Erratum to "Mechanism of Action and Pharmacokinetics of Approved Bispecific Antibodies" [Biomol Ther 32(6), 708-722 (2024)]

NSCLC with activating mutations in the EGFR gene are associated with high response rates to EGFR tyrosine kinase inhibitors (TKIs), such as erlotinib and gefitinib, but invariably acquired resistance emerges over time. A primary cause of resistance is the emergence of secondary mutations in EGFR which neutralize the effectiveness of TKIs. In addition, the cMet pathway is often activated, either through MET gene amplification, overexpression of cMet protein, or an increase in the ligand HGF, to provide a compensatory survival pathway conferring resistance to EGFR TKIs. We have designed a bispecific EGFR-cMet antibody (JNJ-61186372) with a unique set of mechanisms of action resulting in anti-tumor activity in the EGFR mutant setting, with or without cMet pathway activation. We have demonstrated three mechanisms of action that contribute to the activity of JNJ-61186372: 1) inhibition of ligand-induced phosphorylation of both EGFR and cMet, 2) receptor degradation in vivo, and 3) enhanced ADCC activity. JNJ-61186372 inhibited EGF-induced phosphorylation of EGFR in cell lines with either wild-type (WT) EGFR or activating mutations in EGFR. In the same cell lines, JNJ-61186372 inhibited HGF-induced phosphorylation of cMet. JNJ-61186372 also blocked pERK and pAkt with similar IC50 values in EGFR-WT and EGFR mutant cell lines, indicating that downstream signaling pathways were inhibited. Total protein levels of both EGFR and cMet were decreased in xenograft tumor models following treatment with JNJ-61186372 compared to tumors from mice treated with PBS control suggesting that one mechanism by which JNJ-61186372 suppresses EGFR and cMet activity in vivo is through degradation of both receptors. The third mechanism of action is directing immune cells to kill tumor cells. JNJ-61186372 is produced with low levels of fucosylation, which translates to an enhanced antibody-dependent cellular cytotoxicity (ADCC). These three mechanisms of action of JNJ-61186372 provide a distinct preclinical profile for targeting both EGFR and cMET in a single bispecific antibody. JNJ-61186372 demonstrated efficacy in multiple in vivo tumor models with EGFR mutations, including both cell line and patient-derived xenografts. Importantly, JNJ-61186372 effectively inhibited tumor growth in models with mutant EGFR and cMet activation, whereas single agent EGFR inhibitors were less effective. The preclinical data support the clinical development of JNJ-61186372 in patients with lung cancer and other malignancies associated with aberrant EGFR and cMET signaling. Citation Format: Sheri L. Moores, Mark Chiu, Barbara Bushey, Kristen Chevalier, Peter Haytko, Joost Neijssen, Paul Parren, Janine Schuurman, Mark Anderson, Ricardo Attar, Robert Kramer, Matthew V. Lorenzi. Discovery and preclinical pharmacology of JNJ-61186372: A novel bispecific antibody targeting EGFR and cMET. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr DDT01-03. doi:10.1158/1538-7445.AM2014-DDT01-03

Bispecific antibodies represent a significant advancement in therapeutic antibody engineering, offering the ability to simultaneously target two distinct antigens. This dual-targeting capability enhances therapeutic efficacy, especially in complex diseases, such as cancer and autoimmune disorders, where drug resistance and incomplete target coverage are prevalent challenges. Bispecific antibodies facilitate immune cell engagement and disrupt multiple signaling pathways, providing a more comprehensive treatment approach than traditional monoclonal antibodies. However, the intricate structure of bispecific antibodies introduces unique pharmacokinetic challenges, including issues related to their absorption, distribution, metabolism, and excretion, which can significantly affect their efficacy and safety. This review provides an in-depth analysis of the structural design, mechanisms of action, and pharmacokinetics of the currently approved bispecific antibodies. It also highlights the engineering innovations that have been implemented to overcome these challenges, such as Fc modifications and advanced dimerization techniques, which enhance the stability and half-life of bispecific antibodies. Significant progress has been made in bispecific antibody technology; however, further research is necessary to broaden their clinical applications, enhance their safety profiles, and optimize their incorporation into combination therapies. Continuous advancements in this field are expected to enable bispecific antibodies to provide more precise and effective therapeutic strategies for a range of complex diseases, ultimately improving patient outcomes and advancing precision medicine.

Bispecific antibodies can target two different targets simultaneously with a wide application prospect and rapid development in tumor treatment. The main function is to recruit effector cells selectively in order to kill tumor cells or bind tumor-associated growth factor receptor to inhibit cell proliferation. This paper reviews the current situation of bispecific antibodies in design, mechanism of action and the research progress in the clinical cancer treatment. Key words: Bispecific antibody; Molecular targeted therapy; Immunotherapy

Every 2 Weeks or Every 4 Weeks Subcutaneous Injection of Emicizumab in Pediatric Patients with Severe Hemophilia A without Inhibitors: A Multi-Center, Open-Label Study in Japan (HOHOEMI Study)

Introduction: MCLA-129 is an ADCC-enhanced IgG1 bispecific antibody that targets epidermal growth factor receptor (EGFR) and c-MET. The HGF/c-MET pathway is frequently upregulated in tumors that are resistant to EGFR tyrosine kinase inhibitors (TKIs). This study investigated the MCLA-129 mechanism of action in vitro and effectiveness in vivo in an EGFR exon20 insertion (ex20ins) non-small cell lung cancer (NSCLC) model. Methods: To study the effect of MCLA-129 on c-MET and EGFR dimerization, β-galactosidase enzyme complementation assays were performed. Ligand-dependent phosphorylation of EGFR and c-MET was assessed in vitro on the NCI-H1650 NSCLC cell line by immunoblotting. Shotgun mutagenesis epitope mapping was used to identify the binding epitope of MCLA-129 to EGFR. ADCC activity of MCLA-129 was determined in NSCLC cells expressing different levels of EGFR and c-MET using an ADCC reporter assay with high affinity FcγRIII 158V-variant and low affinity 158F-variant effector cells. ADCP was measured with monocyte-derived macrophages incubated with pHrodo-labeled HCC827 NSCLC target cells with readout of tumor cell phagocytosis on the Incucyte® system. For the evaluation of efficacy in vivo in the EGFR exon20ins PDX model, MCLA-129 was administered at 2.5, 8 or 25 mg/kg i.p. weekly. Results: MCLA-129 blocks EGF and HGF binding to EGFR and c-MET, respectively, and prevents receptor dimerization. Consequently, MCLA-129 inhibits phosphorylation of EGFR and c-MET as demonstrated in NCI-H1650 cells. Mutagenesis analysis identified critical binding residues of domain III of EGFR for MCLA-129 binding that are distinct from cetuximab binding. MCLA-129 displayed potent ADCC activity on NSCLC cell lines using effector cells expressing either high or low affinity FcγRIII variant. MCLA-129 demonstrated potent dose-dependent ADCP of HCC827 NSCLC cells. Finally, MCLA-129 led to significant dose-dependent regression of a patient-derived EGFR exon20ins NSCLC tumor in a mouse PDX model. Conclusion: MCLA-129 is a Biclonics® common light chain bispecific antibody with multiple mechanisms of action including potent inhibition of c-MET and EGFR ligand binding, ligand-induced receptor dimerization and phosphorylation, ADCC and ADCP. MCLA-129 demonstrates significant growth inhibition of a patient-derived EGFR exon20ins tumor in a preclinical xenograft model. A phase 1/2 clinical trial of MCLA-129 in solid tumors is ongoing. These data support the further clinical development of MCLA-129 in patients with NSCLC, including NSCLC with EGFR exon20ins, and other solid tumors. Citation Format: David J. de Gorter, Marie O'Connor, Alexandre Deshiere, Martijn van Rosmalen, Szabolcs Fatrai, Jeroen Lammerts van Bueren, Cecile A. Geuijen. Mechanism of action of MCLA-129, a bispecific antibody that targets EGFR and c-MET and impairs growth of EGFR exon 20 insertion mutant non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 336.

GB261, an Fc-Function Enabled and CD3 Affinity De-Tuned CD20/CD3 Bispecific Antibody, Demonstrated a Highly Advantageous Safety/Efficacy Balance in an Ongoing First-in-Human Dose-Escalation Study in Patients with Relapsed/Refractory Non-Hodgkin Lymphoma

Treatment outcomes for multiple myeloma (MM) have improved due to the introduction of autologous stem cell transplantation and novel drugs. However, many patients develop resistance to existing therapies; hence, novel treatment strategies for these patients must be established. Therapeutic antibodies, including daratumumab and isatuximab targeting CD38 and elotuzumab targeting SLAMF7, have been introduced as immunotherapies for MM. These antibodies exert cytotoxic effects on myeloma cells through the activation of effectors such as natural killer cells and complement, and induction of phagocytosis by macrophages. Suppressed anti-tumor immunity may be related to acquisition of drug resistance by myeloma cells in patients with MM. It has been reported that the effect of therapeutic antibodies is through the stimulation of anti-tumor immunity. Thus, as each therapeutic antibody displays its own mechanism of action, therapy based on this mechanism of action should be introduced. Furthermore, chimeric antigen receptor (CAR) T-cell therapy, antibody drug conjugates (ADC), and bispecific antibodies (BsAbs) are gradually being introduced as novel immunotherapies for MM. CAR T-cells with high proliferation levels and persistence in recipients to improve the duration of therapeutic response are currently being developed.

Preclinical Evaluation of ISB 1442, a First-in-Class CD38 and CD47 Bispecific Antibody Innate Cell Modulator for the Treatment of AML and T-ALL

(Background and purpose) A bispecific antibody (BsAb), as a next-generation therapeutic antibody, has two antigen binding sites in an antibody molecule. Many formats have been exploited for decades. Among them, we focused on T cell dependent BsAb (TDB), which can engage and redirect cytotoxic T cells against tumor cells and is expected to be effective in treating refractory cancer. However, although many studies revealed its potent antitumor effect in animal models, it has not been applied to solid tumors yet in clinics. In this context, we think that it is necessary to clarify pharmacokinetics (PK), pharmacodynamics (PD) and mechanism of action (MOA) of TDB, especially within tumor tissue. For this aim, we have decided to develop the molecular imaging-based systems for the evaluation of TDB in order to visualize antigen binding to two target antigens, BsAb delivery or immunological synapse formation. (Materials a& Methods) We used hEx3 which is a humanized IgG-like antibody with bivalent Fv regions against each EGFR and CD3. We applied the TDB to colorectal cancer (CRC) cells with or without KRAS or BRAF mutation. In the first part, we visualized the BsAb binding to two antigens, EGFR on CRC cells and CD3 on T cells. Next, immunological synapse formation as a cutting-edge mechanism involving T cells-killing tumor cells, which is an important MOA of TDB, was visualized in combination with Granzyme staining. In the second part, we evaluated in vitro cytotoxicity and the in vivo antitumor effect of hEx3 in NOD-SCID mice bearing CRC xenografts with KRAS or BRAF mutation following human PBMC administration. (Results) We found that CRC cells were damaged via Granzyme accumulation through immunological synapse. In the ELISA data, we also found that released gamma interferons can damage CRC cells. In vitro and In vivo studies showed that the hEx3 demonstrated a significant cytocidal effect and antitumor activity not only for KRAS wild but also mutant. (Conclusion) we established a molecular imaging-based TDB evaluation system and visualized antigen-binding and immunological synapse formation as a MOA of TDB. Present data indicated that TDB can damage CRC cells in two distinct manners, namely immunological synapse-dependent and -independent manners using cytokines including a gamma interferon. TDB may thus be a promising next generation antibody therapy against CRC regardless KRAS status. Citation Format: Daisuke Kamakura, Masahiro Yasunaga, Ryutaro Asano, Yasuhiro Matsumura. Development of bispecific antibodies using molecular imaging [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2274.

RG6234, a GPRC5DxCD3 T-Cell Engaging Bispecific Antibody, Is Highly Active in Patients (pts) with Relapsed/Refractory Multiple Myeloma (RRMM): Updated Intravenous (IV) and First Subcutaneous (SC) Results from a Phase I Dose-Escalation Study

Objectives: Bispecific antibodies (BsAbs) act through the formation of an immunologic synapse between T-cells (CD3) and a tumor-associated surface antigen (TAA) leading to T-cell activation and serial lysis of tumor cells. The aim of the present study is to explore the mechanism of action (MOA) and the in vitro effect of BsAbs on hematologic samples with the PharmaFlow platform. Methods: Fresh whole bone marrow (BM) and peripheral blood (PB) from 41 samples from 3 different hematologic diseases (31 AML, 3 ALL, and 7 CLL) and two AML cell lines were tested with CD3-CD123 (for AML patients and cell lines) or CD3-CD19 (for ALL and CLL patients) BsAbs in the PharmaFlow platform, an innovative proprietary method that uses flow cytometry (FCM) to efficiently count the number of tumor cells killed by activated T-cells. We analyzed the populations of leukemic cells, activated T-cells, and residual normal cells. Additional key parameters were also used to explore the MOA after BsAb exposure at different time incubations (24h-144h), such as the effective E:T ratio (the number of T cells that kill a number of leukemic cells), real basal E:T ratio, tumor antigen expression, T-cell expansion, and expression of immune checkpoint proteins on target and effector cells before and after cell culture. For some experiments, fluorescence-activated cell sorting (FACS) was performed to evaluate T-cell cytotoxicity after BsAb exposure. Results: Most of the samples demonstrated T-cell activation and effective lysis of tumor cells after BsAb exposure independent of TAA expression and in a dose-response manner. Once sorted, these T cells could kill tumor cells in the absence of BsAb, as well as tumor cells that did not express the TAA target. Interestingly, these activated T cells selectively killed tumor cells with low cytotoxicity in residual normal cells from the same patients. Moreover, differential T-cell cytotoxicity was observed between samples. We observed samples with leukemic resistance or no T-cell activity (especially in CLL with CD3-CD19), as well as others with higher T-cell cytotoxicity and minimal number of activated T cells (especially in AML with CD3-CD123). The integration of all the predictive parameters (E:T ratios, Tumor-Specific Antigen (TSA) expression, etc.) allowed us to generate an in vitro response model and select samples with higher T-cell cytotoxicity after the BsAb exposure. Conclusion: Our findings are consistent with a model where, in addition to the standard MOA inducing tumor cells lysis by proximity, BsAbs can highly enrich cytotoxic clonal T-cell subsets with TSA and induce strong activation and proliferation of T cells capable of killing tumor cells in an effective and selective manner. This differential in vitro T-cell cytotoxicity effect between patients could allow us to select better candidates for adoptive antitumor immunotherapy with BsAbs. Citation Format: Daniel Primo, Pilar Hernandez, Julian Gorrochategui, Maria L. Vicente, David Martinez-Cuadron, Pau Montesinos, Joaquin Mártinez-López, Joan Ballesteros. A novel in vitro approach elucidates a new mode of cytotoxic action of bispecific antibodies on hematologic malignancies [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B012.

Studies have demonstrated that the clinical benefit of PD-1 blockade can be further improved by combination with an αCTLA-4 mAb in some indications. However, this increased activity is commensurate with significant immune related adverse events (irAE's). Therefore, novel approaches are required to uncouple toxicity from anti-tumour efficacy and realise the full potential of this combination. MEDI5752 is a monovalent bispecific human IgG1 monoclonal antibody (mAb) with an engineered fragment crystallisable (Fc) domain to reduce Fc effector function, that specifically binds two clinically validated negative T cell regulators; PD-1 (programmed cell death 1) and CTLA-4 (cytotoxic T-lymphocyte-associated protein 4). MEDI5752 has been designed to suppress the PD-1 pathway and provide modulated CTLA-4 inhibition to uncouple CTLA-4 dependent peripheral toxicity from tumour efficacy. PD-1 expression is a defining feature of tumour infiltrating lymphocytes (TILs). We show that MEDI5752 can saturate CTLA-4 on PD-1+ cells at orders of magnitude lower concentrations than required to saturate CTLA-4 on PD-1- cells. Moreover, our data demonstrate that monovalent targeting of CTLA-4 with MEDI5752 is significantly less potent (15 fold) than bivalent targeting with a parental αCTLA-4 mAb in reporter assays. In contrast, the switch to monovalent targeting of PD-1 has limited effect on potency (within 3-fold compared to a parental αPD-1 mAb) in a PD-1/L1 reporter assay. Together these data demonstrate the potential for MEDI5752 to inhibit CTLA-4 on TILs whilst sparing peripheral T cell populations and reducing toxicity. Furthermore, profiling of MEDI5752 in a range of primary T cell activation assays reveals equivalent activity to a combination of parental PD-1 and CTLA-4 antibodies. MEDI5752 is rapidly internalised upon target binding with kinetics similar to the parental αCTLA-4 mAb reflecting the rapid recycling of this receptor. However, in contrast to an αCTLA-4 mAb (or an αPD-1 mAb), MEDI5752, by tethering CTLA-4 to PD-1, leads to the internalisation and subsequent degradation of PD-1. This novel mechanism of action further differentiates MEDI5752 from a combination of mAb's targeting PD-1 and CTLA-4. MEDI5752 is a novel monovalent bispecific which may provide an improved therapeutic index when compared to a combination of bivalent αPD-1 and αCTLA-4 mAb's, and could provide benefit in cancer indications. Citation Format: Simon J. Dovedi, Yariv Mazor, Matthew Elder, Sumati Hasani, Bo Wang, Suzanne Mosely, Desmond Jones, Anna Hansen, Chuning Yang, Yanli Wu, Ikbel Achour, Nick Durham, Gareth Browne, Thomas Murray, James Hair, Michelle Morrow, Godfrey Rainey, Maria Jure Kunkel, John Gooya, Daniel Freeman, Ronald Herbst, Robert Wilkinson. MEDI5752: A novel bispecific antibody that preferentially targets CTLA-4 on PD-1 expressing T-cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2776.

Non-small cell lung cancers (NSCLCs) with activating mutations in the Epidermal Growth Factor Receptor (EGFR) gene are associated with high response rates (70-80%) to EGFR tyrosine kinase inhibitors (TKIs), such as erlotinib and gefitinib, but most acquire resistance over time through numerous mechanisms. In these patients, the cMet pathway is often activated to compensate and provide resistance to the EGFR targeted monotherapy; this activation can occur by MET gene amplification, overexpression of cMet protein, or an increase in the ligand HGF. We have designed a bispecific EGFR-cMet antibody (JNJ-61186372) with multiple mechanisms of action resulting in anti-tumor activity in the EGFR mutant setting, with or without cMet pathway activation. Controlled Fab-arm exchange was used to produce JNJ-61186372, a technique that allows for efficient large-scale preparation of bispecific antibodies with a regular IgG1 structure. JNJ-61186372 was shown to bind EGFR and cMet and efficiently inhibited ligand-induced phosphorylation of both receptors. In addition to this important mechanism of action, we have engineered the antibody to contain lower than normal fucose levels to increase Fc-dependent effector mechanisms. JNJ-61186372 exhibited antibody dependent cellular cytotoxicity (ADCC) activity in vitro in a range of NSCLC cell lines with EGFR mutations, KRas mutation, and/or amplified MET gene. Furthermore, the low fucose form of JNJ-61186372 demonstrated more effective ADCC activity compared to its normal fucose counterpart. The bispecific JNJ-61186372 antibody showed increased potency (2-3 fold) compared to the combination of monovalent EGFR and monovalent cMet antibodies, demonstrating the beneficial effects of dual targeting in a single molecule. Antibody dependent cell-mediated phagocytosis (ADCP) activity of JNJ-61186372 was also confirmed in vitro. We have also demonstrated that Fc-dependent effector functions contributed to in vivo anti-tumor growth activity of JNJ-61186372, in a xenograft model with EGFR mutations and cMet activation. Our data demonstrate that the bispecific antibody JNJ-61186372, generated using controlled Fab-arm exchange, has in vitro ADCC and ADCP activity in EGFR mutant settings, either with or without cMet pathway activation, and with KRas mutation. In addition, the Fc-dependent effector mechanisms contributed to in vivo anti-tumor efficacy. The dual signaling inhibition of EGFR and cMet pathways by JNJ-61186372, combined with enhanced Fc effector function, may provide multiple mechanisms to combat resistance in EGFR mutant NSCLC patients. Citation Format: Keri L. Soring, Katharine D. Grugan, Randall J. Breszki, Jose Pardinas, Leopoldo Luistro, Barbara Bushey, Joost Neijssen, Paul Parren, Janine Schuurman, Mark Anderson, Ricardo Attar, Matthew V. Lorenzi, Mark Chiu, Sheri Moores. Activity of a bispecific antibody targeting EGFR and cMet with enhanced Fc effector function in EGFR mutant setting with cMet pathway activation. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A11.

Bispecific antibodies have moved from being an academic curiosity with therapeutic promise to reality, with two molecules being currently commercialized (Hemlibra® and Blincyto®) and many more in clinical trials. The success of bispecific antibodies is mainly due to the continuously growing number of mechanisms of actions (MOA) they enable that are not accessible to monoclonal antibodies. One of the earliest MOA of bispecific antibodies and currently the one with the largest number of clinical trials is the redirecting of the cytotoxic activity of T-cells for oncology applications, now extending its use in infective diseases. The use of bispecific antibodies for crossing the blood–brain barrier is another important application because of its potential to advance the therapeutic options for neurological diseases. Another noteworthy application due to its growing trend is enabling a more tissue-specific delivery or activity of antibodies. The different molecular solutions to the initial hurdles that limited the development of bispecific antibodies have led to the current diverse set of bispecific or multispecific antibody formats that can be grouped into three main categories: IgG-like formats, antibody fragment-based formats, or appended IgG formats. The expanded applications of bispecific antibodies come at the price of additional challenges for clinical development. The rising complexity in their structure may increase the risk of immunogenicity and the multiple antigen specificity complicates the selection of relevant species for safety assessment.

Targeted therapy and immunotherapy have changed the landscape of treatment of cancers with respect to longevity and quality of life. Programmed death 1 (PD-1) and vascular endothelial growth factor (VEGF) are very salient targets of anticancer drugs. Dual blockade with bispecific antibodies is a novel mode of treatment of malignancies, which has already been successful in hematological malignancies. Ivonescimab, a novel bispecific antibody that targets PD-1 and VEGF, has been developed in China. It has been found to be beneficial in nonsmall cell lung cancer, biliary tract cancers, and breast cancers for which phase 3 clinical trials have already been discussed in various meetings and publications. The current drug review is regarding the parameters of this novel drug—the mechanism of action, clinical and preclinical trials, and approvals.