Antibody Platform Research Articles

Next-generation cell-penetrating antibodies for tumor targeting and RAD51 inhibition.

Monoclonal antibody therapies for cancer have demonstrated extraordinary clinical success in recent years. However, these strategies are thus far mostly limited to specific cell surface antigens, even though many disease targets are found intracellularly. Here we report studies on the humanization of a full-length, nucleic acid binding, monoclonal lupus-derived autoantibody, 3E10, which exhibits a novel mechanism of cell penetration and tumor specific targeting. Comparing humanized variants of 3E10, we demonstrate that cell uptake depends on the nucleoside transporter ENT2, and that faster cell uptake and superior in vivo tumor targeting are associated with higher affinity nucleic acid binding. We show that one human variant retains the ability of the parental 3E10 to bind RAD51, serving as a synthetically lethal inhibitor of homology-directed repair in vitro. These results provide the basis for the rational design of a novel antibody platform for therapeutic tumor targeting with high specificity following systemic administration.

Development of a novel humanized anti-TSLP monoclonal antibody, QX008N, and exploration of combination therapy of anti-TSLP antibody and anti-IL-4R antibody

BackgroundSevere asthma is a complex and chronic respiratory disease, and current conventional treatments are not effective in controlling the patients’ condition. Thymic stromal lymphopoietin (TSLP) is a key regulatory factor in the initiation and maintenance of asthma. Thus, blocking TSLP during allergic inflammation emerges as a promising therapeutic approach; however, novel anti-TSLP therapies remain to be developed. Furthermore, the importance of other signaling molecules, such as IL-4 and IL-13, should be considered. Moreover, to the best of our knowledge, the inhibitory effect of binding upstream and downstream signaling molecules has not been assessed. PurposeThis study aimed to develop a novel, humanized anti-TSLP antibody and explore the enhancement in its efficacy when combined with anti-IL-4R antibodies to treat asthma. ResultsQX008N, derived from a rabbit antibody platform, exhibits a high affinity for TSLP and superior efficacy in blocking TSLP-induced signaling pathways and inflammation in vitro compared with Tezepelumab. In a cynomolgus monkey asthma model, QX008N ameliorated lung function and reduced the levels of eosinophils and IgE. Moreover, the coadministration of QX008N with anti-IL-4R antibodies enhanced the inhibition of inflammatory mediator production triggered via costimulation in vitro. In mouse asthma models, the simultaneous blockade of TSLP and IL-4R using anti-TL4R and anti-TSLP surrogates surpassed the efficacy of monotherapy. To the best of our knowledge, the therapeutic effect of a combination of anti-TSLP and IL-4R antibodies in an asthma model has not yet been reported. ConclusionThese results furnish comprehensive preclinical evidence for QX008N as an innovative anti-TSLP therapeutic agent and provide a preliminary rationale for the development of combination therapies that simultaneously target the TSLP and IL-4R signaling pathways.

Fostering collaborative care in multiple myeloma: The impact of holistic education on decision-making with antibody and CAR-T platforms.

e21008 Background: The management of multiple myeloma (MM) continues to be transformed by evidence supporting the use of CD38 antibodies and B-cell maturation antigen (BCMA) immunotherapy (chimeric antigen receptor (CAR-T) and bispecific antibodies) in newly diagnosed and relapsed/refractory MM (NDMM and RRMM), respectively. However, real-world evidence and survey data show that these approaches are under-utilized across lines of therapy and that educational outreach to patients on their use is inadequate (Puig N et al. ASH 2022. Abstract 1859; Pink S et al. ISPOR 2022. Abstract HSD44; Terpos E et al. Blood Cancer J. 2021;11:40; Adu Y et al. ASH 2023. Abstract 2418). Methods: To address these gaps, PeerView partnered with HealthTree Foundation for Multiple Myeloma to develop 4 live events and 5 online enduring activities from 06/2022 to 06/2023, to educate clinicians on the evidence and principles for integrating CD38 and BCMA antibody platforms into patient care. The activities’ core objectives focused on improving knowledge and skills for integrating CD38 antibodies and BCMA immunotherapy platforms into sequential MM treatment. Furthermore, through the partnership with HealthTree Foundation, the initiatives featured patient perspectives as well as tools and resources to foster clinician–patient communication on use of antibody and CAR-T therapy. Pre- and post-activity multiple choice questions measured changes in knowledge and skills. Responses from enduring activity learners were compared with those from a matched sample of nonparticipants. In a meta-analysis of outcomes from the 9 activities, learning objectives were grouped by key themes, with changes in knowledge and skills averaged across them. Results: Total learners: 12,248 (hematologist-oncologists/oncologists: 7,345; oncology nurses: 1,247) In all activities, learners’ knowledge and skills improved, with increases of: 35 percentage points (ppt) in knowledge of new evidence and changing practice guidelines supporting BCMA antibody platforms in RRMM (44% to 79%); 36 ppt in skills in treatment planning and evidence-based integration of CD38 and BCMA-targeting antibodies in the management of MM (52% to 88%); 46 ppt in ability to address safety considerations related to the use of antibody therapy and BCMA immunotherapy (39% to 85%); 58 ppt in patient interaction skills (eg, counseling, shared decision-making) when using antibody-based and CAR-T platforms (28% to 86%). Conclusions: Improvements in knowledge and skills, combined with a greater focus on patient-centricity, suggest that learners are poised to more effectively integrate CD38 antibodies and BCMA immunotherapy into guideline-recommended MM care that improves the overall well-being and outcomes of patients with MM.

Development of a 10 g/L process for a difficult-to-express multispecific antibody format using a holistic process development approach

Ichnos has developed a multi-specific antibody platform based on the BEAT® (Bispecific engagement by antibodies based on the T-cell receptor) interface. The increased complexity of the bi- and multi-specific formats generated with this platform makes these molecules difficult-to-express proteins compared to standard monoclonal antibodies (mAbs). This report describes how expression limitations of a bi-specific bi-paratopic BEAT antibody were improved in a holistic approach. An initial investigation allowed identification of a misbalance in the subunits composing the BEAT antibody as the potential root cause. This misbalance was then addressed by a signal peptide optimization, and the overall expression level was increased by the combination of two vector design elements on a single gene vector. Further improvements were made in the selection of cell populations and an upstream (USP) platform process was applied in combination with a cell culture temperature shift. This allowed titer levels of up to 6 g/L to be reached with these difficult-to-express proteins. Furthermore, a high-density seeding process was developed that allowed titers of around 11 g/L for the BEAT antibody, increasing the initial titer by a factor of 10. The approach was successfully applied to a tri-specific antibody with titer levels reaching 10 g/L. In summary, a platform process for difficult-to-express proteins was developed using molecular biology tools, cell line development, upstream process optimization and process intensification.

The EV antibody database: An interactive database of curated antibodies for extracellular vesicle and nanoparticle research

Antibodies are critical tools for research into extracellular vesicles (EVs) and other extracellular nanoparticles (ENPs), where they can be used for their identification, characterization, and isolation. However, the lack of a centralized antibody platform where researchers can share validation results thus minimizing wasted personnel time and reagents, has been a significant obstacle. Moreover, because the performance of antibodies varies among assay types and conditions, detailed information on assay variables and protocols is also of value. To facilitate sharing of results on antibodies that are relevant to EV/ENP research, the EV Antibody Database has been developed by the investigators of the Extracellular RNA Communication Consortium (ERCC). Hosted by the ExRNA Portal (https://exrna.org/resources/evabdb/), this interactive database aggregates and shares results from antibodies that have been tested by research groups in the EV/ENP field. Currently, the EV Antibody Database includes modules for antibodies tested for western Blot, EV Flow Cytometry, and EV Sandwich Assays, and holds 110 records contributed by 6 laboratories from the ERCC. Detailed information on antibody sources, assay conditions, and results is provided, including negative results. We encourage ongoing expert input and community feedback to enhance the database's utility, making it a valuable resource for comprehensive validation data on antibodies and protocols in EV biology.

Structure-Based High-Efficiency Homogeneous Antibody Platform by Endoglycosidase Sz Provides Insights into Its Transglycosylation Mechanism

Structure-Based High-Efficiency Homogeneous Antibody Platform by Endoglycosidase Sz Provides Insights into Its Transglycosylation Mechanism

Abstract 5318: SM2275, A tetraspecific, dual EGFR and PD-L1-targeting, conditionally activating co-stimulatory CD28 and simultaneously blocking PD-1/PD-L1 signaling to promoting fully activated T cell killing effects on tumor cells

Abstract Background: T cells require both signal 1 TCR engagement and signal 2 co-stimulatory signal to achieve complete activation and tumor recognition. CD28 is a signal 2 co-stimulatory receptor and activating CD28 increases T cell sensitivity, cytokine production and T cell survival. PD-1/PD-L1 signaling attenuates the T cells functional activity by inhibiting CD28 co-stimulation in the presence of TCR stimulation. Therefore, we developed SM2275, a tetraspecific VHH antibody against EGFR, PD-L1, CD28 and HSA, for the treatment of EGFR+/PD-L1+ solid tumors. Preclinical study demonstrated promising efficacy and favorable safety profile via a unique mechanism of action. Methods: SM2275 was developed using StarMab’s proprietary Quadbody multifunctional VHH antibody platform. It features two monovalent, low-affinity binding domains for EGFR and PD-L1, along with nanomolar low affinity CD28 binding domains arranged in a tandem format, followed by a half-life extending HSA binding domain. Differential binding affinities of SM2275 to EGFR/PD-L1 single-expressing versus double-expressing cell lines were analyzed by flow cytometry. Avidity-enhanced blockade of PD-1 on EGFR+PD-L1+ double-positive compared to PD-L1+ single-positive cells was analyzed via competitive binding assay and Jurkat PD1 NFAT Luciferase reporter assay. EGFR/PD-L1 dual-target-dependent CD28 activation was confirmed using a primary human T cell IL2 & IFN-γ release assay in the presence of TCR stimulation. In vivo efficacy against EGFR+/PD-L1+ double-positive human tumors were evaluated using a syngeneic tumor transplantation model of MC38-hEGFR/hPDL1 cell line into hPD-L1/hCD28 humanized mice. Results: SM2275 displays dramatically increased affinity for cancer cells co-expressing EGFR/PD-L1 compared to single-expressing cells. This leads to an avidity-enhanced PD-L1 blockade specifically on the surface of EGFR+PD-L1+ double-positive cancer cells, enabling localized reactivation of tumor-infiltrating T cells without systemic immune toxicity. Simultaneously blocking the PD-L1 signaling unleashed the inhibition of CD28 co-stimulation for the full T cell activation. In the presence of SM2275, EGFR/PD-L1 double-positive cancer cells induce significantly enhanced CD28 activation and cytokine secretion compared to single-antigen-expressing cells. In a syngeneic tumor transplantation model, SM2275 effectively inhibits the growth of MC38-hEGFR/hPDL1 tumors in hPD-L1-hCD28 humanized mice, with no apparent cytokine release and immune toxicity. Conclusions: SM2275 is a novel dual-EGFR & PD-L1-targeting, conditional CD28 agonistic antibody. It demonstrated promising efficacy and favorable safety profile in preclinical studies via a unique mechanism of action. Citation Format: Shihao Lu, Xiaodan Liu, Sheila Zhou, Wenjing Song, Huiqin Geng, Simin Yang, Hong Zhou, Siyao Xie, Yi Wei, Xing Zhang, Yanbin Liang. SM2275, A tetraspecific, dual EGFR and PD-L1-targeting, conditionally activating co-stimulatory CD28 and simultaneously blocking PD-1/PD-L1 signaling to promoting fully activated T cell killing effects on tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5318.

Abstract 2714: Developing an mRNA encoded therapeutic antibody platform to simplify manufacturing and reduce time to clinic

Abstract The manufacturing of therapeutic antibodies requires expensive production, often challenging purification, lengthy stability optimization and complex protein characterization that despite continual improvement, keeps the cost of treatment in the clinic high. Alternatively, leveraging gene-based approaches such as mRNA to express therapeutics circumvents many of the manufacturing challenges and instead rely on in situ production of antibodies within a patient, improving the developability and cost of sophisticated, disease-modifying antibodies. Crucially, recent work demonstrates that therapeutic antibodies translated in vivo from mRNA can be readily detected within hours following infusion into pre-clinical models and can persist up to several days or weeks. Peak levels of circulating mRNA-encoded antibodies are comparable to infused recombinant equivalents dosed to patients and have been shown to be within favorable therapeutic ranges in phase I trials. Here we outline an mRNA-LNP based platform to encode and deliver therapeutic antibodies in vivo that can overcome costly and challenging manufacturing and that demonstrates both robust PK/PD kinetics and potency. We describe how to effectively encode a standard-of-care anti-HER2 antibody - Trastuzumab - using LNP-encapsulated modified mRNA and validate using in vitro as well as in vivo efficacy models. Initial in vitro characterization of mRNA encoded antibodies demonstrates robust translation in producer cell lines, reproducible expression kinetics, retention of antigen specificity and HC:LC integrity when analyzed by ELISA and WB. To evaluate whether Trastuzumab-encoding mRNA-produced protein is detectable in vivo, antibody levels were measured in mouse serum after IV administration of a single dose of mRNA formulated LNPs. Peak plasma concentrations of mRNA-encoded trastuzumab were benchmarked against circulating recombinant Trastuzumab in parallel groups, and pharmacokinetics interrogated. Of note, the majority of mRNA encoded antibody was likely produced in the liver as shown by bioluminescence imaging. To demonstrate the efficacy of the platform, the potency of mRNA-encoded Trastuzumab isolated from producer cell lines will be assessed using in vitro cytotoxicity assays with human PBMCs and HER2+ tumor lines to dissect ADCC or direct tumor growth inhibition. Additionally, to determine whether mRNA-encoded Trastuzumab is able to retain anti-tumor activity in vivo, a mouse tumor xenograft model will be established and the mRNA antibody-dependent effects on tumor volume, growth and morbidity-free survival will be examined. Overall, the data demonstrate that mRNA-encoded therapeutic antibodies could provide an effective, alternate strategy for solid tumor treatment and may unlock a strategy to deliver lead biologics at reduced cost and with improved developability. Citation Format: Roxana Redis, Dan Rocca, Rachel Pooley, Matthew Benson, Namrata Jayanth, Eva Oswald, Alexander Hale, Louise Brackenbury, Justin Bryans. Developing an mRNA encoded therapeutic antibody platform to simplify manufacturing and reduce time to clinic [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2714.

Abstract 3190: Twin Fc-immune cell engager (ICE): A novel antibody platform for cancer immunotherapy with enhanced avidity for Fcγ receptors and complement

Abstract Immunoglobulin G (IgG)-based mAbs have become a dominant class of cancer immunotherapy due to their excellent drug-like properties, long half-life, and generally safe profiles. Typical IgG-based therapeutic mAbs have strong binding to target antigens owing to the valency effect of two antigen-binding fragments (Fabs). However, the single crystallizable fragment (Fc) lacks the valency effect to engage immune effector elements. As Fcγ receptors (FcγR) such as FcγRIIa and FcγRIIIa and C1q complement component have low affinity for Fc, IgG-based mAbs require high antibody opsonization density to exhibit avidity for these low affinity Fcγ receptors and C1q. This may also account for the suboptimal efficacy of IgG-based mAbs against tumors with less abundant antigen. To enhance effector functions of IgG1 by overcoming such limitation, we devised a novel platform, Twin Fc-ICE, in which a single Fab region is attached by two Fc regions which allow for high avidity binding to Fcγ receptors and C1q. The two Fc regions of Twin Fc-ICE are arranged in parallel, not in tandem, to adopt natural topologies of multiple Fc regions presented by IgG1-opsonized targets. Supporting the avidity effect of Twin Fc-ICE for FcγR binding, we showed that CTN001, a Twin Fc-ICE with trastuzumab Fab, have markedly increased binding to FcγRs including FcγRI, FcγRIIa and FcγRIIIa compared with trastuzumab. The avid FcγR binding was further demonstrated by flow cytometry analysis of human blood which revealed significantly increased binding of CTN001 relative to trastuzumab for immune cells expressing activating FcγRs such as natural killer (NK) cells, monocytes, and dendritic cells (DCs). The monovalent antigen binding of Twin Fc-ICE requires Fab with high affinity. However, provided that high affinity is achieved, the monovalent Fab adds to the two Fc regions to maximize the Fc load on cancer cells expressing low levels of antigen. Indeed, flow cytometry analysis revealed that treatment of CTN001 alone results in higher Fc load on HER2-expressing cancer cells than combination of two anti-HER2 IgG1 mAbs, trastuzumab and pertuzumab. In consistent with increased Fc load on target cells and avid FcγR binding, CTN001 induces enhanced effector functions such as CDC, ADCC and ADCP against HER2-expressing cancer cells compared with trastuzumab, which will be presented in a separate poster. CTN001 exhibits comparable thermal stability with trastuzumab and can be readily purified to >95% purity with typical purification platforms. The serum half-life of CTN001 at 10 mg/kg intravenous dose in rats is found non-inferior to that of trastuzumab (~12 days versus ~7 days). Taken together, Twin Fc-ICE provides an optimized platform with drug-like properties, which leverages avidity effects of Fc to potentiate therapeutic efficacy of antibody. Citation Format: Eun Shik Choi, Hyunkyu Park, Jiseon Bae, Ki-June Lee, Eunmi Kim, Sujin Yoon, Deokjae Lee, Gi-Hyeok Yang. Twin Fc-immune cell engager (ICE): A novel antibody platform for cancer immunotherapy with enhanced avidity for Fcγ receptors and complement [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3190.

Abstract 183: A novel bispecific antibody macrophage engager (BiME) designed for the treatment of solid tumors

Abstract Background: Anti-CD3-based bispecific T cell engagers (BiTE) showed limited clinical efficacy in solid tumors and caused significant cytokine storm. Similar to BiTE where T cells are activated by CD3 antibody, we constructed a novel bi-specific macrophage engager (BiME) platform where macrophage is activated by anti-SIRPa that is directed to a particular tumor via the targeting of the tumor-associated antigen (TAA), resulting in phagocytosis of the tumor cells. This BiME does not lead to cytokine storm and is particularly applicable for the treatment of solid tumors whose microenvironment contains plenty of macrophages. Tumor-associated macrophages are major component of immune cells in the tumor micro-environment (TME) that express an array of effector molecules leading to the inhibition of anti-tumor immune responses. Signal regulatory protein α (SIRPα) is a myeloid-lineage inhibitory receptor that restricts phagocytosis through engagement of its ligand CD47 expressed on tumors and normal tissues. Compared to anti-CD47 therapeutics, targeting myeloid-restricted SIRPα provides a differential pharmacokinetic, safety, and efficacy profile. Here, we report the construction of a SIRPα antagonist-based bispecific macrophage engager (BiME) platform, a potent tumor-killing bi-specific antibody platform. Methods: We have generated a panel of single domain antibody (sdAb), scFv or Fab antibodies targeting different TAAs and SIRPa. We constructed BiME antibodies using different orientations, ratios, and IgG isotypes of anti-TAA arm and anti-SIRPα arm. These bispecific antibodies were evaluated for homologue binding, and CD47-SIRPα blocking properties by ELISA and FACS. In vitro function activity was determined by phagocytosis assay using human monocyte derived macrophage and mouse bone marrow derived macrophage. In vivo anti-tumor efficacy was tested in a syngeneic tumor model with hSIRPα knock-in mice. The pharmacokinetic (PK) and safety profile were assessed in hSIRPα knock-in mice or cynomolgus monkeys. Results: Through Elpiscience proprietary BiME platform, we have generated a panel of TAA/SIRPα bispecific antibodies with TAA being Claudin18.2, EGFR, PDL1 and DLL-3. The BiME treatment demonstrated strong anti-tumor efficacy in multiple mouse syngeneic tumor models. Mechanistically, both myeloid and T cells were activated and contributed to antitumor activity of BiME bispecific antibody. the BiME bispecific antibodies were well tolerated in non-human primates (NHPs) and there is no hematotoxicity or cytokine release syndrome (CRS) observed after treatment of BiME antibodies. Conclusions: These findings provide novel mechanistic insights into how myeloid and T cells can be uniquely modulated by the bispecific macrophage engagers (BiMEs) and demonstrate the potential and advantage of BiME in future clinical development for solid tumor treatment as compared to BiTE approach. Citation Format: Dawei Sun, Haixia Jiang, Yanan Geng, Yue Wu, Rui Gao, Yefeng Lu, Quan Qiu, Yanfen Hu, Yingchao Liu, Xiaoli Guo, Xiaofeng Niu, Hongtao Lu. A novel bispecific antibody macrophage engager (BiME) designed for the treatment of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 183.

Proteomics for heart failure risk stratification: a systematic review

BackgroundHeart failure (HF) is a complex clinical syndrome with persistently high mortality. High-throughput proteomic technologies offer new opportunities to improve HF risk stratification, but their contribution remains to be clearly defined. We aimed to systematically review prognostic studies using high-throughput proteomics to identify protein signatures associated with HF mortality.MethodsWe searched four databases and two clinical trial registries for articles published from 2012 to 2023. HF proteomics studies measuring high numbers of proteins using aptamer or antibody-based affinity platforms on human plasma or serum with outcomes of all-cause or cardiovascular death were included. Two reviewers independently screened articles, extracted data, and assessed the risk of bias. A third reviewer resolved conflicts. We assessed the risk of bias using the Risk Of Bias In Non-randomized Studies—of Exposure tool.ResultsOut of 5131 unique articles identified, nine articles were included in the review. The nine studies were observational; three used the aptamer platform, and six used the antibody platform. We found considerable heterogeneity across studies in measurement panels, HF definitions, ejection fraction categorization, follow-up duration, and outcome definitions, and a lack of risk estimates for most protein associations. Hence, we proceeded with a systematic review rather than a meta-analysis. In two comparable aptamer studies in patients with HF with reduced ejection fraction, 21 proteins were identified in common for the association with all-cause death. Among these, one protein, WAP four-disulfide core domain protein 2 was also reported in an antibody study on HFrEF and for the association with CV death. We proposed standardized reporting criteria to facilitate the interpretation of future studies.ConclusionsIn this systematic review of nine studies evaluating the association of proteomics with mortality in HF, we identified a limited number of proteins common across several studies. Heterogeneity across studies compromised drawing broad inferences, underscoring the importance of standardized approaches to reporting.

Selection and characterization of a peptide-based complement modulator targeting C1 of the innate immune system.

The human complement pathway plays a pivotal role in immune defence, homeostasis, and autoimmunity regulation, and complement-based therapeutics have emerged as promising interventions, with both antagonistic and agonistic approaches being explored. The classical pathway of complement is initiated when the C1 complex binds to hexameric antibody platforms. Recent structural data revealed that C1 binds to small, homogeneous interfaces at the periphery of the antibody platforms. Here, we have developed a novel strategy for complement activation using macrocyclic peptides designed to mimic the interface between antibodies and the C1 complex. In vitro selection utilizing the RaPID system identified a cyclic peptide (cL3) that binds to the C1 complex via the globular head domains of C1q. Notably, when immobilized on surfaces, cL3 effectively recruits C1 from human serum, activates C1s proteases, and induces lysis of cell-mimetic lipid membranes. This represents the first instance of a peptide capable of activating complement by binding C1 when immobilized. Further characterization and synthesis of deletion mutants revealed a critical cycle size of cL3 essential for C1 binding and efficient complement activation. Importantly, cL3 also demonstrated the ability to inhibit complement-mediated lysis without affecting C1 binding, highlighting its potential as a therapeutic modality to prevent complement-dependent cytotoxicity whilst promoting cellular phagocytosis and cell clearance. In summary, this study introduces the concept of "Peptactins" - peptide-based activators of complement - and underscores the potential of macrocyclic peptides for complement modulation, offering potential advantages over traditional biologicals in terms of size, production, and administration.

Deciphering breast cancer cell heterogeneity: a quantum dot-conjugate approach employing MCF-7 and THP-1 co-cultures

Cancer cell heterogeneity in MCF-7/THP-1 co-cultures was shown using a quantum dot antibody platform (QDλ/Ab), mimicking marker polarization in cancer cells during metastasis establishment.

Vaccination against neoantigens induced in cross-priming cDC1 in vivo

The conventional type 1 dendritic cells (cDC1) play a pivotal role in protective immunity against pathogens and cancer. However, their low frequency in the blood and tissues limits their use in immune therapy. We have recently described a method to vaccinate against neoantigens that are induced in tumor cells by targeted delivery of a TAP siRNA to dendritic cells using a TLR9 binding CpG oligonucleotide. Since TLR9 is also expressed in immune suppressive myeloid populations TLR9 targeting could reduce the effectiveness of this approach. Here, we describe a modular multivalent antibody platform to target the TAP siRNA to resident Clec9a expressing cDC1 and show that it leads to selective and sustained TAP downregulation in cDC1 and inhibits tumor growth in mice more effectively than CpG targeted siRNA. To induce DC maturation an agonistic CD40 antibody was administered to the siRNA treated mice. To obviate the need for a second drug formulation and reduce the risk of toxicity, we exploited the multivalent nature of this targeting platform to co-deliver the TAP siRNA and a DC maturation agent, a CpG containing oligonucleotide, to cDC1 in vivo and show that it was more effective than Clec9a targeting of TAP siRNA in combination with CD40 antibody. This study describes a way to manipulate the function of cDC1 cells in vivo using a broadly applicable antibody-based targeting platform to deliver multiple biological agents to specific cells in vivo to potentiate (immune) therapy and to probe the biology of specific cell types in their natural settings.

A chemical biology/modular antibody platform for ADP-ribosylation signaling

A chemical biology/modular antibody platform for ADP-ribosylation signaling

Complementary methods for monitoring hole-hole homodimer associated with a WuXiBody-based asymmetric bispecific antibody

WuXiBody is a bispecific antibody (bsAb) platform developed by WuXi Biologics. Its key feature is the replacement of one parental antibody's CH1/CL region with the T-cell receptor (TCR) constant domain, which prevents mispairing between non-cognate heavy chain and light chain. In addition, heavy chain heterodimerization in asymmetric WuXiBody molecule is promoted by the knobs-into-holes (KiH) technology. Despite the great success of KiH strategy in improving heterodimer formation, homodimers (especially the hole-hole homodimer) can still be generated at low levels. In general, detection and monitoring of homodimers during KiH bsAb purification are challenging as homodimers share similar physicochemical properties with the target heterodimeric bsAb. Nevertheless, the unique design of WuXiBody allows homodimers to be effectively detected and monitored by multiple methods. In the current work, with an asymmetric WuXiBody case study, we demonstrated that hole-hole homodimer can be effectively monitored by six chromatography methods including hydrophobic interaction chromatography (HIC), reverse phase (RP), cation exchange (CEX), KappaSelect, CaptureSelect CH1-XL and Protein L.

A multi-specific, multi-affinity antibody platform neutralizes sarbecoviruses and confers protection against SARS-CoV-2 in vivo

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has been responsible for a global pandemic. Monoclonal antibodies (mAbs) have been used as antiviral therapeutics; however, these therapeutics have been limited in efficacy by viral sequence variability in emerging variants of concern (VOCs) and in deployment by the need for high doses. In this study, we leveraged the multi-specific, multi-affinity antibody (Multabody, MB) platform, derived from the human apoferritin protomer, to enable the multimerization of antibody fragments. MBs were shown to be highly potent, neutralizing SARS-CoV-2 at lower concentrations than their corresponding mAb counterparts. In mice infected with SARS-CoV-2, a tri-specific MB targeting three regions within the SARS-CoV-2 receptor binding domain was protective at a 30-fold lower dose than a cocktail of the corresponding mAbs. Furthermore, we showed in vitro that mono-specific MBs potently neutralize SARS-CoV-2 VOCs by leveraging augmented avidity, even when corresponding mAbs lose their ability to neutralize potently, and that tri-specific MBs expanded the neutralization breadth beyond SARS-CoV-2 to other sarbecoviruses. Our work demonstrates how avidity and multi-specificity combined can be leveraged to confer protection and resilience against viral diversity that exceeds that of traditional monoclonal antibody therapies.

Evaluation of novel anti-CEACAM6 antibody-based conjugates for radioimmunotheranostics of pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a highly malignant solid tumor that lacks early diagnostic methods. Recently, targeted immunotherapy and radiotherapy have been integrated with radionuclide-antibody conjugate drugs, which can be used for targeted diagnosis and dynamic imaging of tumors. CEACAM6 is overexpressed in pancreatic tumors and is a potential theranostic target for PDAC. We aimed to develop a novel targeted carrier for theranostics of PDAC and other solid tumors. Based on camelid heavy-chain-only antibodies, we developed a CEACAM6-targeting recombinant antibody NY004, and evaluated it as a novel antibody-carrier for imaging and therapy of cancer in tumor models. We labeled NY004 with theranostic nuclides and applied this self-developed antibody platform in diagnostic imaging and antitumor assessment in PDAC models. Through microPET, IHC, and biodistribution assays, targeting and biodistribution of [89Zr]-NY004 in solid tumors including PDAC was examined, and the investigated tumors were all CEACAM6-positive malignancies. We found that NY004 was suitable for use as a drug carrier for radioimmunotheranostics. Our study showed that NY004 was characterized by high targeted uptake and a long retention time in PANC-1 tumors (up to 6days post-injection), with good specificity and high imaging efficiency. Therapeutic evaluation of the radionuclide-labeled antibody drug [177Lu]-NY004 in PDAC tumor-bearing model revealed that NY004 had high and prolonged uptake in tumors, relatively low non-target organ uptake, and good anti-tumor efficacy. As a drug platform for radiotheranostics, CEACAM6-specific antibody NY004 met the requirements of easy-labeling, targeting specificity, and effective persistence in pancreatic adenocarcinoma tissues. • [89Zr]-NY004 has good specificity and high imaging efficiency, and is characterized by high tumor-targeting uptake and a long tumor retention time as a PET molecular imaging tracer. • Therapeutic radionuclide-conjugated antibody drug [177Lu]-NY004 has high uptake and prolonged uptake duration in tumors, low non-target organ uptake, and significant tumor-inhibiting efficacy in PDAC model. • The self-developed antibody structure NY004 is a promising drug platform for radioimmunotheranostics of CEACAM6-positive tumors including pancreatic ductal adenocarcinoma.

Abstract LB210: RenNano® mice: A heavy-chain-only antibody platform for the generation of nanobody therapeutics

Abstract Monoclonal antibodies have been used to successfully treat various diseases, including tumors, autoimmune diseases, and infectious diseases. Traditional antibodies are comprised of a tetramer of two heavy chains and two light chains, totaling 150 kDa in molecular weight. However, the large size of antibodies can limit the therapeutic application; in particular, the penetration of tumors and the blood brain barrier (BBB) is not always feasible. In contrast to traditional antibodies, heavy-chain-only antibodies (HCAbs) are significantly smaller (~75 kDa), as they contain only two heavy chains. Since the heavy chain variable domain of HCAbs (i.e., VHH or single domain antibody, sdAb, or nanobody) is solely responsible for antigen recognition, nanobodies can function independently as a therapeutic molecule, which may be advantageous for penetrating tumors or the BBB. Previously, we generated a fully human antibody mouse platform, RenMabTM, in which the murine heavy chain and kappa light chain variable domains were replaced by the full human heavy chain and kappa light chain V(D)J loci in situ. Here, we have further modified the RenMabTM model to generate a fully human heavy-chain-only antibody mouse model, termed RenNano®. The modified heavy chain constant regions of RenNano® mice allow them to spontaneously produce HCAbs. Flow cytometry and biolayer interferometry confirmed that RenNano®-derived HCAbs can bind antigens without light chains. Despite this reliance on the heavy chain only variable regions for antigen specificity, RenNano® mice can generate antigen-specific antibodies with high affinity (10−8 -10−9 KD) upon immunization with various antigens. In addition, many RenNano®-derived HCAbs exhibited a longer CDR3 length, which could promote the recognition of difficult-to-reach epitopes. Furthermore, RenNano®-derived HCAbs have favorable diversity, and excellent developability properties such as a higher degree of hydrophilicity. Anti-4-1BB HCAbs can also activate 4-1BB-NF-κB signaling in a dose-dependent manner, as demonstrated in reporter assays. In summary, the full human heavy-chain-only antibody mice, RenNano®, can produce human HCAb with high affinity and good efficacy. Thus, RenNano® is a powerful platform to discover HCAb/nanobodies for various therapeutic applications. Citation Format: Yiqing Hu, Qi Zhang, Lijun Zhang, Yabo Zhang, Huizhen Zhao, Jiawei Yao, Liu Yang, Baihong Liu, Shensen Wang, Zhengfang Su, Li Hui, Qingcong Lin, Qiangqiang Ma, Yuelei Shen. RenNano® mice: A heavy-chain-only antibody platform for the generation of nanobody therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB210.

Abstract 1864: An improved trispecific antibody (TsAb) platform for optimally engaging T cells for the treatment of solid tumor malignancies

Abstract T-cell recruiting bispecific antibodies have been shown to be clinically effective in treating hematological malignancies. However, their application against solid tumors is limited by several challenges, including a narrow therapeutic window due to cytokine release syndrome (CRS) induced toxicity, limited number of functional T cells, and an immunosuppressive tumor microenvironment (TME). Engagement of CD28 co-stimulatory domain can promote T cell fitness and sustained activation to overcome T -cell anergy. Here, we describe the development of a novel tri-specific antibody (TsAb) platform which is designed to optimally engage T cells via co-stimulation of both CD3 and of CD28 to achieve more effective tumor cell killing without increased CRS induction. To that end, we identified three selected formats (TsAb-1, TsAb-2 and TsAb-3) based on improved properties of the antibody formats, such as safety, efficacy, stability and developability. These formats have been developed to target three different well-established solid tumor-associated antigens (TAAs) as proof of concept. The binding activity of TsAbs to TAA, CD3 and CD28 was evaluated by ELISA and flow cytometry. Tumor cell cytotoxicity was assessed and secretion of cytokines by immune cells using a tumor cell-PBMC co-culture assay. Effective engagement of CD3 and CD28 was evaluated by flow analysis of CD25 Bcl-xL expression, respectively. Additionally, flow immunophenotyping was performed to characterize the differentiation of memory T cell subsets. Our results show that while all three TsAb formats displayed comparable cytotoxicity, TsAb-3 triggered immune cell activation only upon TAA engagement and T cell synapse formation. TsAb-3 showed > 90% purity, no CRS induction in PBMCs alone or in coculture with low TAA-expressing tumor cells. Additionally, tethered TsAb-3 format was unable to stimulate PBMCs in the absence of TAA. Upon further analysis of T cell phenotype differentiation, TsAb-3 showed enhanced CD25+ T cell activation, Bcl-xL induction and increased T-effector and memory cell phenotypes on day 3 and day 7. In conclusion, our data support further development of TsAb as a versatile and improved T cell engager therapeutics for the treatment of solid malignancies. These versatile formats can be utilized not only in T cell engager therapeutics, but also in rationale combinations with immune check point inhibitors. Citation Format: Reshmi Nair, Veena Somasundaram, Arvind Goswami, Jaya Bhatnagar, Sourav Paul, Chaitali Dey, Kalyanaraman Vaidyanathan, Rajkumar Paul, Sankar Periasamy, Mary Helen KG, Mahalakshmi NV, Abhishek Sinha, Rachel Salazar, Avanish K. Varshney. An improved trispecific antibody (TsAb) platform for optimally engaging T cells for the treatment of solid tumor malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1864.