The Antibody Society

the official website of the antibody society

An international non-profit supporting antibody-related research and development.

Crizanlizumab-tmca (Adakveo) approved by FDA

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On November 15, 2019, the U.S. Food and Drug Administration approved crizanlizumab-tmca (Adakveo) as a treatment to reduce the frequency of vaso-occlusive crisis (VOC), which occurs when blood circulation is obstructed by sickled red blood cells, for patients age 16 years and older. Crizanlizumab is a humanized antibody directed against P-selectin, which contributes to the pathogenesis of sickle cell disease, including vaso-occlusive events and hemolytic anemia. Crizanlizumab was granted Orphan Drug designation in the US and European Union for the treatment of VOC in patients with sickle cell disease, as well as FDA’s Breakthrough Therapy designation for prevention of VOCs in patients of all genotypes with sickle cell disease. A marketing application for crizanlizumab is undergoing review by the European Medicines Agency.

FDA’s approval was based on Phase 2 results from the SUSTAIN study (NCT01895361), which demonstrated that crizanlizumab provided significant benefit over placebo, such as:  1) the percentage of crizanlizumab-treated patients (5 mg/kg) who did not experience any vaso-occlusive crisis (VOC) was higher compared to those treated with placebo (36% vs 17%, P=0.010); 2) 45% reduction in the median annual rate of VOCs leading to health care visits in patients with or without hydroxyurea therapy compared to placebo (1.63 vs 2.98, P=0.010); 3) 42% reduction in median annual rate of days hospitalized versus placebo (4.00 vs 6.87 P=0.45), and 4) A three-fold longer median time to first VOC vs placebo (4.07 vs 1.38 months, P< 0.001). [1, 2]

1. Novartis. FDA accepts file and accelerates review of Novartis sickle cell disease medicine crizanlizumab (SEG101). July 16, 2019 press release.

2. Kutlar A, Kanter J, Liles DK, Alvarez OA, Cançado RD, Friedrisch JR, Knight-Madden JM, Bruederle A, Shi M, Zhu Z, et al. Effect of crizanlizumab on pain crises in subgroups of patients with sickle cell disease: A SUSTAIN study analysis. Am J Hematol. Am J Hematol. 2019 Jan;94(1):55-61. doi: 10.1002/ajh.25308.

Interested in more information about US- or EU- approved antibody therapeutics? The Antibody Society maintains a comprehensive table of approved mAb therapeutics and those in regulatory review in the EU or US in the Web Resources section of our website. 

“Building Scientific Communities – Lessons Learned with AIRR”

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AIRR-C Executive Sub-committee member Christian Busse gave a talk at the deRSE2019 (Konferenz für ForschungssoftwareentwicklerInnen in Deutschland – Conference for software engineers in Germany) on building scientific communities using the AIRR-C as an example.

Check out his talk here: https://doi.org/10.5446/42486.

Feeding drug development programs with sufficient antibody

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Author: Nick Hutchinson, Fujifilm Diosynth Biotechnologies

The demand for antibody and antibody-related therapeutics continues to increase. [1] The United States Food and Drug Administration has approved ~ 100 antibody therapeutics for a wide range of treatments. Nearly 600 antibody drugs are in clinical trials, [1] with ~75 of these in pivotal Phase 2 or Phase 3 studies.

Small or even virtual companies are developing many of these molecules. Technical teams working within these organizations must understand the activities needed to successfully commercialize the drugs. One critical activity is establishment of production strategies capable of supplying the material requirements of pre-clinical development, toxicology studies, clinical trials and then, if successful, market demand.

Patients cannot benefit from life-saving medicines if the drug’s launch is delayed due to lack of the material required for each phase of development. Furthermore, companies that miss clinical milestones suffer from delayed investments, thus reducing the opportunity to reach the clinic in a timely manner and capture market share, which lowers future revenues.

Many start-up biotech firms have a laser-like focus on the pre-clinical development of their antibody candidates, but sooner or later they must consider a manufacturing strategy that enables pre-clinical or clinical programs to stay on track.

Is manufacturability an obstacle to development?

One question drug developers should consider is the extent to which the manufacturability of the candidate is likely to be problematic and jeopardise material supply. Many of the standard, full-length antibodies have well-understood properties and are relatively easy to manufacture, allowing timely delivery to the clinic. However, there is an increasing number of modalities within this product class, [2] e.g., bispecifics, Fc-fusions and antigen-binding fragments, which may present additional production challenges. These can include challenges such as low expression from cell lines suitable for use in manufacturing, poor stability during purification processes or the need for non-standard analytical methods.

One company I spoke to, for example, knew that they needed to increase the productivity of their cell cultures from below 0.5 g/L to greater than 3 g/L in order for the product to be commercially viable. Another company developing a monoclonal antibody explained that they needed a titer of ~ 10 g/L to ensure production efficiency was sufficiently high to allow them to be price competitive. A third company found that the isoelectric point of their Fc-fusion molecule was relatively low and they needed a tailored purification process for their product.

Companies developing standard IgG1, IgG2 or IgG4 products can leverage manufacturing platforms. [3] These allow production of different monoclonal antibodies with the required quality specifications and at high productivity with little process development. They offer a significant time- and cost-saving over the alternative, i.e, developing new processes for each new candidate. Companies with a pipeline of products may choose to invest in their own manufacturing platform, but, for many early-stage biotech companies it makes little sense to spend investors’ cash on production assets when there is considerable uncertainty around the likely success of a program. For this reason, many will outsource process development and manufacturing to a contract development and manufacturing organization (CDMO), many of whom will have their own established platform processes.

Early material supplies of antibody candidates

Cell line development scientists can generate stable, clonal cell banks derived from a production-ready host cell line in as little as 10 weeks following transfection. Cell cultures with transfectant pools can produce tens to hundreds of grams of material in as little as eight weeks following transfection. Scientists developing antibody therapeutics can use this antibody for their pre-clinical activities and initial formulation development experiments. In our experience, even at the pre-clinical stage, the drug development process can consume substantial amounts of material. Accurately determining material requirements at this stage will help ensure sufficient antibody is available.

Preclinical material supply might be met with bench-scale bioreactors, but we have worked on programs where the material requirements were sufficiently large that a 200-L mammalian cell culture run was needed, even though the cell line gave a high titer. This clearly demonstrated the utility of having a platform process because no additional process development on either the bioreactor conditions or the purification steps was needed. Expert developers of cell lines know that their host cell line will grow to high cell density under their platform conditions, and will select clones that combine high productivity with the desired product quality profile using high-throughput screening technologies.

Process development scientists operating platform processes typically allocate time, which would previously have been dedicated to manufacturing development, to the refinement of operating parameters and studies of manufacturing robustness that increase the likelihood of that full-scale production lots will be successfully released.

Supplying Toxicology and Early Clinical Material

Pilot-scale batches allow companies to predict large-scale manufacturing performance and refine scale-dependant process parameters. Companies often use material from the pilot-scale batch for toxicology work, stability studies and for generating reference standard, against which the first batch for clinical use can be released. It generally takes 6 – 8 months to reach this stage from the start of cell line development, yielding hundreds of grams of antibody, if not more.

For many companies, the demand for clinical-grade drug, manufactured to current Good Manufacturing Practices (GMP), can be met using bioreactors no larger in volume than 2000-L. The initial batch can be released within 12-14 months from the start of cell line development. Each batch can supply between 1 to 10 kilograms of antibody.

Modern, high-throughput manufacturing facilities provide enormous amounts of capacity such that with a robust, high-titer cell line no further scale-up may be required and firms can commercialize their product within the same facility they used for clinical lots. Others elect to scale-up still further to large-scale stainless steel manufacturing facilities, especially if the market demand is high and the overall process productivity is modest. More recently, firms are considering going to market with manufacturing processes that utilize smaller bioreactors operated in a continuous, perfusion mode. We believe such processes can yield over 15 kg of antibody from a 500-L bioreactor over a 4-week period. Deciding which approach to adopt is never easy because of uncertainties around factors such as dose requirements, overall market demand and competitive pressures. Experienced CDMOs will support customers through this decision-making process and will be able to provide invaluable advice.

In conclusion, many small biotech companies with new antibody drug assets can mitigate risks to drug development and commercialization timelines by thoroughly understanding the material supply requirements for preclinical, toxicology and clinical studies. Once they know this, they can determine how the need can be met by manufacturing organizations during process development and GMP production operations as part as an over-arching strategy for product commercialization.

[1] Kaplon H, Reichert JM. Antibodies to watch in 2019. MAbs. 2019;11(2):219-238. doi: 10.1080/19420862.2018.1556465.

[2] Scott M, Clark N. Next generation antibody therapeutics: Antibody fragments, dual-targeting strategies, and beyond… . European Pharmaceutical Review. 2009.

[3] Shukla AA, Wolfe LS, Mostafa SS, Norman C. Evolving trends in mAb production processes. Bioengineering & Translational Medicine. 2017;] 2(1): 58–69. doi: 10.1002/btm2.10061

 

Most read from mAbs, Nov/Dec 2019

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The Antibody Society is pleased to be affiliated with mAbs, a multi-disciplinary journal dedicated to advancing the art and science of antibody research and development. We hope you enjoy these summaries based on the abstracts of the most read papers published in a recent issue.

All the articles are open access; PDFs can be freely downloaded by following the links below.

Issue 11.8 (Nov/Dec 2019)

Insights into the IgG heavy chain engineering patent landscape as applied to IgG4 antibody development

In this new Perspective, Dumet et al., present the results from their study of the patent landscape of IgG4 Fc engineering, i.e., patents claiming modifications in the heavy chain. Thirty-seven relevant patent families were identified, comprising hundreds of IgG4 Fc variants focusing on removal of residual effector functions (since IgG4s bind to FcγRI and weakly to other FcγRs), half-life enhancement and IgG4 stability. Given the number of expired or soon to expire major patents in those 3 areas, companies developing blocking antibodies now have, or will in the near future, access to free tools to design silenced, half-life extended and stable IgG4 antibodies.

Antibody discovery and engineering by enhanced CRISPR-Cas9 integration of variable gene cassette libraries in mammalian cells

Parola et al. describe an antibody engineering and screening approach where complete variable light (VL) and heavy (VH) chain cassette libraries are stably integrated into the genome of hybridoma cells by enhanced Cas9-driven homology-directed repair (HDR), resulting in their surface display and secretion. By developing an improved HDR donor format that utilizes in situ linearization, they were able to achieve >15-fold improvement of genomic integration, resulting in a screening workflow that only requires a simple plasmid electroporation. This proved suitable for different applications in antibody discovery and engineering. By integrating and screening an immune library obtained from the variable gene repertoire of an immunized mouse, they isolated a diverse panel of >40 unique antigen-binding variants. They also successfully performed affinity maturation by directed evolution screening of an antibody library based on random mutagenesis, leading to the isolation of several clones with affinities in the picomolar range.

DuoMab: a novel CrossMab-based IgG-derived antibody format for enhanced antibody-dependent cell-mediated cytotoxicity

In this new Report,  Sustmann et al. present a generic approach to generate two novel IgG-derived antibody formats that are based on a modification of the CrossMab technology. MoAbs harbor two heavy chains (HCs) resulting in one binding entity and one Fc, whereas DuoMabs are composed of four HCs harboring two binding entities and two Fc regions linked at a disulfide-bridged hinge. The latter bivalent format is characterized by avidity-enhanced target cell binding while simultaneously increasing the ‘Fc-load’ on the surface. DuoMabs were shown to be producible in high yield and purity and bind to surface cells with affinities comparable to IgGs. The increased Fc load directed at the surface of target cells by DuoMabs modulates their ADCC competency toward target cells, making them attractive for applications that require or are modulated by FcR interactions.

Single-step Protein A and Protein G avidity purification methods to support bispecific antibody discovery and development

Heavy chain (Hc) heterodimers represent a majority of bispecific antibodies (bsAbs) under clinical development. Although recent technologies achieve high levels of Hc heterodimerization (HD), traces of homodimer contaminants are often present, and as a consequence robust purification techniques for generating highly pure heterodimers in a single step are needed. Ollier et al. describe two different purification methods that exploit differences in Protein A (PA) or Protein G (PG) avidity between homo- and heterodimers. Differential elution between species was enabled by removing PA or PG binding in one of the Hcs of the bsAb. The PA method allowed the avidity purification of heterodimers based on the VH3 subclass, which naturally binds PA and interferes with separation, by using a combination of IgG3 Fc and a single amino acid change in VH3, N82aS. The PG method relied on a combination of three mutations that completely disrupts PG binding, M428G/N434A in IgG1 Fc and K213V in IgG1 CH1. Both methods achieved a high level of heterodimer purity as single-step techniques without Hc HD (93–98%). Since PA and PG have overlapping binding sites with the neonatal Fc receptor (FcRn), they investigated the effects of the engineering both in vitro and in vivo. Mild to moderate differences in FcRn binding and Fc thermal stability were observed, but these did not significantly change the serum half-lives of engineered control antibodies and heterodimers. The methods are conceptually compatible with various Hc HD platforms such as BEAT® (Bispecific Engagement by Antibodies based on the T cell receptor), in which the PA method has already been successfully implemented.