multiple libraries of scFv sequences. On out-of-distribution (refers to the fact that the out-of-distribution sequnes are blind to the algorithm) sequences, we show that a sufficiently simple CNN model performs better than general pre-trained language models trained on diverse protein sequences (average Spearman correlation coefficient, $$\rho $$ρ, of 0.4 as opposed to 0.15). On the other hand, an antibody-specific language model performs comparatively better than the CNN model on the same task ($$\rho = $$ρ= 0.52). Further, we demonstrate that for an independent mAb with available thermal melting temperatures for 20 experimentally characterized thermostable mutations, these models trained on TS50 data could identify 18 residue positions and 5 identical amino-acid mutations showing remarkable generalizability. Our results suggest that such models can be broadly applicable for improving the biological characteristics of antibodies. Further, transferring such models for alternative physicochemical properties of scFvs can have potential applications in optimizing large-scale production and delivery of mAbs or bsAbs.
... Interactions between IGF-1R and EGFR signaling have been proven to contribute to the development of resistance to anti-EGFR therapies. Moreover, the dual inhibition of EGFR/HER2 and IGF-1R signaling did produce stronger antitumor effects than either monotherapies in pancreatic cancer, ovarian cancer, colon cancer, etc. [30][31][32][33]. Therefore, we examined the antitumor activity of the EGFR/HER2 inhibitor (gefitinib and lapatinib) combined with the IGF-1R inhibitor (linsitinib) on the ESCC. ...
Both the epidermal growth factor receptor (EGFR) and insulin-like growth factor 1 receptor (IGF-1R) have been implicated in the development of cancers, and the increased expression of both receptors has been observed in esophageal cancer. However, the tyrosine kinase inhibitors of both receptors have thus far failed to provide clinical benefits for esophageal cancer patients. Studies have confirmed the complicated crosstalks that exist between the EGFR and IGF-1R pathways. The EGFR and IGF-1R signals act as mutual compensation pathways, thereby conveying resistance to EGFR or IGF-1R inhibitors when used alone. This study evaluated the antitumor efficacy of the EGFR/HER2 inhibitors, gefitinib and lapatinib, in combination with the IGF-1R inhibitor, linsitinib, on the esophageal squamous cell carcinoma (ESCC). Gefitinib or lapatinib, in combination with linsitinib, synergistically inhibited the proliferation, migration, and invasion of ESCC cells, caused significant cell cycle arrest, and induced marked cell apoptosis. Their combination demonstrated stronger inhibition on the activation of EGFR, HER2, and IGF-1R as well as the downstream signaling molecules. In vivo, the addition of linsitinib to gefitinib or lapatinib also potentiated the inhibition effects on the growth of xenografts. Our results suggest the next clinical exploration of the combination of gefitinib or lapatinib with linsitinib in the treatment of ESCC patients.
... While dysfunctional FcRn interactions have been noted to negatively impact mAb PK, previous studies of other BsAbs showed no connectivity to FcRn as causative in rapid clearance observations [11,14,16,[34][35][36]. To the best of our knowledge, the data presented herein is the first report connecting an altered FcRn release to BsAb PK. ...
Bispecific antibodies (BsAb) that engage multiple pathways are a promising therapeutic strategy to improve and prolong the efficacy of biologics in complex diseases. In the early stages of discovery, BsAbs often exhibit a broad range of pharmacokinetic (PK) behavior. Optimization of the neonatal Fc receptor (FcRn) interactions and removal of undesirable physiochemical properties have been used to improve the ‘pharmacokinetic developability’ for various monoclonal antibody (mAb) therapeutics, yet there is a sparsity of such information for BsAbs. The present work evaluated the influence of FcRn interactions and inherent physiochemical properties on the PK of two related single chain variable fragment (scFv)-based BsAbs. Despite their close relation, the two BsAbs exhibit disparate PK in cynomolgus monkeys with BsAb-1 having an aberrant clearance of ~2 mL/h/kg and BsAb-2 displaying a an ~10-fold slower clearance (~0.2 mL/h/kg). Evaluation of the physiochemical characteristics of the molecules, including charge, non-specific binding, thermal stability, and hydrophobic properties, as well as FcRn interactions showed some differences. In-depth drug disposition results revealed that a substantial disparity in the complete release from FcRn at a neutral pH is a primary factor contributing to the rapid clearance of the BsAb-1 while other biophysical characteristics were largely comparable between molecules.
... BsAbs are an emerging class of recombinant therapeutic protein drugs that can be engineered to selectively target, modulate and interconnect biologic activities of otherwise separately acting surface receptors and ligands in a pre-designed manner [28]. Moreover, tetravalent bsAbs are known to have significantly enhanced avidity towards cells that simultaneously express both target antigens of interest, as they have up to four binding sites available for enhancement of functional interactions [29]. Thus, use of the tetravalent molecular format of bsAb CD73xEpCAM may result in an enhanced avidity towards carcinoma cells and carcinoma-derived EVs due to its capacity for multivalent interactions with the co-exposed CD73 and EpCAM molecules. ...
Tumor-derived extracellular vesicles (EVs) carry potent immunosuppressive factors that affect the antitumor activities of immune cells. A significant part of the immunoinhibitory activity of EVs is attributable to CD73, a GPI-anchored ecto-5′-nucleotidase involved in the conversion of tumor-derived proinflammatory extracellular ATP (eATP) to immunosuppressive adenosine (ADO). The CD73-antagonist antibody oleclumab inhibits cell surface-exposed CD73 and is currently undergoing clinical testing for cancer immunotherapy. However, a strategy to selectively inhibit CD73 exposed on EVs is not available. Here, we present a novel bispecific antibody (bsAb) CD73xEpCAM designed to bind with high affinity the common EVs surface marker EpCAM and concurrently inhibit CD73. Unlike oleclumab, bsAb CD73xEpCAM potently inhibited the immunosuppressive activity of EVs from CD73pos/EpCAMpos carcinoma cell lines and patient-derived colorectal cancer cells. Taken together, selective blockade of EVs-exposed CD73 by bsAb CD73xEpCAM may be useful as an alternate or complementary targeted approach in cancer immunotherapy.
... A BsAb can bridge its two target antigens and bring them into close proximity [36]. A BsAb can retarget the effector cells, e.g., T cells, against tumor cells by simultaneously binding to cell surface antigens expressed from both cells and thus significantly activate the antitumor activities of effector cells [37][38][39]. A BsAb can also ligate two different receptors on the same cell and change intracellular downstream signaling [40]. ...
Immune checkpoint blockade has shown significant clinical benefit in multiple cancer indications, but many patients are either refractory or become resistant to the treatment over time. HER2/neu oncogene overexpressed in invasive breast cancer patients associates with more aggressive diseases and poor prognosis. Anti-HER2 mAbs, such as trastuzumab, are currently the standard of care for HER2-overexpressing cancers, but the response rates are below 30% and patients generally suffer relapse within a year. In this study we developed a bispecific antibody (BsAb) simultaneously targeting both PD1 and HER2 in an attempt to combine HER2-targeted therapy with immune checkpoint blockade for treating HER2-positive solid tumors. The BsAb was constructed by fusing scFvs (anti-PD1) with the effector-functional Fc of an IgG (trastuzumab) via a flexible peptide linker. We showed that the BsAb bound to human HER2 and PD1 with high affinities (EC50 values were 0.2 and 0.14 nM, respectively), and exhibited potent antitumor activities in vitro and in vivo. Furthermore, we demonstrated that the BsAb exhibited both HER2 and PD1 blockade activities and was effective in killing HER2-positive tumor cells via antibody-dependent cellular cytotoxicity. In addition, the BsAb could crosslink HER2-positive tumor cells with T cells to form PD1 immunological synapses that directed tumor cell killing without the need of antigen presentation. Thus, the BsAb is a new promising approach for treating late-stage metastatic HER2-positive cancers.
... The dual-targeting capacity may render BsAbs advantageous over conventional monospecific antibodies. Development of single BsAb is far less complex and potentially more cost effective than developing two or more individual mAbs [22]. In this investigation, we have generated a tetravalent anti-EGFR/VEGFR2 BsAb with IgG-like format, which has ability to simultaneously block EGFR and VEGFR2 signaling pathways and demonstrate superior anti-tumor activity in vitro and in vivo models on TNBC. ...
Simple Summary
Triple-negative breast cancer (TNBC) accounts for approximately 10–20% of all diagnosed breast cancers and is often associated with a poor prognosis. There is therefore an urgent need to develop novel and targeted therapeutic approaches against TNBC. Epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor 2 (VEGFR2) are prominent therapeutic protein targets that are frequently overexpressed in TNBC. In this investigation, we developed a novel bispecific antibody (BsAb) targeting EGFR and VEGFR2 (designated as anti-EGFR/VEGFR2 BsAb) and investigate its anti-tumor activity using TNBC cellular and xenograft mouse models. Data from these studies indicate that anti-EGFR/VEGFR2 BsAb elicited more comprehensive anti-tumor activity via multiple mechanisms of action, including direct inhibition of EGFR and VEGFR2 in TNBC cells, and disruption of autocrine and paracrine pathways in TNBC and endothelial cells, compared to the individual parental mAbs. Our data suggest that this novel BsAb warrants further investigation as a targeted antibody therapeutic to treat TNBC.
Abstract
Both EGFR and VEGFR2 frequently overexpress in TNBC and cooperate with each other in autocrine and paracrine manner to enhance tumor growth and angiogenesis. Therapeutic mAbs targeting EGFR (cetuximab) and VEGFR2 (ramucirumab) are approved by FDA for numerous cancer indications, but none of them are approved to treat breast cancers. TNBC cells secrete VEGF-A, which mediates angiogenesis on endothelial cells in a paracrine fashion, as well as promotes cancer cell growth in autocrine manner. To disrupt autocrine/paracrine loop in TNBC models in addition to mediating anti-EGFR tumor growth signaling and anti-VEGFR2 angiogenic pathway, we generated a BsAb co-targeting EGFR and VEGFR2 (designated as anti-EGFR/VEGFR2 BsAb), using publicly available sequences in which cetuximab IgG backbone is connected to the single chain variable fragment (scFv) of ramucirumab via a glycine linker. Physiochemical characterization data shows that anti-EGFR/VEGFR2 BsAb binds to both EGFR and VEGFR2 in a similar binding affinity comparable to parental antibodies. Anti-EGFR/VEGFR2 BsAb demonstrates in vitro and in vivo anti-tumor activity in TNBC models. Mechanistically, anti-EGFR/VEGFR2 BsAb not only directly inhibits both EGFR and VEGFR2 in TNBC cells but also disrupts autocrine mechanism in TNBC xenograft mouse model. Furthermore, anti-EGFR/VEGFR2 BsAb inhibits ligand-induced activation of VEGFR2 and blocks paracrine pathway mediated by VEGF secreted from TNBC cells in endothelial cells. Collectively, our novel findings demonstrate that anti-EGFR/VEGFR2 BsAb inhibits tumor growth via multiple mechanisms of action and warrants further investigation as a targeted antibody therapeutic for the treatment of TNBC.
... Besides, these components are included in excess of infection intervention and crosstalk between flag falls [46]. Likewise, upregulation of option receptors and pathway exchanging is regularly identified with imperviousness to treatment [47]. The hindrance of a few targets or various locales on one target is related with enhanced restorative adequacy. ...
The improvements of counter acting antibody generation systems of various types of immunoglobulins have been created on a vast scale. Miscellaneous scientific tools and skills used to design the most efficient and accurate method. The hybridoma innovation opened a new era in the production of antibodies against target antigens of desirable pathogens, life-threatening infections including immune system issue and various intense poisons. Despite that, these clinical acculturated or chimeric murine antibodies have a few constraints and complexities. The study aims to review and explain the advanced antibody engineering to enhance the innovative potential of antibodies. Therefore, our major effort focusing to defeat the current challenges, late advances in hereditary building methods and phage display system that permitted the creation of exceedingly particular recombinant antibodies. These antibodies have been built in the chase for novel remedial medications furnished with improved immune protective capacities. That will potential connects with the resistant effector's capacities; compel advancement of combination proteins, proficient tumor and tissue entrance and high-liking antibodies coordinated against moderated targets. Propelled counteracting agent designing systems have broad applications in the fields of immunology, biotechnology, diagnostics and helpful prescriptions. Even so, there is constrained learning with respect to element immune response improvement approaches. Along these lines, this study reaches outside of our ability to comprehend traditional polyclonal and monoclonal antibodies. Besides, late advances in immunizer designing systems together with counteracting agent sections, show advances, immunomodulation and expansive utilization of antibodies are examined to upgrade creative neutralizer generation in the expedition for a more advantageous future for people.
... 5 To evaluate the EFab designs in controlling light chain pairing, several expression vectors were built using the two Fabs M60-A02 (anti-epidermal growth factor receptor (EGFR)) and C06 (anti-IGF1R), which have been described previously. 20 The test molecules were tagged with green fluorescent protein (GFP) (30 kDa, light chain of C06) or human serum albumin (HSA) (66 kDa, heavy chain of Fab 1 or EFab, M60-A02) to enable simple differentiation of correct versus incorrect Fab pairs by migration on non-reducing SDS-PAGE ( Figure 2, for reducing SDS-PAGE, see Figure S1). The proteins were generated by transient expression in Chinese hamster ovary (CHO) cells. ...
... Another pair tested, M60-A02/C06, did not exhibit simultaneous binding, although a M60-A02/C06 bispecific was previously reported to bind both antigens simultaneously in a tetravalent bispecific format, with two C06 scFvs on the C-term of the Fc. 20 The lack of binding observed here is likely a result of steric hindrance caused by the asymmetric bispecific format we used to study EFab domain substitution. Substitution of the EFab led to a decrease in thermal stability of the Fabs as measured by DSC. ...
Bispecific antibody therapeutics can expand the functionality of a conventional monoclonal antibody drug because they can bind multiple antigens. However, their great potential is counterbalanced by the challenges faced in their production. The classic asymmetric bispecific containing an Fc requires the expression of four unique chains – two light chains and two heavy chains; each light chain must pair with its correct heavy chain, which then must heterodimerize to form the full bispecific. The light-chain pairing problem has several solutions, some of which require engineering and optimization for each bispecific pair. Here, we introduce a technology called EFab Domain Substitution, which replaces the CH2 of IgE for one of the CL/CH1 domains into one arm of an asymmetric bispecific to encourage the correct pairing of the light chains. EFab Domain Substitution provides very robust correct pairing while maintaining antibody function and is effective for many variable domains. We report its effect on the biophysical properties of an antibody and the crystal structure of the EFab domain substituted into the adalimumab Fab (PDBcode 6CR).
