Alternative splicing for tuneable expression of protein subunits at desired ratios.

The controlled expression of two or more proteins at a defined and stable ratio remains a substantial challenge, particularly in the bi- and multispecific antibody field. Achieving an optimal ratio of protein subunits can facilitate the assembly of multimeric proteins with high efficiency and minimize the production of by-products. In this study, we propose a solution based on alternative splicing, enabling the expression of a tunable and predefined ratio of two distinct polypeptide chains from the same pre-mRNA under the control of a single promoter. The pre-mRNA used in this study contains two open reading frames situated on separate exons. The first exon is flanked by two copies of the chicken troponin intron 4 (cTNT-I4) and is susceptible to excision from the pre-mRNA by means of alternative splicing. This specific design enables the modulation of the splice ratio by adjusting the strength of the splice acceptor. To illustrate this approach, we developed constructs expressing varying ratios of GFP and dsRED and extended their application to multimeric proteins such as monoclonal antibodies, achieving industrially relevant expression levels (>1 g/L) in a 14-day fed-batch process. The stability of the splice ratio was confirmed by droplet digital PCR in a stable pool cultivated over a 28-day period, while product quality was assessed via intact mass analysis, demonstrating absence of product-related impurities resulting from undesired splice events. Furthermore, we showcased the versatility of the construct by expressing two subunits of a bispecific antibody of the BEAT® type, which contains three distinct subunits in total.

Geometric Antibody Engineering Reveals the Spatial Factor on the Efficacy of Bispecific T Cell Engagers.

Bispecific antibodies (BsAbs) represent an emerging class of biologics that can recognize two different antigens or epitopes. T-cell engagers (TcEs) bind two targets in trans on the cell surface of the effector and target cell to induce proximal immune effects, opening exciting windows for immunotherapies. To date, the engineering of BsAbs has been mainly focused on tuning the molecular weight and valency. However, the effects of spatial factors on the biological functions of BsAbs have been less explored due to the lack of biochemical methods to precisely manipulate protein geometry. Here, we studied the geometric effects of the TcEs. First, by genetically inserting rigidly designed ankyrin repeat proteins into TcEs, we revealed that the efficacy progressively decreased as the spacer distance of the two binding domains increased. Then, we constructed 26 pairs of TcEs with the same size but varying orientations using click chemistry-mediated conjugation at different mutation sites. We found that linear ligation sites play a minor role in modulating cell-killing efficacy. Next, we rendered the TcEs' advanced topology by cyclization chemistry using the SpyTag/SpyCatcher pair or sortase ligation approaches. Cyclized TcEs were generally more potent than their linear counterparts. Particularly, sortase A cyclized TcEs, bearing a minimal tagging motif, exhibited better cell-killing efficacy in vitro and improved stability both in vitro and in vivo compared to the linear TcE. This work combines modern bioconjugation chemistry and protein engineering tools for antibody engineering, shedding light on the elusive spatial factors of BsAbs functionality.

Utilizing targeted integration CHO pools to potentially accelerate the GMP manufacturing of monoclonal and bispecific antibodies.

Monoclonal antibodies (mAbs) are effective therapeutic agents against many acute infectious diseases including COVID-19, Ebola, RSV, Clostridium difficile, and Anthrax. mAbs can therefore help combat a future pandemic. Unfortunately, mAb development typically takes years, limiting its potential to save lives during a pandemic. Therefore "pandemic mAb" timelines need to be shortened. One acceleration tool is "deferred cloning" and leverages new Chinese hamster ovary (CHO) technology based on targeted gene integration (TI). CHO pools, instead of CHO clones, can be used for Phase I/II clinical material production. A final CHO clone (producing the mAb with a similar product quality profile and preferably with a higher titer) can then be used for Phase III trials and commercial manufacturing. This substitution reduces timelines by ~3 months. We evaluated our novel CHO TI platform to enable deferred cloning. We created four unique CHO pools expressing three unique mAbs (mAb1, mAb2, and mAb3), and a bispecific mAb (BsAb1). We then performed single-cell cloning for mAb1 and mAb2, identifying three high-expressing clones from each pool. CHO pools and clones were inoculated side-by-side in ambr15 bioreactors. CHO pools yielded mAb titers as high as 10.4 g/L (mAb3) and 7.1 g/L (BsAb1). Subcloning yielded CHO clones expressing higher titers relative to the CHO pools while yielding similar product quality profiles. Finally, we showed that CHO TI pools were stable by performing a 3-month cell aging study. In summary, our CHO TI platform can increase the speed to clinic for a future "pandemic mAb."

Robust production of monovalent bispecific IgG antibodies through novel electrostatic steering mutations at the CH1-Cλ interface.

Bispecific antibodies represent an increasingly large fraction of biologics in therapeutic development due to their expanded scope in functional capabilities. Asymmetric monovalent bispecific IgGs (bsIgGs) have the additional advantage of maintaining a native antibody-like structure, which can provide favorable pharmacology and pharmacokinetic profiles. The production of correctly assembled asymmetric monovalent bsIgGs, however, is a complex engineering endeavor due to the propensity for non-cognate heavy and light chains to mis-pair. Previously, we introduced the DuetMab platform as a general solution for the production of bsIgGs, which utilizes an engineered interchain disulfide bond in one of the CH1-CL domains to promote orthogonal chain pairing between heavy and light chains. While highly effective in promoting cognate heavy and light chain pairing, residual chain mispairing could be detected for specific combinations of Fv pairs. Here, we present enhancements to the DuetMab design that improve chain pairing and production through the introduction of novel electrostatic steering mutations at the CH1-CL interface with lambda light chains (CH1-Cλ). These mutations work together with previously established charge-pair mutations at the CH1-CL interface with kappa light chains (CH1-Cκ) and Fab disulfide engineering to promote cognate heavy and light chain pairing and enable the reliable production of bsIgGs. Importantly, these enhanced DuetMabs do not require engineering of the variable domains and are robust when applied to a panel of bsIgGs with diverse Fv sequences. We present a comprehensive biochemical, biophysical, and functional characterization of the resulting DuetMabs to demonstrate compatibility with industrial production benchmarks. Overall, this enhanced DuetMab platform substantially streamlines process development of these disruptive biotherapeutics.

Functional integration of protein A binding ability to antibody fragments for convenient and tag-free purification.

Although the development of small therapeutic antibodies is important, the affinity tags used for their purification often result in heterogeneous production and immunogenicity. In this study, we integrated Staphylococcus aureus protein A (SpA) binding ability into antibody fragments for convenient and tag-free purification. SpA affinity chromatography is used as a global standard purification method for conventional antibodies owing to its high binding affinity to the Fc region. SpA also has a binding affinity for some variable heavy domains (VH) classified in the VH3 subfamily. Through mutagenesis based on alignment and structural modeling results using the SpA-VH3 cocrystal structure, we integrated the SpA-binding ability into the anti-CD3 single-chain Fv. Furthermore, we applied this mutagenesis approach to more complicated small bispecific antibodies and successfully purified the antibodies using SpA affinity chromatography. The antibodies retained their biological function after purification. Integration of SpA-binding ability into conventional antibody fragments simplifies the purification and monitoring of the production processes and, thus, is an ideal strategy for accelerating the development of small therapeutic antibodies. Furthermore, because of its immunoactivity, the anti-CD3 variable region with SpA-binding ability is an effective building block for developing engineered cancer therapeutic antibodies without the Fc region.

CLDN18.2 BiTE Engages Effector and Regulatory T Cells for Antitumor Immune Response in Preclinical Models of Pancreatic Cancer.

BACKGROUND & AIMS: BiTE (bispecific T-cell engager) immune therapy has demonstrated clinical activity in multiple tumor indications, but its influence in the tumor microenvironment remains unclear. CLDN18.2 is overexpressed in solid tumors including gastric cancer (GC) and pancreatic ductal adenocarcinoma (PDAC), both of which are characterized by the presence of immunosuppressive cells, including regulatory T cells (Tregs) and few effector T cells (Teffs). METHODS: We evaluated the activity of AMG 910, a CLDN18.2-targeted half-life extended (HLE) BiTE molecule, in GC and PDAC preclinical models and cocultured Tregs and Teffs in the presence of CLDN18.2-HLE-BiTE. RESULTS: AMG 910 induced potent, specific cytotoxicity in GC and PDAC cell lines. In GSU and SNU-620 GC xenograft models, AMG 910 engaged human CD3+ T cells with tumor cells, resulting in significant antitumor activity. AMG 910 monotherapy, in combination with a programmed death-1 (PD-1) inhibitor, suppressed tumor growth and enhanced survival in an orthotopic Panc4.14 PDAC model. Moreover, Treg infusion enhanced the antitumor efficacy of AMG 910 in the Panc4.14 model. In syngeneic KPC models of PDAC, treatment with a mouse surrogate CLDN18.2-HLE-BiTE (muCLDN18.2-HLE-BiTE) or the combination with an anti-PD-1 antibody significantly inhibited tumor growth. Tregs isolated from mice bearing KPC tumors that were treated with muCLDN18.2-HLE-BiTE showed decreased T cell suppressive activity and enhanced Teff cytotoxic activity, associated with increased production of type I cytokines and expression of Teff gene signatures. CONCLUSIONS: Our data suggest that BiTE molecule treatment converts Treg function from immunosuppressive to immune enhancing, leading to antitumor activity in immunologically "cold" tumors.

Chemical generation of checkpoint inhibitory T cell engagers for the treatment of cancer.

Bispecific T cell engagers (BiTEs), a subset of bispecific antibodies (bsAbs), can promote a targeted cancer cell's death by bringing it close to a cytotoxic T cell. Checkpoint inhibitory T cell engagers (CiTEs) comprise a BiTE core with an added immunomodulatory protein, which serves to reverse cancer-cell immune-dampening strategies, improving efficacy. So far, protein engineering has been the main approach to generate bsAbs and CiTEs, but improved chemical methods for their generation have recently been developed. Homogeneous fragment-based bsAbs constructed from fragment antigen-binding regions (Fabs) can be generated using click chemistry. Here we describe a chemical method to generate biotin-functionalized three-protein conjugates, which include two CiTE molecules, one containing an anti-PD-1 Fab and the other containing an immunomodulatory enzyme, Salmonella typhimurium sialidase. The CiTEs' efficacy was shown to be superior to that of the simpler BiTE scaffold, with the sialidase-containing CiTE inducing substantially enhanced T cell-mediated cytotoxicity in vitro. The chemical method described here, more generally, enables the generation of multi-protein constructs with further biological applications.

Surfaceome Profiling Suggests Potential of Anti-MUC1×EGFR Bispecific Antibody for Breast Cancer Targeted Therapy.

Breast cancer (BC) treatment has traditionally been challenging due to tumor heterogeneity. Bispecific antibodies (bsAbs) offer a promising approach for overcoming these challenges by targeting multiple specific epitopes. In the current study, we designed a new bsAb against the most common BC cell surface proteins (SPs). To achieve this, we analyzed RNA-sequencing data to identify differentially expressed genes, which were further evaluated using Gene Ontology enrichment, Hidden Markov Models, clinical trial data, and survival analysis to identify druggable gene-encoding cell SPs. Based on these analyses, we constructed and expressed a bsAb targeting the mucin 1 (MUC1) and epidermal growth factor receptor (EGFR) proteins, which are the dominant druggable gene-encoding cell SPs in BC. The recombinant anti-MUC1×EGFR bsAb demonstrated efficient production and high specificity for MUC1 and EGFR + cell lines and BC tissue. Furthermore, the bsAb significantly reduced the proliferation and migration of BC cells. Our results suggested that simultaneous targeting with bsAbs could be a promising targeted therapy for improving the overall efficacy of BC treatment.

A non-genetic engineering platform for rapidly generating and expanding cancer-specific armed T cells.

BACKGROUND: Cancer-specific adoptive T cell therapy has achieved successful milestones in multiple clinical treatments. However, the commercial production of cancer-specific T cells is often hampered by laborious cell culture procedures, the concern of retrovirus-based gene transfection, or insufficient T cell purity. METHODS: In this study, we developed a non-genetic engineering technology for rapidly manufacturing a large amount of cancer-specific T cells by utilizing a unique anti-cancer/anti-CD3 bispecific antibody (BsAb) to directly culture human peripheral blood mononuclear cells (PBMCs). The anti-CD3 moiety of the BsAb bound to the T cell surface and stimulated the differentiation and proliferation of T cells in PBMCs. The anti-cancer moiety of the BsAb provided these BsAb-armed T cells with the cancer-targeting ability, which transformed the naïve T cells into cancer-specific BsAb-armed T cells. RESULTS: With this technology, a large amount of cancer-specific BsAb-armed T cells can be rapidly generated with a purity of over 90% in 7 days. These BsAb-armed T cells efficiently accumulated at the tumor site both in vitro and in vivo. Cytotoxins (perforin and granzyme) and cytokines (TNF-α and IFN-γ) were dramatically released from the BsAb-armed T cells after engaging cancer cells, resulting in a remarkable anti-cancer efficacy. Notably, the BsAb-armed T cells did not cause obvious cytokine release syndrome or tissue toxicity in SCID mice bearing human tumors. CONCLUSIONS: Collectively, the BsAb-armed T cell technology represents a simple, time-saving, and highly safe method to generate highly pure cancer-specific effector T cells, thereby providing an affordable T cell immunotherapy to patients.

Generation of robust bispecific antibodies through fusion of single-domain antibodies on IgG scaffolds: a comprehensive comparison of formats.

Bispecific antibodies (bsAbs) enable dual binding of different antigens with potential synergistic targeting effects and innovative therapeutic possibilities. The formation of bsAbs is, however, often dependent on complex engineering strategies with a high risk of antibody chain mispairing leading to contamination of the final product with incorrectly assembled antibody species. This study demonstrates formation of bsAbs in a generic and conceptually easy manner through fusion of single-domain antibodies (sdAbs) onto IgG scaffolds through flexible 10 amino acid linkers to form high-quality bsAbs with both binding functionalities intact and minimal product-related impurities. SdAbs are attractive fusion partners due to their small and monomeric nature combined with antigen-binding capabilities comparable to conventional human antibodies. By systematically comparing a comprehensive panel of symmetric αPD-L1×αHER2 antibodies, including reversely mirrored antigen specificities, we investigate how the molecular geometry affects production, stability, antigen binding and CD16a binding. SdAb fusion of the heavy chain was generally preferred over light chain fusion for promoting good expression and high biophysical stability as well as maintaining efficient binding to both antigens. We find that N-terminal sdAb fusion might sterically hinder antigen-binding to the Fv region of the IgG scaffold, whereas C-terminal fusion might disturb antigen-binding to the fused sdAb. Our work demonstrates a toolbox of complementary methods for in-depth analysis of key features, such as in-solution dual antigen binding, thermal stability, and aggregation propensity, to ensure high bsAb quality. These techniques can be executed at high-throughput and/or with very low material consumption and thus represent valuable tools for bsAb screening and development.

Construction of a novel TROP2/CD3 bispecific antibody with potent antitumor activity and reduced induction of Th1 cytokines.

Many cancers, including triple-negative breast cancer, overexpress TROP2 on the surface of tumor cells. TROP2 has become a promising tumor associated antigen for the development of novel antibody-based targeted therapy. Herein, we constructed a novel bispecific antibody with the ability to simultaneously target TROP2 on the tumor surface and bind to CD3 to activate T cells. Given that the excessive production of Th1 cytokines induced by CD3-mediated T-cell overactivation may lead to toxicity in the clinic, we devised a strategy to modify this CD3-induced T cell activation by a two-step reduction in the bispecific antibody binding affinity for CD3 to a level that retained the ability of the bispecific antibody to effectively inhibit tumor growth while greatly reducing the amount of Th1 cytokines secreted by T cells. Thus, we provide insight into the design of T cell engagers that exhibit a promising toxicity profile while retaining inhibitory effects on tumor growth.

Antibody engineering and its therapeutic applications.

As a natural function, antibodies defend the host from infected cells and pathogens by recognizing their pathogenic determinants. Antibodies (Abs) gained wide acceptance with an enormous impact on human health and have predominantly captured the arena of bio-therapeutics and bio-diagnostics. The scope of Ab-based biologics is vast, and it is likely to solve many unmet clinical needs in future. The majority of attention is now devoted to developing innovative technologies for manufacturing and engineering Abs, better suited to satisfy human needs. The advent of Ab engineering technologies (AET) led to phenomenal developments leading to the generation of Abs-/Ab-derived molecules with desirable functional properties proportional to their expanding requirements. Evolution brought by AET, from the naturally occurring Ab forms to several advanced Ab formats and derivatives, was much needed as it is of great interest to the pharmaceutical industry. Thus, numerous advancements in AET have propelled success in therapeutic Ab development, along with the potential for ever-increasing improvements. Unique characteristics of Abs, such as its diversity, specificity, structural integrity and an array of possible applications, together inspire continuous innovation in the field. Overall, the AET could assist in conquer of several limitations of Abs in terms of their applicability in the field of therapeutics, diagnostics and research; AET has so far led to the production of next-generation Abs, which have revolutionized these arenas. Here in this review, we discuss the various distinguished engineering platforms for Ab development and the progress in modern therapeutics by the so-called "next-generation Abs."

IgG-like bispecific antibodies with potent and synergistic neutralization against circulating SARS-CoV-2 variants of concern.

Monoclonal antibodies are a promising approach to treat COVID-19, however the emergence of SARS-CoV-2 variants has challenged the efficacy and future of these therapies. Antibody cocktails are being employed to mitigate these challenges, but neutralization escape remains a major challenge and alternative strategies are needed. Here we present two anti-SARS-CoV-2 spike binding antibodies, one Class 1 and one Class 4, selected from our non-immune human single-chain variable fragment (scFv) phage library, that are engineered into four, fully-human IgG-like bispecific antibodies (BsAb). Prophylaxis of hACE2 mice and post-infection treatment of golden hamsters demonstrates the efficacy of the monospecific antibodies against the original Wuhan strain, while promising in vitro results with the BsAbs demonstrate enhanced binding and distinct synergistic effects on neutralizing activity against circulating variants of concern. In particular, one BsAb engineered in a tandem scFv-Fc configuration shows synergistic neutralization activity against several variants of concern including B.1.617.2. This work provides evidence that synergistic neutralization can be achieved using a BsAb scaffold, and serves as a foundation for the future development of broadly reactive BsAbs against emerging variants of concern.

HIV-1 bispecific antibody iMab-N6 exhibits enhanced breadth but not potency over its parental antibodies iMab and N6.

The recently published AMP trial (HVTN 703/HPTN 081 and HVTN704/HPTN 085) results have validated broad neutralising antibodies (bNAbs) as potential anti-HIV-1 agents. However, single bNAb preparations are unlikely to cope with the onslaught of existing and de novo resistance mutations, thus necessitating the use of bNAb combinations to achieve clinically relevant results. Specifically engineered antibodies incorporating two bNAbs into a single antibody structure have been developed. These bispecific antibodies (bibNAbs) retain the benefits of bNAb combinations, whilst several conformations exhibit improved neutralisation potency over the parental bNAbs. Here we report on the engineering of a bibNAb comprising of an HIV-1 spike targeting bNAb N6 and a host CD4 targeting antibody ibalizumab (iMab). Antibodies were expressed in HEK293T cells and purified by protein-A affinity chromatography followed by size exclusion chromatography to achieve homogenous, monomeric, bibNAb preparations. Antibody purity was confirmed by SDS-PAGE whilst epitope specificity and binding were confirmed by ELISA. Finally, antibody breadth and potency data were generated by HIV-1 neutralisation assay (n = 21, inclusive of the global panel). iMab-N6 exhibited better neutralisation breadth (100% coverage) in comparison to its parental bNAbs iMab (90%) and N6 (95%). This is encouraging as exceptional neutralisation breadth is necessary for HIV-1 treatment or prevention. Unfortunately, iMab-N6 did not exhibit any enhancement in potency over the most potent parental antibody, iMab (p = 0.1674, median IC50 of 0.0475 µg/ml, and 0.0665 µg/ml respectively) or the parental combination, iMab + N6 (p = 0.1964, median IC50: combination 0.0457 µg/ml). This result may point to a lack of dual engagement of the bibNAb Fab moieties necessary for potency enhancement. Against the previously reported bibNAbs; iMab-CAP256, 10E08-iMab, and PG9-iMab; iMab-N6 was the lowest performing bibNAb. The re-engineering of iMab-N6 to enhance its potency, while retaining breadth, is a worthwhile endeavour due to its clinical potential.

The Arrival of Gene Therapy for Patients with Hemophilia A.

Historically, the standard of care for hemophilia A has been intravenous administration of exogenous factor VIII (FVIII), either as prophylaxis or episodically. The development of emicizumab, a humanized bispecific monoclonal antibody mimicking activated FVIII, was a subsequent advance in treatment. However, both exogenous FVIII and emicizumab require repeated and lifelong administration, negatively impacting patient quality of life. A recent breakthrough has been the development of gene therapy. This allows a single intravenous treatment that could result in long-term expression of FVIII, maintenance of steady-state plasma concentrations, and minimization (or possibly elimination) of bleeding episodes for the recipient's lifetime. Several gene therapies have been assessed in clinical trials, with positive outcomes. Valoctocogene roxaparvovec (an adeno-associated viral 5-based therapy encoding human B domain-deleted FVIII) is expected to be the first approved gene therapy in European countries, including Italy, in 2022. Some novel challenges exist including refining patient selection criteria, managing patient expectations, further elucidation of the durability and variability of transgene expression and long-term safety, and the development of standardized 'hub and spoke' centers to optimize and monitor this innovative treatment. Gene therapy represents a paradigm shift, and may become a new reference standard for treating patients with hemophilia A.

CHO cathepsin B identified as the protease responsible for a target bispecific antibody fragmentation.

In a previous work we demonstrated that CHO protease caused fragmentation of an expressed bispecific antibody (bsAb) and this detrimental host cell protein (HCP) can be effectively removed through an optimized Protein A wash step. In addition, preliminary evidence suggested that the responsible protease belongs to the threonine or cysteine protease family. In the current study, this protease was further identified as cathepsin B. First, we identified several CHO proteases in the further fractionated Protein A wash using liquid chromatography-tandem mass spectrometry (LC-MS/MS), and this allowed us to select four candidate proteases. Next, by examining the cleavage pattern of each individual protease and comparing it with that observed during purification, cathepsin B was identified as the protease responsible for the observed bsAb fragmentation.

Lineage Conversion in Pediatric B-Cell Precursor Acute Leukemia under Blinatumomab Therapy.

We report incidence and deep molecular characteristics of lineage switch in 182 pediatric patients affected by B-cell precursor acute lymphoblastic leukemia (BCP-ALL), who were treated with blinatumomab. We documented six cases of lineage switch that occurred after or during blinatumomab exposure. Therefore, lineage conversion was found in 17.4% of all resistance cases (4/27) and 3.2% of relapses (2/63). Half of patients switched completely from BCP-ALL to CD19-negative acute myeloid leukemia, others retained CD19-positive B-blasts and acquired an additional CD19-negative blast population: myeloid or unclassifiable. Five patients had KMT2A gene rearrangements; one had TCF3::ZNF384 translocation. The presented cases showed consistency of gene rearrangements and fusion transcripts across initially diagnosed leukemia and lineage switch. In two of six patients, the clonal architecture assessed by IG/TR gene rearrangements was stable, while in others, loss of clones or gain of new clones was noted. KMT2A-r patients demonstrated very few additional mutations, while in the TCF3::ZNF384 case, lineage switch was accompanied by a large set of additional mutations. The immunophenotype of an existing leukemia sometimes changes via different mechanisms and with different additional molecular changes. Careful investigation of all BM compartments together with all molecular -minimal residual disease studies can lead to reliable identification of lineage switch.

Bispecific Antibody Expressed by an Oncolytic Herpes Simplex Virus Type 2 Can Transform Heterologous T Cells Into Uniform Tumor Killer Cells.

BsAb (bispecific antibody)-armed oncolytic viruses (OVs) are effective in regulating tumor microenvironment. However, oHSV2 (oncolytic herpes simplex virus type 2) expressing immune checkpoints targeting BsAb molecules are not reported. Here, we generated oHSV2-armed PD-L1/CD3 BsAb and established pharmacodynamic evaluation models, which suggested that our oHSV2-BsAb molecules have an improved oncolytic potency in vitro and in vivo. The oHSV2 viruses armed with BsAb molecules targeting programmed cell death ligand 1 (PD-L1)/CD3 or CD19/CD3 (oHSV2-PD-L1/CD3-BsAb or oHSV2-CD19/mCD3-BsAb) were constructed; besides inducing oncolysis in virus-infected tumor cells, the modified oncolytic virus oHSV2-PD-L1/CD3-BsAb can also activate peripheral blood mononuclear cells (PBMCs) by releasing PD-L1/CD3 BsAb and thereby induce PBMC-mediated killing of PD-L1-positive tumor cells, regardless of PD-L1 expression level. The expressed PD-L1/CD3 BsAb can upregulate the activation markers of T cells in PBMCs and induce different cytokine secretion. The activation of T cells and the enrichment of related immune regulatory pathways are further confirmed by proteomics. It also demonstrated that the OVs or PBMCs could upregulate PD-L1 expression on the surface of tumor cells through transforming "cold tumors" with low PD-L1 expression into "hot tumors" with high PD-L1 expression, which can facilitate the targeting of BsAb molecules and enhance the effect of oncolysis. oHSV2-PD-L1/CD3-BsAb or oHSV2-CD19/mCD3-BsAb showed an enhanced oncolytic effect in vitro and in vivo compared to backbone virus oHSV2-GFP. Our results showed that the newly designed oHSV2-BsAb had enhanced therapeutic effects against solid tumors and provided a new option of immunotherapy.

An engineered bispecific human monoclonal antibody against SARS-CoV-2.

The global severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic requires effective therapies against coronavirus disease 2019 (COVID-19), and neutralizing antibodies are a promising therapy. A noncompeting pair of human neutralizing antibodies (B38 and H4) blocking SARS-CoV-2 binding to its receptor, ACE2, have been described previously. Here, we develop bsAb15, a bispecific monoclonal antibody (bsAb) based on B38 and H4. bsAb15 has greater neutralizing efficiency than these parental antibodies, results in less selective pressure and retains neutralizing ability to most SARS-CoV-2 variants of concern (with more potent neutralizing activity against the Delta variant). We also selected for escape mutants of the two parental mAbs, a mAb cocktail and bsAb15, demonstrating that bsAb15 can efficiently neutralize all single-mAb escape mutants. Furthermore, prophylactic and therapeutic application of bsAb15 reduced the viral titer in infected nonhuman primates and human ACE2 transgenic mice. Therefore, this bsAb is a feasible and effective strategy to treat and prevent severe COVID-19.

Far-Red Light Triggered Production of Bispecific T Cell Engagers (BiTEs) from Engineered Cells for Antitumor Application.

Bispecific T-cell engagers (BiTEs), which have shown potent antitumor activity in humans, are emerging as one of the most promising immunotherapeutic strategies for cancer treatment in recent years. However, the clinical application of BiTEs nowadays has been hampered by their short half-life in the circulatory system due to their low molecular weight and rapid renal clearance. Inevitable continuous infusion of BiTEs has become a routine operation in order to achieve effective treatment, although it is costly, inconvenient, time-consuming, and even painful for patients in some cases. To develop an on-demand, tunable, and reversible approach to overcome these limitations, we assembled a transcription-control device into mammalian cells based on a bacterial far-red light (FRL) responsive signaling pathway to drive the expression of a BiTE against Glypican 3 (GPC3), which is a highly tumor-specific antigen expressed in most hepatocellular carcinomas (HCC). As demonstrated in in vitro experiments, we proved that the FRL sensitive device spatiotemporally responded to the control of FRL illumination and produced a therapeutic level of BiTEs that recruited and activated human T cells to eliminate GPC3 positive tumor cells. By functionally harnessing the power of optogenetics to remotely regulate the production of BiTEs from bioengineered cells and demonstrating its effectiveness in treating tumor cells, this study provides a novel approach to achieve an in vivo supply of BiTEs, which could be potentially applied to other formats of bispecific antibodies and facilitate their clinical applications.