Parental mAb Research Articles

Characterization of monoclonal antibody charge variants under near-native separation conditions using nanoflow sheath liquid capillary electrophoresis-mass spectrometry

BackgroundMonoclonal antibodies (mAbs) undergo multiple post-translational modifications (PTMs) during production and storage, resulting for instance in charge and oxidized variants. PTMs need to be assessed as critical quality attributes to assure protein quality and safety. Capillary zone electrophoresis (CZE) enables efficient charge-based separation. The CZE method developed by He et al. (2011) is currently applied routinely in the pharmaceutical industry for profiling charge heterogeneity of mAbs. However, as the method relies on a non-volatile background electrolyte (BGE), it cannot be directly hyphenated with mass spectrometry (MS), hampering the identification of separated charge variants. ResultsThis study presents a CZE-UV/MS method using a neutral static capillary coating of hydroxypropyl methylcellulose combined with a volatile BGE at pH 5.0 to allow for MS-compatible mAb charge variant separations. The effect of several parameters, including pH and concentration of the BGE, applied voltage, and injected mAb concentrations on separation performance was investigated using a panel of commercially available mAbs. The optimized method was evaluated with IgG1 and IgG4 mAbs of varying pI (7.4–9.2) and degrees of heterogeneity. Basic and acidic variants were separated from the parent mAb using a BGE of 50 mM acetic acid adjusted to pH 5.0 with ammonium hydroxide. The relative abundances of charge variants determined with the new method showed a good correlation with the corresponding relative levels obtained with the method of He et al. CZE-MS coupling was accomplished using the nanoCEasy, a low-flow sheath liquid interface, which enabled the identification and quantitation of basic, acidic, and incomplete pyroglutamate variants, and glycoforms of the tested mAbs. SignificanceThis manuscript describes a new CZE-MS method that permits heterogeneity assessment of mAbs under MS-compatible conditions, providing charge variant separation.

EmbB and EmbC regulate the sensitivity of Mycobacterium abscessus to echinomycin.

Treatment of Mycobacterium abscessus (Mab) infections is very challenging due to its intrinsic resistance to most available drugs. Therefore, it is crucial to discover novel anti-Mab drugs. In this study, we explored an intrinsic resistance mechanism through which Mab resists echinomycin (ECH). ECH showed activity against Mab at a minimum inhibitory concentration (MIC) of 2 µg/ml. A ΔembC strain in which the embC gene was knocked out showed hypersensitivity to ECH (MIC: 0.0078-0.0156 µg/ml). The MICs of ECH-resistant strains screened with reference to ΔembC ranged from 0.25 to 1 µg/ml. Mutations in EmbB, including D306A, D306N, R350G, V555I, and G581S, increased the Mab's resistance to ECH when overexpressed in ΔembC individually (MIC: 0.25-0.5 µg/ml). These EmbB mutants, edited using the CRISPR/Cpf1 system, showed heightened resistance to ECH (MIC: 0.25-0.5 µg/ml). The permeability of these Mab strains with edited genes and overexpression was reduced, as evidenced by an ethidium bromide accumulation assay, but it remained significantly higher than that of the parent Mab. In summary, our study demonstrates that ECH exerts potent anti-Mab activity and confirms that EmbB and EmbC are implicated in Mab's sensitivity to ECH. Mutation in EmbB may partially compensate for a loss of EmbC function.

Broadening sarbecovirus neutralization with bispecific antibodies combining distinct conserved targets on the receptor binding domain

ABSTRACT Monoclonal neutralizing antibodies (mAbs) are considered an important prophylactic against SARS-CoV-2 infection in at-risk populations and a strategy to counteract future sarbecovirus-induced disease. However, most mAbs isolated so far neutralize only a few sarbecovirus strains. Therefore, there is a growing interest in bispecific antibodies (bsAbs) which can simultaneously target different spike epitopes and thereby increase neutralizing breadth and prevent viral escape. Here, we generate and characterize a panel of 30 novel broadly reactive bsAbs using an efficient controlled Fab-arm exchange protocol. We specifically combine some of the broadest mAbs described so far, which target conserved epitopes on the receptor binding domain (RBD). Several bsAbs show superior cross-binding and neutralization compared to the parental mAbs and cocktails against sarbecoviruses from diverse clades, including recent SARS-CoV-2 variants. BsAbs which include mAb COVA2–02 are among the most potent and broad combinations. As a result, we study the unknown epitope of COVA2–02 and show that this mAb targets a distinct conserved region at the base of the RBD, which could be of interest when designing next-generation bsAb constructs to contribute to a better pandemic preparedness.

Comparison of single-chain variable fragments and monoclonal antibody against dihydroartemisinin

Immunoassay relies on antibodies, but traditional antibodies such as monoclonal antibody (mAb) require animal immunization and complex procedures. Single-chain variable fragment (scFv) becomes a potential alternative to mAb with advantages of the low cost, rapid and easy prepared. In the present study, we prepared scFvs against dihydroartemisinin (DHA) based on E. coli and HEK293T cell expression system, named MBP-scFv and scFv-Fc, respectively. Their properties were compared with the parent mAb. The calculated affinity constants of mAb, MBP-scFv and scFv-Fc were 2.1 × 108 L mol−1, 2.2 × 107 L mol−1 and 1.6 × 108 L mol−1, respectively. The half inhibitory concentration (IC50) of mAb, MBP-scFv and scFv-Fc were 1.16 ng mL−1, 2.15 ng mL−1 and 6.57 ng mL−1, respectively. Both the scFv showed less sensitive than the mAb based on the IC50. The cross-reactivities of MBP-scFv for artemisinin and artesunate exhibited similarities to the mAb, yet the cross-reactivities of scFv-Fc for these compounds exceeded those of the mAb significantly. The stability of the scFvs was ascertained to be maintained for over 5 days at room temperature, and for more than a month at both 4 °C and − 20 °C. After that, the indirect competitive enzyme-linked immunosorbent assays (icELISAs) based on the scFv from E. coli were used to detect the DHA content in eight drug samples, and the result was consistent with ultra-performance liquid chromatography simultaneously. Although scFv can be used for quantitative determination of drugs, but it still cannot completely replace mAb in immunoassay without evolution and modification.

Enhancement of complement-dependent cytotoxicity by linking factor-H derived short consensus repeats 19-20 to CD20 antibodies.

Antibody-mediated complement-dependent cytotoxicity (CDC) on malignant cells is regulated by several complement control proteins, including the inhibitory complement factor H (fH). fH consists of 20 short consensus repeat elements (SCRs) with specific functional domains. Previous research revealed that the fH-derived SCRs 19-20 (SCR1920) can displace full-length fH on the surface of chronic lymphocytic leukemia (CLL) cells, which sensitizes CLL cells for e.g. CD20-targeting therapeutic monoclonal antibody (mAb) induced CDC. Therefore, we constructed lentiviral vectors for the generation of cell lines that stably produce mAb-SCR-fusion variants starting from the clinically approved parental mAbs rituximab, obinutuzumab and ofatumumab, respectively. Flow-cytometry revealed that the modification of the mAbs by the SCRs does not impair the binding to CD20. Increased in vitro lysis potency compared to their parental mAbs was corroborated by showing specific and dose dependent target cell elimination by CDC when compared to their parental mAbs. Lysis of CLL cells was not affected by the depletion of NK cells, suggesting that antibody-dependent cellular cytotoxicity plays a minor role in this context. Overall, this study emphasizes the crucial role of CDC in the elimination of CLL cells by mAbs and introduces a novel approach for enhancing CDC by directly fusing fH SCR1920 with mAbs.

Humanization of Pan-HLA-DR mAb 44H10 Hinges on Critical Residues in the Antibody Framework.

Reducing the immunogenicity of animal-derived monoclonal antibodies (mAbs) for use in humans is critical to maximize therapeutic effectiveness and preclude potential adverse events. While traditional humanization methods have primarily focused on grafting antibody Complementarity-Determining Regions (CDRs) on homologous human antibody scaffolds, framework regions can also play essential roles in antigen binding. Here, we describe the humanization of the pan-HLA-DR mAb 44H10, a murine antibody displaying significant involvement of the framework region in antigen binding. Using a structure-guided approach, we identify and restore framework residues that directly interact with the antigen or indirectly modulate antigen binding by shaping the antibody paratope and engineer a humanized antibody with affinity, biophysical profile, and molecular binding basis comparable to that of the parental 44H10 mAb. As a humanized molecule, this antibody holds promise as a scaffold for the development of MHC class II-targeting therapeutics and vaccines.

Bispecific antibodies targeting two glycoproteins on SFTSV exhibit synergistic neutralization and protection in a mouse model

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease with a high fatality rate of up to 30% caused by SFTS virus (SFTSV). However, no specific vaccine or antiviral therapy has been approved for clinical use. To develop an effective treatment, we isolated a panel of human monoclonal antibodies (mAbs). SF5 and SF83 are two neutralizing mAbs that recognize two viral glycoproteins (Gn and Gc), respectively. We found that their epitopes are closely located, and we then engineered them as several bispecific antibodies (bsAbs). Neutralization and animal experiments indicated that bsAbs display more potent protective effects than the parental mAbs, and the cryoelectron microscopy structure of a bsAb3 Fab-Gn-Gc complex elucidated the mechanism of protection. In vivo virus passage in the presence of antibodies indicated that two bsAbs resulted in less selective pressure and could efficiently bind to all single parental mAb-escape mutants. Furthermore, epitope analysis of the protective mAbs against SFTSV and RVFV indicated that they are all located on the Gn subdomain I, where may be the hot spots in the phleboviruses. Collectively, these data provide potential therapeutic agents and molecular basis for the rational design of vaccines against SFTSV infection.

A dual-targeting approach using a human bispecific antibody against the receptor-binding domain of the Middle East Respiratory Syndrome Coronavirus

The emergence of the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) has posed a significant global health concern due to its severe respiratory illness and high fatality rate. Currently, despite the potential for resurgence, there are no specific treatments for MERS-CoV, and only supportive care is available. Our study aimed to address this therapeutic gap by developing a potent neutralizing bispecific antibody (bsAb) against MERS-CoV. Initially, we isolated four human monoclonal antibodies (mAbs) that specifically target the MERS-CoV receptor-binding domain (RBD) using phage display technology and an established human antibody library. Among these four selected mAbs, our intensive in vitro functional analyses showed that the MERS-CoV RBD-specific mAb K111.3 exhibited the most potent neutralizing activity against MERS-CoV pseudoviral infection and the molecular interaction between MERS-CoV RBD and human dipeptidyl peptidase 4. Consequently, we engineered a novel bsAb, K207.C, by utilizing K111.3 as the IgG base and fusing it with the single-chain variable fragment of its non-competing pair, K111.1. This engineered bsAb showed significantly enhanced neutralization potential against MERS-CoV compared to its parental mAb. These findings suggest that K207.C may serve as a potential candidate for effective MERS-CoV neutralization, further highlighting the promise of the bsAb dual-targeting approach in MERS-CoV neutralization.

Cadherin-17 as a target for the immunoPET of adenocarcinoma

PurposeCadherin-17 (CDH17) is a calcium-dependent cell adhesion protein that is overexpressed in several adenocarcinomas, including gastric, colorectal, and pancreatic adenocarcinoma. High levels of CDH17 have been linked to metastatic disease and poor prognoses in patients with these malignancies, fueling interest in the protein as a target for diagnostics and therapeutics. Herein, we report the synthesis, in vitro validation, and in vivo evaluation of a CDH17-targeted 89Zr-labeled immunoPET probe.MethodsThe CDH17-targeting mAb D2101 was modified with an isothiocyanate-bearing derivative of desferrioxamine (DFO) to produce a chelator-bearing immunoconjugate — DFO-D2101 — and flow cytometry and surface plasmon resonance (SPR) were used to interrogate its antigen-binding properties. The immunoconjugate was then radiolabeled with zirconium-89 (t1/2 ~ 3.3 days), and the serum stability and immunoreactive fraction of [89Zr]Zr-DFO-D2101 were determined. Finally, [89Zr]Zr-DFO-D2101’s performance was evaluated in a trio of murine models of pancreatic ductal adenocarcinoma (PDAC): subcutaneous, orthotopic, and patient-derived xenografts (PDX). PET images were acquired over the course of 5 days, and terminal biodistribution data were collected after the final imaging time point.ResultsDFO-D2101 was produced with a degree of labeling of ~ 1.1 DFO/mAb. Flow cytometry with CDH17-expressing AsPC-1 cells demonstrated that the immunoconjugate binds to its target in a manner similar to its parent mAb, while SPR with recombinant CDH17 revealed that D2101 and DFO-D2101 exhibit nearly identical KD values: 8.2 × 10−9 and 6.7 × 10−9 M, respectively. [89Zr]Zr-DFO-D2101 was produced with a specific activity of 185 MBq/mg (5.0 mCi/mg), remained >80% stable in human serum over the course of 5 days, and boasted an immunoreactive fraction of >0.85. In all three murine models of PDAC, the radioimmunoconjugate yielded high contrast images, with high activity concentrations in tumor tissue and low uptake in non-target organs. Tumoral activity concentrations reached as high as >60 %ID/g in two of the cohorts bearing PDXs.ConclusionTaken together, these data underscore that [89Zr]Zr-DFO-D2101 is a highly promising probe for the non-invasive visualization of CDH17 expression in PDAC. We contend that this radioimmunoconjugate could have a significant impact on the clinical management of patients with both PDAC and gastrointestinal adenocarcinoma, most likely as a theranostic imaging tool in support of CDH17-targeted therapies.

Abstract LB425: A tumor microenvironment-targeting CD98-directed ADC confers robust anti-tumor activity in multiple cancers with favorable pharmacokinetics and safety profiles in preclinical models

Abstract CD98, a subunit of the LAT1/CD98 amino acid transporter complex, is intimately involved in cell adhesion, migration, proliferation, and signal transduction by enhancing amino acid transport and amplifying integrin signaling. CD98 is expressed at aberrantly high levels in many solid tumors and hematological malignancies, and this is frequently associated with poor prognosis, highlighting its potential as a cancer therapy target. However, CD98 is also expressed in various normal tissues hence complicating the development of antibody-based therapies owing to lack of tumor selectivity, poor pharmacokinetics (PK), and safety issues. We previously reported a pH-dependent anti-CD98 monoclonal antibody (mAb) that selectively targets the acidic tumor microenvironment (Tian, X., et al. Nat Biomed Eng 7(1): 8-23.). In the present report, we further developed that mAb into an antibody-drug conjugate (ADC; HH018-sesutecan), in which the mAb is conjugated to a topoisomerase I inhibitor-based linker-drug, sesutecan, at a drug:antibody ratio (DAR) of approximately 8. Sesutecan previously demonstrated promising characteristics in preclinical studies with multiple targets and conferred an encouraging benefit:risk profile in the clinic with a FRα-directed ADC (rinatabart sesutecan). HH018-sesutecan exhibited similar pH-dependent binding as its parental mAb in vitro, and markedly improved PK compared to the non-pH-dependent ADC control in CD98-humanized C57BL/6 mice. HH018-sesutecan demonstrated potent cytotoxicity against CD98-expressing cancer cell lines, strong bystander effect, as well as dose-dependent anti-tumor activity in mouse models representing multiple tumor types (colorectal cancer, ovarian cancer, renal cell carcinoma, lymphoma, hypopharyngeal carcinoma, and oral adeno-squamous carcinoma). Notably, sustained tumor regression was achieved with a single dose of 3-5 mg/kg in ~8 tumor models. A pilot toxicity study was conducted for HH018-sesutecan at two repeat-dose of 30 mg/kg in cynomolgus monkeys. HH018-sesutecan was well-tolerated with the principal toxicity (myelosuppression) being payload driven and reversible, accompanied with a favorable PK profile (half-life of 7-9 days). In summary, our studies demonstrate that the pH-dependent CD98-directed HH018- sesutecan exerts wide-ranging and potent anti-tumor efficacy, as well as favorable PK and safety profiles in preclinical models. HH018-sesutecan is a promising agent for further development in a broad range of CD98-expressing tumors. Citation Format: Fang Yang, Lei Wang, Xinxin Tian, Xuan Qiu, Jianhe Chen, Wenke Qi, Guangyu Li, Haidong Liu, Juan Liu, Suping Huang, Bin Ye, Qilin Wu, Baiteng Zhao, Wenhui Li, Jianhua Sui, Zhu Chen. A tumor microenvironment-targeting CD98-directed ADC confers robust anti-tumor activity in multiple cancers with favorable pharmacokinetics and safety profiles in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB425.

Abstract 2050: Preclinical characterization of PRO1106, a novel and promising SLITRK6-directed sesutecan ADC

Abstract SLITRK6 is a type I transmembrane member of the SLITRK family of proteins and is highly expressed in certain epithelial tumors including urothelial, lung, breast cancer and glioblastoma, with minimal-to-low expression in normal tissues. Recent studies with a vedotin-based SLITRK6-directed ADC, AGS15E, have offered clinical proof-of-concept of harnessing this target with the ADC modality for addressing high unmet need at least in bladder cancer. PRO1106 is comprised of a proprietary humanized SLITRK6-targeting IgG1 (dubbed as “mAb”) and the proprietary topoisomerase 1 inhibitor-based linker-drug, sesutecan. Sesutecan previously demonstrated promising characteristics in preclinical studies with multiple targets and conferred an encouraging benefit:risk profile in the clinic with a FRα-directed ADC (rinatabart sesutecan). PRO1106 and the parent mAb exhibited strong binding (with low nanomolar affinity) to recombinant human or cynomolgus monkey SLITRK6 on bio-layer interferometry. No binding was observed with human SLITRK1 or SLITRK4 or in additional negative-binding assays. Potent cellular binding (with sub-nanomolar EC50) was also observed for the parent mAb and ADC with bladder, head and neck, and brain cancer cell lines as well as SLITRK6-overexpressing 293F cells. Target-binding was superior to sirtratumab (the parent antibody in AGS15E) and the binding epitope appeared to be distinct from that of sirtratumab in cross-block studies. PRO1106 displayed rapid internalization and elicited robust cytotoxicity (IC50 in the sub-nanomolar to low nanomolar range) in SLITRK6-expressing tumor cells but not target-negative cells. PRO1106 produced strong tumor growth inhibition in multiple cell-derived xenograft (CDX) models in mice representing bladder cancer and esophageal squamous cell carcinoma. PRO1106 showed a stable PK with much higher exposure compared to an in-house synthesized analog of AGS15E in rats. In an exploratory PK study in cynomolgus monkeys, PRO1106 at 5 mg/kg exhibited extended PK with minimal release of exatecan in circulation and with no apparent toxicity observed. In summary, PRO1106 is an ADC built on a clinically validated linker-drug directed at a clinically validated target, with promising data in preclinical pharmacology studies. As SLITRK6 expression in skin is negligible, PRO1106 may offer an exciting new option for patients with bladder cancer where the current standard-of-care ADC therapy (enfortumab vedotin) confers significant skin-related toxicity burden as well as for patients with additional cancer types. Citation Format: Wanwan Shen, Haotian Lu, Yang Xiao, Xuan Qiu, Haidong Liu, Lei Wang, Zhu Chen. Preclinical characterization of PRO1106, a novel and promising SLITRK6-directed sesutecan ADC [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 2050.

Abstract 2621: Preclinical Efficacy of DM002, a bispecific HER3 × MUC1 antibody-drug conjugate with a novel DNA topoisomerase I inhibitor, in solid tumor models

Abstract MUC1 is a known tumor-associated antigen (TAA), but previous anti-MUC1 agents have shown limited clinical efficacy due to neutralization by the soluble N-terminal autoproteolytic product of MUC1. More recently, HER3-targeted ADCs, such as Patritumab deruxtecan, have shown encouraging early clinical activity in a variety of tumors. However, the majority of patients did not exhibit response to paritumab deruxtecan treatment. Therefore, improved therapies targeting these TAAs are needed. As HER3 and MUC1 are co-expressed in a wide range of cancer types, we hypothesized that simultaneous targeting of HER3 and MUC1 with a bispecific antibody-drug conjugate (bsADC) could potentially lead to increased efficacy, reduced resistance and improved safety in the clinical setting. We generated an anti-HER3 × MUC1 bispecific antibody using the fully human common light chain antibody transgenic mice (RenLite®). The HER3 × MUC1 bsAb showed improved cell binding and internalization in vitro compared to the parental mAb, suggesting synergy between the two arms. We then conjugated the HER3xMUC1 bsAb with vcMMAE for proof-of-concept studies, or with our novel DNA topoisomerase I inhibitor linker/payload conjugates (BLD1102) to make DM002-BLD1102 bsADC. Both bsADCs potently inhibited the growth of HER3+MUC1+ PDX tumors. DM002-vcMMAE also showed greater in vivo efficacy than its parental mAb ADCs, consistent with their in vitro internalization activity. DM002-BLD1102 also showed strong anti-tumor activity in DM002-vcMMAE resistant PDX models, suggesting the superiority of this new DNA topoisomerase I inhibitor linker/payload over the classical vcMMAE linker/payload. In conclusion, the HER3 × MUC1 bsAb is a promising agent for the treatment of HER3 and MUC1 positive tumors. Citation Format: Yifu Zhang, Chengzhang Shang, Nannan Wang, Gao An, Chaoshe Guo, W. Frank An, Yi Yang. Preclinical Efficacy of DM002, a bispecific HER3 × MUC1 antibody-drug conjugate with a novel DNA topoisomerase I inhibitor, in solid tumor models [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 2621.

Abstract 6580: A Novel EGFR x cMET bispecific ADC PRO1286 demonstrated broad antitumor activity and promising tolerability in preclinical models

Abstract EGFR and cMET are frequently co-expressed on tumor cells, often upregulated as the escape mechanism for each other, and both well-validated targets in oncology. Small molecule TKIs and amivantamab have been approved for non-small cell lung cancer with relevant actionable genomic alterations (AGA), and monoclonal antibodies offer treatment options for EGFR-expressing head and neck and colorectal cancers without AGA. Yet high unmet need in these tumors still exists. We sought to develop an EGFR and cMET dual-targeting ADC using our proprietary technology platform with carefully chosen design features for the many tumors expressing EGFR and cMET with or without AGA. PRO1286 is comprised of a monovalent bispecific human IgG1 (dubbed as “Bsab”) with fine-tuned affinity on EGFR and cMET, and the proprietary topoisomerase 1 inhibitor-based linker-drug, sesutecan. Sesutecan previously demonstrated promising characteristics in preclinical studies with multiple targets and conferred an encouraging benefit:risk profile in the clinic with a FRα-directed ADC (rinatabart sesutecan). PRO1286 at DAR8 displays high hydrophilicity and excellent stability and developability in vitro. PRO1286 binds selectively and specifically to each target with a low nanomolar EC50 in the CHO-K1 cells overexpressing each target. PRO1286 exhibited efficient internalization (~50-80% internalized in 4 hrs) and strong cytotoxicity (sub-nanomolar IC50) against a broad panel of tumor cell lines with higher potency than the parent mAbs. PRO1286 produced marked and dose dependent anti-tumor activity in mouse xenograft models encompassing a wide range of target expression levels, tumor types (head and neck, lung, esophageal, gastric, colorectal), and genomic landscape (with or without AGA in EGFR or cMET; with diverse additional co-mutations). Sustained tumor growth inhibition or complete remission was observed in several xenograft models after treatment with a single dose (2 mg/kg) of PRO1286. PRO1286 was more efficacious than benchmarking molecules in the mouse studies. In an exploratory toxicity study in cynomolgus monkeys, PRO1286 was well tolerated at 30 mg/kg with the main toxicity being payload-driven and residing in bone marrow. PRO1286 exhibited a stable PK profile that was similar to that of the Bsab in rats and favorable PK characteristics in monkeys. In summary, PRO1286 demonstrated promising physiochemical properties, PK/PD, antitumor activity, and tolerability in preclinical studies. It may carry an expanded therapeutic index compared to other EGFR- and/or cMET-directed ADCs. It may also differentiate from existing EGFR- and/or cMET-targeting agents on broad coverage of EGFR and cMET-expressing tumors with or without the respective AGAs. Effort is under way to advance a DAR optimized molecule to the clinic for the treatment of many EGFR- or cMET-expressing solid tumors. Citation Format: Yang Xiao, Haiyu Huang, Jianhe Yin, Xuan Qiu, Lei Wang, Haidong Liu, Baiteng Zhao, Zhu Chen. A Novel EGFR x cMET bispecific ADC PRO1286 demonstrated broad antitumor activity and promising tolerability in preclinical models [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 6580.

Empowering SARS-CoV-2 variant neutralization with a bifunctional antibody engineered with tandem heptad repeat 2 peptides.

With the global pandemic and the continuous mutations of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the need for effective and broadly neutralizing treatments has become increasingly urgent. This study introduces a novel strategy that targets two aspects simultaneously, using bifunctional antibodies to inhibit both the attachment of SARS-CoV-2 to host cell membranes and viral fusion. We developed pioneering IgG4-(HR2)4 bifunctional antibodies by creating immunoglobulin G4-based and phage display-derived human monoclonal antibodies (mAbs) that specifically bind to the SARS-CoV-2 receptor-binding domain, engineered with four heptad repeat 2 (HR2) peptides. Our in vitro experiments demonstrate the superior neutralization efficacy of these engineered antibodies against various SARS-CoV-2 variants, ranging from original SARS-CoV-2 strain to the recently emerged Omicron variants, as well as SARS-CoV, outperforming the parental mAb. Notably, intravenous monotherapy with the bifunctional antibody neutralizes a SARS-CoV-2 variant in a murine model without causing significant toxicity. In summary, this study unveils the significant potential of HR2 peptide-driven bifunctional antibodies as a potent and versatile strategy for mitigating SARS-CoV-2 infections. This approach offers a promising avenue for rapid development and management in the face of the continuously evolving SARS-CoV-2 variants, holding substantial promise for pandemic control.

Development of a Bispecific IgG1 Antibody Targeting BCMA and PDL1.

We designed, produced, and purified a novel IgG1-like, bispecific antibody (bsAb) directed against B-cell maturation antigen (BCMA), expressed by multiple myeloma (MM) cells, and an immune checkpoint inhibitor (ICI), PDL1, expressed in the MM microenvironment. The BCMA×PDL1 bsAb was fully characterized in vitro. BCMA×PDL1 bound specifically and simultaneously, with nM affinity, to both native membrane-bound antigens and to the recombinant soluble antigen fragments, as shown by immunophenotyping analyses and surface plasmon resonance (SPR), respectively. The binding affinity of bsAb for PDL1 and BCMA was similar to each other, but PDL1 affinity was about 10-fold lower in the bsAb compared to parent mAb, probably due to the steric hindrance associated with the more internal anti-PDL1 Fab. The bsAb was also able to functionally block both antigen targets with IC50 in the nM range. The bsAb Fc was functional, inducing human-complement-dependent cytotoxicity as well as ADCC by NK cells in 24 h killing assays. Finally, BCMA×PDL1 was effective in 7-day killing assays with peripheral blood mononuclear cells as effectors, inducing up to 75% of target MM cell line killing at a physiologically attainable, 6 nM, concentration. These data provide the necessary basis for future optimization and in vivo testing of this novel bsAb.

The origins of nonideality exhibited by monoclonal antibodies and Fab fragments in human serum.

The development of therapeutic antibodies remains challenging, time-consuming, and expensive. A key contributing factor is a lack of understanding of how proteins are affected by complex biological environments such as serum and plasma. Nonideality due to attractive or repulsive interactions with cosolutes can alter the stability, aggregation propensity, and binding interactions of proteins in solution. Fluorescence correlation spectroscopy (FCS) can be used to measure apparent second virial coefficient (B2,app ) values for therapeutic and model monoclonal antibodies (mAbs) that capture the nature and strength of interactions with cosolutes directly in undiluted serum and similar complex biological media. Here, we use FCS-derived B2,app measurements to identify the components of human serum responsible for nonideal interactions with mAbs and Fab fragments. Most mAbs exhibit neutral or slightly attractive interactions with intact serum. Generally, mAbs display repulsive interactions with albumin and mildly attractive interactions with IgGs in the context of whole serum. Crucially, however, these attractive interactions are much stronger with pooled IgGs isolated from other serum components, indicating that the effects of serum nonideality can only be understood by studying the intact medium (rather than isolated components). Moreover, Fab fragments universally exhibited more attractive interactions than their parental mAbs, potentially rendering them more susceptible to nonideality-driven perturbations. FCS-based B2,app measurements have the potential to advance our understanding of how physiological environments impact protein-based therapeutics in general. Furthermore, incorporating such assays into preclinical biologics development may help de-risk molecules and make for a faster and more efficient development process.

A protective human antibody against respiratory syncytial virus by targeting a prefusion epitope across sites IV and V of the viral fusion glycoprotein

Respiratory syncytial virus (RSV) is one of the leading pathogens that cause lower respiratory tract infections in infants and the elderly. Passive immunoprophylaxis with monoclonal antibody (mAb) has been approved to prevent morbidity and mortality from RSV infection in infants. Here we report the isolation of two neutralizing mAbs against RSV from convalescent children by prefusion form of fusion (F) glycoprotein as bait. One mAb RV11 exhibited good potency in neutralization of RSV strains from both A and B subtypes in cell-based assay, and protected mice from RSV infection in vivo. An RV11 escape mutant was identified, which contains an S443P mutation in F protein. Crystal structure showed the RV11 bound to a conserved prefusion epitope across the antigenic sites IV and V of the F glycoprotein. RV11 showed a strong synergistic effect when combined with two RSV antivirals, an F-targeting small molecular inhibitor ziresovir and a site Ø neutralizing mAb D25 (the parental mAb for nirsevimab). The study extended our knowledge to the neutralizing and protective epitopes of RSV, and the mAb RV11 deserves further development for clinical translation.

A novel bispecific antibody dual-targeting approach for enhanced neutralization against fast-evolving SARS-CoV-2 variants.

The emergence of new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has caused unprecedented health and socioeconomic crises, necessitating the immediate development of highly effective neutralizing antibodies. Despite recent advancements in anti-SARS-CoV-2 receptor-binding domain (RBD)-specific monoclonal antibodies (mAbs) derived from convalescent patient samples, their efficacy against emerging variants has been limited. In this study, we present a novel dual-targeting strategy using bispecific antibodies (bsAbs) that specifically recognize both the SARS-CoV-2 RBD and fusion peptide (FP), crucial domains for viral attachment to the host cell membrane and fusion in SARS-CoV-2 infection. Using phage display technology, we rapidly isolated FP-specific mAbs from an established human recombinant antibody library, identifying K107.1 with a nanomolar affinity for SARS-CoV-2 FP. Furthermore, we generated K203.A, a new bsAb built in immunoglobulin G4-(single-chain variable fragment)2 forms and demonstrating a high manufacturing yield and nanomolar affinity to both the RBD and FP, by fusing K102.1, our previously reported RBD-specific mAb, with K107.1. Our comprehensive in vitro functional analyses revealed that the K203.A bsAb significantly outperformed the parental RBD-specific mAb in terms of neutralization efficacy against SARS-CoV-2 variants. Furthermore, intravenous monotherapy with K203.A demonstrated potent in vivo neutralizing activity without significant in vivo toxicity in a mouse model infected with a SARS-CoV-2 variant. These findings present a novel bsAb dual-targeting strategy, directed at SARS-CoV-2 RBD and FP, as an effective approach for rapid development and management against continuously evolving SARS-CoV-2 variants.

A NOVEL IMMUNOSTIMULATORY PD-L1/OX40 TETRAVALENT BISPECIFIC ANTIBODY FOR CANCER IMMUNOTHERAPY

Abstract Single agent immune checkpoint therapy has shown substantial and durable clinical activity in many tumor types; however, only a fraction of the patients could benefit from this approach. To improve beyond the anti-PD-1/PD-L1 treatment options, bispecific antibodies (BsAb) that combines PD-L1 blockade and conditional co-stimulatory receptor activation simultaneously in one molecule have been developed and demonstrated superior anti-tumor activity in pre-clinical models. However, many of these PD-L1 based BsAb faced challenge in clinical development due to insufficient activity or unexpected toxicity. Here, we demonstrated that OX40 might be a more suitable partner for PD-L1 based BsAb design than other agonistic targets (CD27 and 4-1BB, etc.) currently in clinical studies. A novel Fc silenced tetravalent PD-L1/OX40 (EMB-09) BsAb targeting optimal OX40 binding epitope has been developed based on EpimAb’s proprietary FIT-Ig® technology. Results showed that EMB-09 maintained the parental mAb binding characteristic and retained the functional properties of each parental mAb including OX40 agonistic as well as PD-L1/PD1 inhibitory pathways. In addition, EMB-09 induced OX40 activation only in the context of PD-L1 engagement. Concurrent PD-L1/PD-1 blockade and OX40 co-stimulation by EMB-09 led to synergistic activation of T cell in vitro and exerted superior anti-tumor activity in mouse tumor models compared to anti-PD-L1 mAb. The underlining mechanism was extensively analyzed, which indicated an increased CD8+ tumor-infiltrating T-cells (TIL) as well as enhanced CD8 TIL activation status upon EMB-09 treatment. Additionally, EMB-09 was well tolerated in cynomolgus monkeys at high dose levels with a favorable safety and PK profile in a GLP-TOX study. In conclusion, as a PD-L1/OX40 BsAb with a novel biology mechanism, EMB-09 demonstrated a markedly improved anti-tumor activity compared to anti-PD-L1 mAb. The first-in-human clinal study of EMB-09 has been initiated (NCT05263180).

Potent broadly neutralizing antibody VIR-3434 controls hepatitis B and D virus infection and reduces HBsAg in humanized mice

Chronic hepatitis B is a global public health problem, and coinfection with hepatitis delta virus (HDV) worsens disease outcome. Here, we describe a hepatitis B virus (HBV) surface antigen (HBsAg)-targeting monoclonal antibody (mAb) with the potential to treat chronic hepatitis B and chronic hepatitis D. HBsAg-specific mAbs were isolated from memory B cells of HBV vaccinated individuals. Invitro neutralization was determined against HBV and HDV enveloped with HBsAg representing eight HBV genotypes. Human liver-chimeric mice were treated twice weekly with a candidate mAb starting 3 weeks post HBV inoculation (spreading phase) or during stable HBV or HBV/HDV coinfection (chronic phase). From a panel of human anti-HBs mAbs, VIR-3434 was selected and engineered for pre-clinical development. VIR-3434 targets a conserved, conformational epitope within the antigenic loop of HBsAg and neutralized HBV and HDV infection with higher potency than hepatitis B immunoglobulins invitro. Neutralization was pan-genotypic against strains representative of HBV genotypes A-H. In the spreading phase of HBV infection in human liver-chimeric mice, a parental mAb of VIR-3434 (HBC34) prevented HBV dissemination and the increase in intrahepatic HBV RNA and covalently closed circular DNA. In the chronic phase of HBV infection or co-infection with HDV, HBC34 treatment decreased circulating HBsAg by >1 log and HDV RNA by >2 logs. The potently neutralizing anti-HBs mAb VIR-3434 reduces circulating HBsAg and HBV/HDV viremia in human liver-chimeric mice. VIR-3434 is currently in clinical development for treatment of patients with chronic hepatitis B or D. Chronic infection with hepatitis B virus and co-infection with hepatitis D virus place approximately 290 million individuals worldwide at risk of severe liver disease and cancer. Available treatments result in low rates of functional cure or require lifelong therapy that does not eliminate the risk of liver disease. We isolated and characterized a potent human antibody that neutralizes hepatitis B and D viruses and reduces infection in a mouse model. This antibody could provide a new treatment for patients with chronic hepatitis B and D.