A new approach for generating bispecific antibodies based on a common light chain format and the stable architecture of human immunoglobulin G1.

Bispecific antibodies combine two different antigen-binding sites in a single molecule, enabling more specific targeting, novel mechanisms of action, and higher clinical efficacies. Although they have the potential to outperform conventional monoclonal antibodies, many bispecific antibodies have issues regarding production, stability, and pharmacokinetic properties. Here, we describe a new approach for generating bispecific antibodies using a common light chain format and exploiting the stable architecture of human immunoglobulin G1 We used iterative experimental validation and computational modeling to identify multiple Fc variant pairs that drive efficient heterodimerization of the antibody heavy chains. Accelerated stability studies enabled selection of one Fc variant pair dubbed "DEKK" consisting of substitutions L351D and L368E in one heavy chain combined with L351K and T366K in the other. Solving the crystal structure of the DEKK Fc region at a resolution of 2.3 Å enabled detailed analysis of the interactions inducing CH3 interface heterodimerization. Local shifts in the IgG backbone accommodate the introduction of lysine side chains that form stabilizing salt-bridge interactions with substituted and native residues in the opposite chain. Overall, the CH3 domain adapted to these shifts at the interface, yielding a stable Fc conformation very similar to that in wild-type IgG. Using the DEKK format, we generated the bispecific antibody MCLA-128, targeting human EGF receptors 2 and 3. MCLA-128 could be readily produced and purified at industrial scale with a standard mammalian cell culture platform and a routine purification protocol. Long-term accelerated stability assays confirmed that MCLA-128 is highly stable and has excellent biophysical characteristics.

Translational PK-PD modeling analysis of MCLA-128, a HER2/HER3 bispecific monoclonal antibody, to predict clinical efficacious exposure and dose.

Introduction MCLA-128 is a bispecific monoclonal antibody targeting the HER2 and HER3 receptors. Pharmacokinetics (PK) and pharmacodynamics (PD) of MCLA-128 have been evaluated in preclinical studies in cynomolgus monkeys and mice. The aim of this study was to characterize the PK and PD of MCLA-128 and to predict a safe starting dose and efficacious clinical dose for the First-In-Human study. Methods A PK-PD model was developed based on PK data from cynomolgus monkeys and tumor growth data from a mouse JIMT-1 xenograft model. Allometric scaling was used to scale PK parameters between species. Simulations were performed to predict the safe and efficacious clinical dose, based on AUCs, receptor occupancies and PK-PD model simulations. Results MCLA-128 PK in cynomolgus monkeys was described by a two-compartment model with parallel linear and nonlinear clearance. The xenograft tumor growth model consisted of a tumor compartment with a zero-order growth rate and a first-order dying rate, both affected by MCLA-128. Human doses of 10 to 480 mg q3wk were predicted to show a safety margin of >10-fold compared to the cynomolgus monkey AUC at the no-observed-adverse-effect-level (NOAEL). Doses of ≥360 mg resulted in predicted receptor occupancies above 99% (Cmax and Cave). These doses showed anti-tumor efficacy in the PK-PD model. Conclusions This analysis predicts that a flat dose of 10 to 480 mg q3wk is suitable as starting dose for a First-in-Human study with MCLA-128. Flat doses ≥360 mg q3wk are expected to be efficacious in human, based on receptor occupancies and PK-PD model simulations.

Validation of the rapid fluorescent focus inhibition test for rabies virus-neutralizing antibodies in clinical samples.

Monoclonal antibodies are successful biologics in treating a variety of diseases, including the prevention or treatment of viral infections. CL184 is a 1:1 combination of two human monoclonal IgG1 antibodies (CR57 and CR4098) against rabies virus, produced in the PER.C6 human cell line. The two antibodies are developed as replacements of human rabies immune globulin (HRIG) and equine rabies immune globulin (ERIG) in postexposure prophylaxis (PEP). The rapid fluorescent focus inhibition test (RFFIT) is a cell-based virus neutralization assay which is usually performed to determine the biological potency of a vaccine and to measure the levels of protection against rabies in humans and animals. In order to confirm the suitability of this assay as a pharmacodynamic assay, we conducted a validation using both HRIG- and CL184-spiked serum samples and sera from vaccinated donors. The validation results met all analytical acceptance criteria and showed that HRIG and CL184 serum concentrations can be compared. Stability experiments showed that serum samples were stable in various suboptimal conditions but that rabies virus should be handled swiftly once thawed. We concluded that the assay is suitable for the measurement of polyclonal and monoclonal rabies neutralizing antibodies in clinical serum samples.

Qualitative and semiquantitative analysis of composite mixtures of antibodies by native mass spectrometry.

Native mass spectrometry was evaluated for the qualitative and semiquantitative analysis of composite mixtures of antibodies representing biopharmaceutical products coexpressed from single cells. We show that by using automated peak fitting of the ion signals in the native mass spectra, we can quantify the relative abundance of each of the antibodies present in mixtures, with an average accuracy of 3%, comparable to a cation exchange chromatography based approach performed in parallel. Moreover, using native mass spectrometry we were able to identify, separate, and quantify 9 antibodies present in a complex mixture of 10 antibodies, whereas this complexity could not be unraveled by cation exchange chromatography. Native mass spectrometry presents a valuable alternative to existing analytical methods for qualitative and semiquantitative profiling of biopharmaceutical products. It provides both the identity of each species in a mixture by mass determination and the relative abundance through comparison of relative ion signal intensities. Native mass spectrometry is a particularly effective tool for characterization of heterogeneous biopharmaceutical products such as bispecific antibodies and antibody mixtures.

A human CD137×PD-L1 bispecific antibody promotes anti-tumor immunity via context-dependent T cell costimulation and checkpoint blockade.

Immune checkpoint inhibitors demonstrate clinical activity in many tumor types, however, only a fraction of patients benefit. Combining CD137 agonists with these inhibitors increases anti-tumor activity preclinically, but attempts to translate these observations to the clinic have been hampered by systemic toxicity. Here we describe a human CD137xPD-L1 bispecific antibody, MCLA-145, identified through functional screening of agonist- and immune checkpoint inhibitor arm combinations. MCLA-145 potently activates T cells at sub-nanomolar concentrations, even under suppressive conditions, and enhances T cell priming, differentiation and memory recall responses. In vivo, MCLA-145 anti-tumor activity is superior to immune checkpoint inhibitor comparators and linked to recruitment and intra-tumor expansion of CD8 + T cells. No graft-versus-host-disease is observed in contrast to other antibodies inhibiting the PD-1 and PD-L1 pathway. Non-human primates treated with 100 mg/kg/week of MCLA-145 show no adverse effects. The conditional activation of CD137 signaling by MCLA-145, triggered by neighboring cells expressing >5000 copies of PD-L1, may provide both safety and potency advantages.

MCLA-117, a CLEC12AxCD3 bispecific antibody targeting a leukaemic stem cell antigen, induces T cell-mediated AML blast lysis.

Objective: We report the characterization of MCLA-117, a novel T cell-redirecting antibody for acute myeloid leukaemia (AML) treatment targeting CD3 on T cells and CLEC12A on leukaemic cells. In AML, CLEC12A is expressed on blasts and leukaemic stem cells. Methods: The functional capacity of MCLA-117 to redirect resting T cells to eradicate CLEC12APOS tumor cells was studied using human samples, including primary AML samples. Results: Within the normal hematopoietic compartment, MCLA-117 binds to cells expressing CD3 and CLEC12A but not to early myeloid progenitors or hematopoietic stem cells. MCLA-117 induces T cell activation (EC50 = 44 ng/mL), T cell proliferation, mild pro-inflammatory cytokine release, and redirects T cells to lyse CLEC12APOS target cells (EC50 = 68 ng/mL). MCLA-117-induced targeting of normal CD34POS cells co-cultured with T cells spares erythrocyte and megakaryocyte differentiation as well as preserves mono-myelocytic lineage development. In primary AML patient samples with autologous T cells, MCLA-117 robustly induced AML blast killing (23-98%) at low effector-to-target ratios (1:3-1:97). Conclusion: These findings demonstrate that MCLA-117 efficiently redirects T cells to kill tumour cells while sparing the potential of the bone marrow to develop the full hematological compartment and support further clinical evaluation as a potentially potent treatment option for AML.

C-type lectin-like molecule-1: a novel myeloid cell surface marker associated with acute myeloid leukemia.

Acute myeloid leukemia (AML) has a poor prognosis due to treatment-resistant relapses. A humanized anti-CD33 antibody (Mylotarg) showed a limited response rate in relapsed AML. To discover novel AML antibody targets, we selected a panel of single chain Fv fragments using phage display technology combined with flow cytometry on AML tumor samples. One selected single chain Fv fragment broadly reacted with AML samples and with myeloid cell lineages within peripheral blood. Expression cloning identified the antigen recognized as C-type lectin-like molecule-1 (CLL-1), a previously undescribed transmembrane glycoprotein. CLL-1 expression was analyzed with a human anti-CLL-1 antibody that was generated from the single chain Fv fragment. CLL-1 is restricted to the hematopoietic lineage, in particular to myeloid cells present in peripheral blood and bone marrow. CLL-1 is absent on uncommitted CD34(+)/CD38(-) or CD34(+)/CD33(-) stem cells and present on subsets of CD34(+)/CD38(+) or CD34(+)/CD33(+) progenitor cells. CLL-1 is not expressed in any other tissue. In contrast, analysis of primary AMLs demonstrated CLL-1 expression in 92% (68 of 74) of the samples. As an AML marker, CLL-1 was able to complement CD33, because 67% (8 of 12) of the CD33(-) AMLs expressed CLL-1. CLL-1 showed variable expression (10-60%) in CD34(+) cells in chronic myelogenous leukemia and myelodysplastic syndrome but was absent in 12 of 13 cases of acute lymphoblastic leukemia. The AML reactivity combined with the restricted expression on normal cells identifies CLL-1 as a novel potential target for AML treatment.

Human monoclonal antibody as prophylaxis for SARS coronavirus infection in ferrets.

SARS coronavirus continues to cause sporadic cases of severe acute respiratory syndrome (SARS) in China. No active or passive immunoprophylaxis for disease induced by SARS coronavirus is available. We investigated prophylaxis of SARS coronavirus infection with a neutralising human monoclonal antibody in ferrets, which can be readily infected with the virus. Prophylactic administration of the monoclonal antibody at 10 mg/kg reduced replication of SARS coronavirus in the lungs of infected ferrets by 3.3 logs (95% CI 2.6-4.0 logs; p<0.001), completely prevented the development of SARS coronavirus-induced macroscopic lung pathology (p=0.013), and abolished shedding of virus in pharyngeal secretions. The data generated in this animal model show that administration of a human monoclonal antibody might offer a feasible and effective prophylaxis for the control of human SARS coronavirus infection.

A proteomic approach to tumour target identification using phage display, affinity purification and mass spectrometry.

Tumour-associated cell surface markers are potential targets for antibody-based therapies. We have obtained a panel of myeloid cell binding single chain variable fragments (scFv) by applying phage display selection on myeloid cell lines followed by a selection round on freshly isolated acute myeloid leukaemia (AML) blasts using flow cytometry. To identify the target antigens, the scFv were recloned and expressed in an IgG(1) format and tested for their ability to immunoprecipitate cell surface proteins. The IgGs that reacted with distinct cell membrane extractable proteins were used in large-scale affinity purification of the target antigen followed by mass-spectrometry-based identification. Well-characterised cell surface antigens, such as leukocyte antigen-related receptor protein tyrosine phosphatase (LAR PTP) and activated leukocyte adhesion molecule (ALCAM) in addition to several unknown proteins, like ATAD3A, were identified. These experiments demonstrate that phage antibody selection in combination with affinity chromatography and mass spectrometry can be exploited successfully to identify novel antibody target molecules on malignant cells.

A human monoclonal antibody cocktail as a novel component of rabies postexposure prophylaxis.

The currently recommended treatment for individuals exposed to rabies virus is the combined administration of rabies vaccine and rabies immune globulin (RIG). This review sets out the criteria used to guide development of a cocktail of human monoclonal antibodies as a replacement for RIG. Using this process as a model, the general requirements for development of safe and efficacious monoclonal antibody alternatives to currently used polyclonal serum products are discussed.