The measurement and control of high-risk host cell proteins for polysorbate degradation in biologics formulation

Non-ionic surfactant polysorbates (PS), including PS-80 and PS-20, are commonly used in the formulation of biotherapeutic products for both preventing surface adsorption and acting as stabilizer against protein aggregation. Trace levels of residual host cell proteins (HCPs) with lipase or esterase enzymatic activity have been shown to degrade polysorbates in biologics formulation. The measurement and control of these low-abundance, high-risk HCPs for polysorbate degradation is an industry-wide challenge to achieve desired shelf-life of biopharmaceuticals in liquid formulation, especially for high-concentration formulation product development. Here, we reviewed the challenges, recent advances and future opportunities of analytical method development, risk assessment and control strategies for polysorbate degradation during formulation development with a focus on enzymatic degradation. Continued efforts to advance our understanding of polysorbate degradation in biologics formulation will help develop high-quality medicines for patients.

Longitudinal waning of mRNA vaccine-induced neutralizing antibodies against SARS-CoV-2 detected by an LFIA rapid test

While mRNA vaccines against SARS-CoV-2 were highly efficacious against severe illness and hospitalization, they seem to be less effective in preventing infection months after vaccination, especially with the Delta variant. Breakthrough infections might be due to higher infectivity of the variants, relaxed protective measures by the general public in “COVID-19 fatigue”, and/or waning immunity post-vaccination. Determining the neutralizing antibody levels in a longitudinal manner may address this issue, but technical complexity of classic assays precludes easy detection and quick answers. We developed a lateral flow immunoassay NeutraXpress™ (commercial name of the test kit by Antagen Diagnostics, Inc.), and tested fingertip blood samples of subjects receiving either Moderna or Pfizer vaccines at various time points. With this device, we confirmed the reported clinical findings that mRNA vaccine-induced neutralizing antibodies quickly wane after 3–6 months. Thus, using rapid tests to monitor neutralizing antibody status could help identify individuals at risk, prevent breakthrough infections and guide social behavior to curtail the spread of COVID-19. Statement of Significance. Mounting evidence suggests that mRNA vaccine-induced neutralizing antibody titres against SARS-CoV-2 wane in 3–6 months. Quick identification of fully vaccinated persons with high risk of breakthrough infections is key to control the COVID-19 pandemic. The described LFIA device having a control/sample dual-lane design serves this purpose with successful field-test data.

Augmenting recombinant antibody production in HEK293E cells: Optimising transfection and culture parameters

Background Optimising recombinant antibody production is important for cost-effective therapeutics and diagnostics. With impact on commercialisation, higher productivity beyond laboratory scales is highly sought, where efficient production can also accelerate antibody characterisations and investigations. Methods Investigating HEK293E cells for mammalian antibody production, various transfection and culture parameters were systematically analysed for antibody light chain production before evaluating them for whole antibody production. Transfection parameters investigated include seeding cell density, the concentration of the transfection reagent and DNA, complexation time, temperature, and volume, as well as culture parameters such as medium replacement, serum deprivation, use of cell maintenance antibiotic, incubation temperature, medium volume, post-transfection harvest day and common nutrient supplements. Results Using 2 mL adherent HEK293E cell culture transfections with 25 kDa linear Polyethylenimine in the most optimised parameters, we demonstrated a ~ 2-fold production increase for light chain alone and for whole antibody production reaching 536 and 49 μg respectively in a cost-effective manner. With the addition of peptone, κ light chain increased by ~ 4-fold to 1032 μg while whole antibody increased to a lesser extent by ~ 2.5-fold to 51 μg, with benefits potentially for antibodies limited by their light chains in production. Conclusions Our optimised findings show promise for a more efficient and convenient antibody production method through transfection and culture optimisations that can be incorporated to scale up processes and with potential transferability to other mammalian-based recombinant protein production using HEK293E cells. Statement of Significance Recombinant antibody production is crucial for antibody research and development. Systematically investigating transfection and culture parameters such as PEI/DNA concentrations, complexation time, volume, and temperature, supplements, etc., we demonstrated a ~ 4-fold light chain alone production increase to 1032 μg and a 2.5-fold whole antibody production increase to 51 μg from 2 mL transfections.

CellCelector™ as a platform in isolating primary B cells for antibody discovery

Background The most robust strategy in antibody discovery is the use of immunized animals and the ability to isolate and immortalize immune B-cells to hybridoma for further interrogation. However, capturing the full repertoire of an immunized animal is labor intensive, time consuming, and limited in throughput. Therefore, techniques to directly mine the antibody repertoire of primary B-cells are of great importance in antibody discovery. Methods In the current study, we present a method to isolate individual antigen specific primary B-cells using the CellCellector™ single-cell isolation platform from XenoMouse® (XM) immunized with a recombinant therapeutic protein, EGFR. We screened a subset of CD138+ B-cells and identified 238 potential EGFR specific B-cells from 1,189 antibody secreting cells (ASCs) and isolated 94 by CellCellector. Results We identified a diverse set of heavy chain CDR sequences and cloned and expressed 20 into a standard human IgG1 antibody format. We further characterized and identified 13 recombinant antibodies that engage soluble and native forms of EGFR. By extrapolating the method to all 400,000 CD138+ B-cells extracted from one EGFR immunized XM, a potential 1,196 unique EGFR-specific antibodies could be discovered. Conclusions CellCelector allows for interrogating the B-cell pool directly and isolating B-cells specific to the therapeutic target of interest. Furthermore, antibody sequences recovered from isolated B-cells engage the native and recombinant target, demonstrating the CellCellector can serve as a platform in antibody discovery.

Structural arrangement of the VH and VL domains in the COBRA™ T-cell engaging single chain diabody

Background COBRA™ (COnditional Bispecific Redirected Activation) T-cell engagers are designed to target solid tumors as a single polypeptide chain prodrug that becomes activated by proteolysis in the tumor microenvironment. One COBRA molecule comprises seven Ig domains: three single-domain antibodies (sdAbs) recognizing a tumor target or human serum albumin (HSA), and CD3ε-binding VH and VL and their inactivated counterparts, VHi and VLi. Pairing of VH and VL, and VLi and VHi, into scFvs is prevented by shortened inter-domain linkers. Instead, VH and VL are expected to interact with VLi and VHi, respectively, thus making a diabody whose binding to CD3ε on the T-cells is impaired. Methods We analyzed the structure of an EGFR COBRA in solution using negative stain electron microscopy (EM) and small-angle X-ray scattering (SAXS). Results We found that this EGFR COBRA forms stable monomers with a very dynamic interdomain arrangement. At most, only five domains at a time appeared ordered, and only one VH-VL pair was found in the Fv orientation. Non-enzymatic post-translational modifications suggest that the CDR3 loops in the VL-VHi pair are exposed but are buried in the VH-VLi pair. The MMP9 cleavage rate of the prodrug when bound to recombinant EGFR or HSA is not affected, indicating positioning of the MMP9-cleavable linker away from the EGFR and HSA binding sites. Conclusion Here we propose a model for EGFR COBRA where VH and VLi form an Fv, and VL and VHi do not, possibly interacting with other Ig domains. SAXS and MMP9 cleavage analyses suggest that all COBRA molecules tested have a similar structural architecture.

US FDA approved therapeutic antibodies with high concentration formulation: Summaries and perspectives

Since 1986 when we first witnessed the approval of monoclonal antibody (mAb) Orthoclone OKT3 by the US FDA, FDA has approved 103 therapeutic antibody drugs in the past 35 years for marketing. Thirty four (34) of these 103 therapeutic antibody drugs (accounting for one third of the total FDA approved antibody therapeutics) are formulated with high protein concentration (100 mg/mL or above). These 34 high concentration antibodies are the focus of this article. The dosage forms of these 34 antibodies are analyzed and discussed in this article. The highest protein concentration of these approved mAbs is 200 mg/mL. The dominant administration route is subcutaneous (76%). Our analysis indicates that it may be rational to implement a platform formulation containing polysorbate, histidine and sucrose to accelerate high concentration formulation development for antibody drugs. The top players/sponsors of high concentration formulation are identified as Roche including its subsidiaries Genentech and Chugai, Novartis, Sanofi, Amgen, GSK, Johnson & Johnson including its subsidiary Janssen, and Regeneron. The FDA approval numbers are significantly increased since 2015 which account for 76% of the total approval number, i.e., 26 out of 34 highly concentrated antibodies. Thus, we believe that the high concentration formulations of antibody drugs will be the future trend of therapeutic antibody formulation development, regardless of the challenges of highly concentrated protein formulations.

Anti-tumor efficacy of an MMAE conjugated antibody targeting cell surface TACE/ADAM17-cleaved Amphiregulin in breast cancer

Background The Epidermal Growth Factor Receptor ligand, Amphiregulin, is a key proliferative effector of estrogen receptor signaling in breast cancer and also plays a role in other malignancies. Amphiregulin is a single-pass transmembrane protein proteolytically processed by TACE/ADAM17 to release the soluble EGFR ligand, leaving a residual transmembrane stalk that is subsequently internalized. Methods Using phage display we identified antibodies that selectively recognize the residual transmembrane stalk of cleaved Amphiregulin. Conjugation with fluorescence labels and monomethyl auristatin E (MMAE) was used to study their intracellular trafficking and anti-cancer effects, respectively. Results We report the development of an antibody drug conjugate, GMF-1A3-MMAE, targeting an AREG neo-epitope revealed following ADAM17-mediated cleavage. The antibody does not interact with uncleaved Amphiregulin, providing a novel means of targeting cells with high rates of Amphiregulin shedding. Using fluorescent dye conjugation, we demonstrated that the antibody is internalized by cancer cells in a manner dependent on the presence of cell surface cleaved Amphiregulin. Antibodies conjugated with MMAE were cytotoxic in vitro and induced rapid regression of established breast tumor xenografts in immunocompromised mice. We further demonstrate that these antibodies recognize the Amphiregulin neo-epitope in formalin fixed paraffin embedded tumor tissue, suggesting their utility as a companion diagnostic for patient selection. Conclusions This ADC targeting Amphiregulin has potential utility in the treatment of breast and other tumors in which proteolytic Amphiregulin shedding is a frequent event.

Development of an integrated structural biology platform specialized for sub-100 kDa protein complexes to support biologics discovery and rational engineering

Background Developing a biologic medicine requires successful decision making during selection and optimization in addition to the pool of candidates at early research stages. Knowing structural information and binding patterns between drug target and discovery candidates greatly increases the probability of success. Methods With the cryo-EM resolution revolution and rapid development of computational software, we have evaluated and integrated various tools in structural biology and the computation field and established a highly cost-effective platform which allows us to obtain fast and accurate structural information for nearly all our biologics projects with a close to 100% success rate and as fast as weeks turn-around time. Results Here we report four case studies selected from 38 different protein structures and share how we integrate cryo-EM structure determination, computational structure modeling, and molecular dynamics simulation. With proper decision making and strategic planning, the platform allows us to obtain quality results within days to weeks, including sub-100 kDa complexes which are usually considered a challenge due to their small size. Conclusions Our utilization of this differential approach and multiple software packages allows us to manage priorities and resources to achieve goals quickly and efficiently. We demonstrate how to effectively overcome particle orientation bias by altering complex composition. In several of our examples, we use glycan density to facilitate interpretation of low-resolution 3D reconstruction and epitope mapping. Protein information plays an important role in our cryo-EM projects, especially in cases where we see significant challenges in obtaining high-resolution 3D maps.

Biological activity validation of a computationally designed rituximab/CD3 T cell engager targeting CD20+ cancers with multiple mechanisms of action

ABSTRACT Background Bispecific T cell engaging antibodies (TEAs) with one arm targeting a cancer antigen and another arm binding to CD3 have demonstrated impressive efficacy in multiple clinical studies. However, establishing a safety/efficacy balance remains challenging. For instance, some TEAs have severe safety issues. Additionally, not all patients or all cancer cells of one patient respond equally to TEAs. Methods Here, we developed a next-generation bispecific TEA with better safety/efficacy balance and expanded mechanisms of action. Using the computer aided antibody design strategy, we replaced heavy chain complementarity-determining regions (HCDRs) in one Rituximab arm with HCDRs from a CD3 antibody and generated a novel CD20/CD3 bispecific antibody. Results After series of computer aided sequence optimization, the lead molecule, GB261, showed great safety/efficacy balance both in vitro and in animal studies. GB261 exhibited high affinity to CD20 and ultra-low affinity to CD3. It showed comparable T cell activation and reduced cytokine secretion compared to a benchmark antibody (BM). ADCC and CDC caused by GB261 only killed CD20+ cells but not CD3+ cells. It exhibited better RRCL cell killing than the BM in a PBMC engrafted, therapeutic treatment mouse model and good safety in cynomolgus monkeys. Conclusions Thus, GB261 is a promising novel TEA against CD20+ cancers.

Perspectives on the development of antibody-drug conjugates targeting ROR1 for hematological and solid cancers

Antibody–drug conjugates (ADCs) are targeted therapeutics generated by conjugation of cytotoxic small molecules to monoclonal antibodies (mAbs) via chemical linkers. Due to their selective delivery of toxic payloads to antigen-positive cancer cells, ADCs demonstrate wider therapeutic indexes compared to conventional chemotherapy. After decades of intensive research and development, significant advances have been made in the field, leading to a total of ten FDA-approved ADCs to treat cancer patients. Currently, ~ 80 ADCs targeting different antigens are under clinical evaluation for treatment of either hematological or solid malignancies. Notably, 3 ADCs targeting the same oncofetal protein, ROR1, have attracted considerable attention when they were acquired or licensed successively in the fourth quarter of 2020 by 3 major pharmaceutical companies. Apparently, ROR1 has emerged as an attractive target for cancer therapy. Since all the components of ADCs, including the antibody, linker, and payload, as well as the conjugation method, play critical roles in ADC’s efficacy and performance, their choice and combination will determine how far they can be advanced. This review summarizes the design and development of current anti-ROR1 ADCs and highlights an emerging trend to target ROR1 for cancer therapy.