Novel Antibody Therapeutic Modalities eBook - Oct 2020

We take an in-depth look at the novel antibody therapeutic modalities being developed and how they are tackling new non-cancer targets.

Novel Antibody Therapeutic Modalities

Introduction & Contents

Novel Antibody Therapeutics

Antibody Therapeutics eBook Series

Within antibody engineering and therapeutics, the industry is working on a number of different modalities, including monoclonal antibodies (mAbs), protein therapeutics, bispecifics, immuno-oncology checkpoints, antibody-drug conjugates (ADCs) and CAR-T/cellular therapies. At the same time, work on these modalities is increasingly exploring new non-cancer targets.

Over the following pages, we explore these novel antibody modalities, including lessons and strategies within the world of conjugation, the search for perfection in bispecifics, a snapshot of the current scope of the industry, and how therapeutic development is being affected by the rapid spread of COVID-19.

Jump to any article using the contents on the following page, or at any time using the Contents menu in the top left. There you can also download this eBook as a PDF.

1. Strategies to Innovate the Production of Next Generation Bioconjugates

From ADC development to tumor-targeting, there are still many lessons to be learned in the world of conjugation. This whitepaper reviews the latest common challenges and novel strategies for accelerating bioconjugates, across preclinical, research & development and CMC considerations.

2. Podcast: How to Create the Ultimate Bispecific

What does it take to create the perfect bispecific? That's what we asked Volker Schellenberger, President and Chief Technology Officer at Amunix Pharmaceuticals in a very first episode of the Life Sciences podcast.

3. Antibody Therapeutic Modalities Report 2020

In May 2020, the Antibody Engineering & Therapeutics conference series conducted a study of industry professionals from around the world. This final report based on 166 survey responses reveals unique insights into the development of and approach to each of these novel modalities.

4. Antibody Therapeutic Development in Response to COVID-19

With the rapid spread of COVID-19 in 2020, there is a race to develop both a vaccine and therapies to treat the virus. Multiple traditional and off-the-shelf approaches are being clinically tested and validated for COVID-19 treatment. In this paper, we will evaluate the antibody therapies for COVID-19 currently in clinical trials and preclinical development.

Strategies to Innovate the Production of Next Generation Bioconjugates

By Rashad Mammadov

Pre-clinical Challenges

Antibody, Site, Linker & Payload combinations to optimize ADCs

Antibody

The therapeutic window of an ADC highly depends on its (monoclonal) antibody component that determines the destiny of cytotoxic payload conjugated to it. Therefore, the antibody should have high target specificity and low cross-reactivity in order to limit undesired toxicity. As such clones are determined, it is critical to establish their homogeneity. This requires robust analytical methods (liquid chromatography/mass spectrometry) for characterization of the antibody. Otherwise, the heterogenous antibody profile can render the ADC prone to failure.

Another challenge in ADC development is the immunogenicity against the antibody. First-generation ADCs utilized antibodies of mouse origin which caused their failure because they were rejected by the human immune system. Later generations have chimeric, humanized, or even human antibodies instead to solve this problem. Apart from avoiding immune cells, the antibody should have enough stability to have long circulating life.

Chemical properties of the antibody is also a barrier in ADC development as they decide how many and which type of linkers can be conjugated to the antibody and to which part. Combinations of these parameters also significantly influence the stability of ADC.

In order to deliver cytotoxic payloads into tumor cells, antibody-antigen interaction should lead to efficient internalization of ADC. In the same context, having good retention after binding to target cells would serve to that end.

In some cases, the antibody binding to its target receptor may induce signaling and produce cytotoxic effect itself through mechanisms such as antibody-dependent cell-mediated cytotoxicity (ADCC), as it happens in the case of trastuzumab emtansine (Kadcyla). Such independent functioning of an antibody is not always considered beneficial for ADC efficacy, since it may reduce internalization of ADC into tumor cells. Such two-pronged killing (antibody and payload) is also a cause for concern since it may be too toxic for healthy cells.

Lastly, antibody isotype selection needs careful planning. Subclasses of IgG - IgG1, IgG2, or IgG4 (mostly IgG1) - is used in current ADC development. IgG3 is not employed as it has fast plasma clearance. The isotype selection defines the difficulty of conjugating linkers to antibody backbone. Antibody isotype also determines the potential immune effector functions such as ADCC.

Site

In an ideal case, a target antigen should be highly expressed in the tumor (with homogeneous expression across the tumor cells), while having minimal or no expression in normal tissues. As an example, HER2 receptor, which is targeted by trastuzumab emtansine (T-DM1), have 100-fold higher expression in the tumor cells than the healthy cells. Accordingly, T-DM1 provides the greatest benefit to the patients with the highest expression of HER2.

Then, it should be internalized efficiently by receptor-mediated endocytosis and not recycled back to the surface. Non-internalized ADCs exert toxicity on neighboring cells. Also, its expression should not get downregulated. In this context, epitope on the target protein also can be important. For example, it was reported that different epitopes of the HER-2 receptor show differences in terms of internalization and degradation of antibody-antigen complex.

Antigens expressed on the surface of the tumor cells are preferred, as they are easier targets for circulating ADC. However, this may not be always possible or in some cases internalization may be hindered due to different reasons such as the high interstitial tumor pressure. For those cases, ADC could be targeted against antigens in the tumor microenvironment.

Shedding of the antigen could be a significant problem as free antigens wandering within the circulation will bind the ADC antibody and compromise its efficacy. Therefore, the target antigen should have minimal shedding.

Payload

In ADC design, one of the questions is that how many cytotoxic drugs will be conjugated to each antibody. In other words, what will be the drug‐to‐antibody ratio (DAR)? If DAR is lower than optimal, that will limit the efficacy of the ADC. However, when DAR is too high, ADC becomes unstable. Altered pharmacokinetic properties of ADC in that case reduces half-life and increases systemic toxicity. The optimal DAR depends on other ADC components as it ranges between 2 and 4 in clinically approved ADC systems.

Payload should be highly potent in terms of cytotoxicity because research shows that at best 0.01% of injected monoclonal antibody (or ADC) binds to target tumor cells. Moreover, optimal DAR also limits the amount of payload that is delivered into tumor cells. As a result, ADC payloads should have IC50 in nanomolar and even picomolar range. Consistently, classical chemotherapy drugs failed to show clinical benefit under ADC framework.

Resistance mechanisms that cancer cells develop against cytotoxic drugs should also be considered as a preclinical challenge. These mechanisms may include increased expression of efflux pumps that remove drugs from cells and altered microtubule composition among others. Exploration of the susceptibility of the payload to drug resistance is crucial. Appropriate drugs with minimal susceptibility should be chosen.

Hydrophobicity/Hydrophilicity of drugs is another issue in ADC design. More lipophilic drugs tend to pass through the cell membrane and kill neighboring cells. This phenomenon is named as bystander effect. It may confer advantage for the treatment of certain solid tumors as their cells have heterogeneous expression of ADC target protein. However, in other cases bystander effect may cause off-target toxicity.

The other challenges in finding an appropriate drug are its stability in blood, solubility in water, and possession of chemical functionalities to conjugate it to a linker molecule.

It is obvious that finding appropriate drugs that meet these criteria is challenging. Currently, two main classes of cytotoxic drugs are used in ADC development. Microtubule inhibitors block assembly of tubulins and cause cell cycle arrest at mitotic phase. Auristatins and Maytansinoids are microtubule inhibitors that are used in three FDA approved ADCs (Adcetris, Kadcyla, and Polivy). The other class includes DNA damaging drugs. Calicheamicin, a drug used in two FDA approved ADCs (Besponsa and Mylotarg), binds DNA’s minor groove and induces double-strand DNA breaks and rapid cell death. Since they could function independently from cell cycle progression, DNA damaging drugs could be used against cancer stem cells which have lower proliferation rate.

Linker

Linker connects cytotoxic payload to monoclonal antibody, hence its properties play a significant role in pharmacokinetics and therapeutic window of ADC. The major preclinical challenge to the ADC efficacy and therapeutic index is high deconjugation rate. Ideally, it should have enough stability that allows ADC to circulate in blood without releasing the payload and causing systemic toxicity. On the other hand, they should release payload once they are inside the target cell. The two types of linkers used in ADC structure are non-cleavable and cleavable.

Non-cleavable linkers provide more stability to ADC and the release of payload happens by lysosomal degradation of the antibody. Even after degradation, payload is released as attached to the linker and the terminal amino acid residue of the antibody. Thus non-cleavable linkers are optimal for drugs that preserve their potency when bearing the moieties left from the degradation. For example, Trastuzumab emtansine (Kadcyla) has non-cleavable thioether linker its maytansinoid-based DM1 drug and antibody. Conversely, MMAE used in the Brentuximab vedotin (Adcetris) is a protein-based cytotoxic drug and has optimal potency in its unconjugated form hence the linker used with this drug is cleavable.

Cleavable linkers release their payload when they meet certain physiological stimulus present in their target site. For example, acid-sensitive linkers (e.g. hydrazone-based linker of Gemtuzumab ozogamicin/Mylotarg) are cleaved in the low pH environment of lysosomes/endosomes. However, these linkers have shown a certain level of plasma instability. As another example, valine-citrulline linker in Brentuximab vedotin is a protease-sensitive linker that is cleaved by cathepsins in lysosomes. Lastly, disulfide linkers used in the design of some ADCs are cleaved by high glutathione concentration found in tumor cells.

Linker’s hydrophilicity/hydrophobicity also affects ADC therapeutic index from many aspects. One of them is bystander effect, that, as it is mentioned above, may be beneficial against solid tumors. Depending on whether you want to enhance or reduce this effect, linker design may change. Non-cleavable linkers that stay with the payload together with a charged amino acid may prevent payload’s passage through membranes thus lowering bystander effect. This effect may be enhanced by using non-polar, more hydrophobic linkers. On the other hand, increased hydrophobicity is associated with high plasma clearance. Moreover, as the linker-drug molecule gets more hydrophobic, it becomes more prone to the work of MDR1 efflux pumps. Therefore, the question of which non-cleavable linker to select depends on which type of cancer cell you are targeting (i.e. does it have a drug-resistance mechanism), whether it is a solid tumor, and which drug you are using as a payload. Cleavable linkers allow drugs to leave alone thus here drug’s chemical properties determine its final destiny.

References

Beck, A., Goetsch, L., Dumontet, C. et al. Strategies and challenges for the next generation of antibody–drug conjugates. Nat Rev Drug Discov 16, 315–337 (2017).

Chau, C. H. et al. Antibody–drug conjugates for cancer. The Lancet, 394, 10200, 793 - 804 (2019).

Diamantis, N., Banerji, U. Antibody-drug conjugates—an emerging class of cancer treatment. Br J Cancer 114, 362–367 (2016).

Hughes, B. Antibody–drug conjugates for cancer: poised to deliver?. Nat Rev Drug Discov 9, 665–667 (2010).

Improved Targeting for Solid Tumors

Bispecific ADCs

As we mentioned among preclinical challenges, it is mostly essential for an ADC to be delivered into lysosomes to release the cytotoxic payload. This is vital for the only approved ADC to treat a solid tumor, T-DM1 (Kadcyla), since it carries a non-cleavable linker. However, HER2 receptor, the target antigen for T-DM1, despite its selective expression on breast tumor cells is found to recycle back with high rate after internalization. Overall, this leads to only a small proportion of T-DM1 to reach lysosomes. This phenomenon may stand behind the limited efficacy of T-DM1 that involve its failure to show a benefit in a gastric cancer trial, whereas trastuzumab (unarmed antibody) passed approval. Also, in Phase II and III clinical trials for breast cancer it failed to show higher clinical benefit than standard therapies.

Its benefit seems to be restricted to breast cancer patients with high HER2 expression. Bispecific antibody technology delivers promising results in the way to solve this problem. These antibodies have two paratopes or they can recognize two different antigens, while conventional antibodies are “monospecific” (although bivalent). In the context of enhancing lysosomal trafficking, two particular bispecific ADC - based strategies stands out.

The first bispecific antibody has each paratope recognizing different epitopes on the same antigen. Such bispecific ADCs have been shown to induce receptor clustering on the target cells and increased internalization, lysosomal delivery and on-target cytotoxicity. ZW49 developed by Zymeworks is a bispecific ADC that recognizes two different epitopes on HER2. It is currently in Phase I for the treatment of biliary tract cancer.

Another approach, named as “drag and degrade”, is based on getting help from a receptor that regularly and efficiently traffics to lysosomes. Here, one of the paratopes of the antibody recognizes a “fast-internalizing” receptor, the other recognizes a tumor-specific antigen. HER2/CD63 and HER2/Prolactin receptor bispecific ADCs have been shown to enhance lysosomal delivery and on-target cytotoxicity when compared to HER2 monospecific ADC.

Both targets of bispecific ADC may not have similar contributions. Various parameters such as the affinity of the individual arms, the density of the target, the overall avidity and the valency of the bispecific format determines tumor selectivity. A study testing EGFR/cMet ADC showed that having low affinity EGFR paratope allowed to show toxicity against tumor cells while sparing normal keratinocytes which have moderate level EGFR expression.

Strategies to Improve the Therapeutic Window: Smaller Formats

Antibodies have relatively larger sizes that hinder the efficient delivery of ADCs to sequestered targets such as the interior of the solid tumors. The importance of this phenomenon is best reminded by the fact that most of the marketed ADCs are approved for the treatment of hematological cancers. Therefore, in recent years, attention is directed towards the use of smaller formats than antibody as a targeting unit of the conjugate. These include antibody fragments, peptides, scaffolds (e.g. centyrins), and even small molecules. Some of these are discussed in the next chapter with clinical applications.

Certain features of smaller format conjugates can potentially improve the therapeutic window. First, they have higher tumor penetration than ADCs due to their smaller physical radius and hence faster diffusion and extravasation coefficients. Second, they have a high plasma clearance rate that may reduce off-target toxicities. However, this may be a disadvantage as well since their concentration in tumors also decreases rapidly. That could be improved via repeated injections or further engineering to enhance their circulation time.

Smaller formats could especially make a difference by increasing the tolerated concentration via minimizing adverse effects. The lack of the Fc domain enables one to avoid cross-reactivity with Fc-receptors in healthy cells and this could improve the tolerability of higher doses. Fc domain of ADCs causes thrombocytopenia and neutropenia via binding to Fc receptors on non-target cells.

References

Comer F., Gao C., Coats S. (2018) Bispecific and Biparatopic Antibody Drug Conjugates. In: Damelin M. (eds) Innovations for Next-Generation Antibody-Drug Conjugates. Cancer Drug Discovery and Development. Humana Press, Cham.

Maruani A. (2018) Bispecifics and antibody–drug conjugates: A positive synergy. Drug Discovery Today: Technologies, 30, 55-61.

https://www.zymeworks.com/pipeline

Deonarain, M.P.; Yahioglu, G.; Stamati, I.; Pomowski, A.; Clarke, J.; Edwards, B.M.; Diez-Posada, S.; Stewart, A.C. Small-Format Drug Conjugates: A Viable Alternative to ADCs for Solid Tumours? Antibodies 2018, 7, 16.

He, R.; Finan, B.; Mayer, J.P.; DiMarchi, R.D. Peptide Conjugates with Small Molecules Designed to Enhance Efficacy and Safety. Molecules 2019, 24, 1855.

Novel Payloads and Delivery Vehicles

Antibody Fragment–Drug Conjugates

Antibody fragments have caught much interest as they constitute only a portion of an antibody, but still retains the paratopes necessary to bind to the target antigen. Employing this technology could minimize immunogenicity and heterogeneity problems, while their smaller size seems to provide better solid tissue penetration. Various engineered formats of antibody fragments fused to cytotoxic payloads have been proposed up to date, some of which have made strides through clinical studies.

Moxetumomab pasudotox (Lumoxity) is an antibody fragment - drug conjugate has been approved recently by the FDA “for the treatment of adult patients with relapsed or refractory hairy cell leukemia (HCL) who have received at least two prior systemic therapies, including treatment with a purine nucleoside analog.” Its targeting component is a disulfide stabilized variable fragment (dsFv) of a monoclonal antibody that recognizes CD22 on malignant B cells. dsFv is fused to a 38-kDa fragment of Pseudomonas exotoxin A (PE38) that interferes with protein synthesis in cells and inducing cell death eventually.

Oportuzumab monatox (Vicinium) employs a slightly different antibody fragment, named as single-chain variable fragment (scFv), that is conjugated to a Pseudomonas exotoxin A fragment. scFv fragments are generated by conjugating N and C terminals of variable regions of the heavy (VH) and light chains (VL) with a short linker peptide. Vicinium targets Epithelial Cell Adhesion Molecule (EpCAM) with the help of its scFv. EpCAM is overexpressed in >98% of high-grade non-muscle invasive bladder cancer (NMIBC), while minimally expressed in healthy bladder tissue. Vicinium was successful in Phase 1 and 2 studies for Bacillus Calmette-Guérin (BCG)-unresponsive, high grade non-muscle invasive bladder cancer (NMIBC). It showed a good safety profile and the complete response (CR) rate at 3 months in 29- 40% of subjects. Now, it is tested in a Phase 3 study to treat BCG-unresponsive NMIBC.

The action mechanisms of Lumoxity and Vicinium rely on receptor-mediated endocytosis and subsequent cleavage of the peptide linker with endosomal proteases that release the cytotoxic payload. However, the anti-cancer mechanism of the Daromun differs from them in that it is expected to stimulate the anti-tumoral effects of immune cells.

Daromun is a combination of two different immunocytokine drugs, Darleukin and Fibromun. Darleukin is a conjugate between a diabody (i.e. a noncovalent dimer of 2 scFv) and two IL-2 molecules. On the other hand, Fibromun is a conjugate of an scFv and a single TNFalpha molecule. Both of their targeting moiety recognizes the extra-domain B of fibronectin has been shown to facilitate tumor accumulation. On the other hand, cytokines are expected to induce local immune cells, especially T cells. Currently, it is tested as an intralesional therapy in patients with fully resectable stage IIIB/C melanoma in a Phase III trial.

Centyrin Conjugates

Centyrins are proteins based on a fibronectin type III (FN3) domain sequence from human tenascin. They have simple structures (~100 amino acids) that lack disulfide bonds and glycosylation so that they can be produced in homogenous batches. They have the potential to overcome limitations of antibodies. While antibodies can bind one or two antigens, centyrins can be engineered via genetic fusion to bind multiple targets. Furthermore, they can be conjugated to cytotoxic drugs, oligonucleotides, and nanoparticles for their targeted delivery. They have good thermal and low pH stability. Also, they are highly soluble enabling to prepare solutions of high concentration.

Centyrins have much smaller size (10 kDa) than antibodies (~1/15th of an antibody) that can provide better penetration and concentration in solid tumors. For the same reason they have short in vivo half-life as they are cleared through renal filtration. This may reduce liver toxicity that is associated with ADC clearance. Half-life of centyrin can be extended by adding moieties specific for serum proteins such as albumin binding domain.

Centyrins have no cysteine residues naturally, and this allows to incorporate cysteines at specific sites for controlled conjugation of cytotoxic/drug payloads. A study, which used high-throughput methods to test the tolerance of each residue of centyrin (total ~100 residues), found that cysteine mutations in 26 sites did not have significant negative effects on biophysical properties or biological activity.

Peptide Drug Conjugates

Peptide Drug Conjugates (PDC) employ peptides, much smaller biological molecules (~1-3 kDa) than antibodies and centyrins, as a targeting unit. Hence, they can penetrate the tumors faster than larger formats while plasma clearance rates are higher. PDCs are composed of a peptide molecule covalently conjugated to a drug molecule by using various linker chemistries. Peptide conjugation helps small molecule drugs to surmount challenges such as poor aqueous solubility, drug-drug interaction, fast metabolism, and cellular impermeability. The amino acid sequence of the peptide part can be custom-tailored to increase physicochemical properties of the conjugate (e.g. including charged/hydrophilic amino acids to increase solubility) or make the peptide mimic receptor binding domain of the proteins with an aim of targeting it to specific cells.

Since chemical synthesis of peptides allow higher molecular diversity and accuracy than what antibody production allows, better structural precision and optimization is possible. Having low molecular weight, PDCs allow purification with HPLC and obtaining homogenous products. All these facilitate commercial synthesis and compliance with regulatory requirements.

177Lu-Dotatate (Lutathera) is a PDC approved by FDA for the treatment of gastroenteropancreatic neuroendocrine tumors (GEP-NETs). The peptide part of 177Lu-Dotatate is a somatostatin analogue and shows high affinity to SSTR2, a somatostatin receptor that is overexpressed in many tumors. Somatostatin naturally inhibits growth hormone secretion and its activity suppress growth of cancer cells. Hence, besides improving targeting of the drug to tumor cells, peptide of 177Lu-Dotatate itself is thought to induce anti-tumor signaling as well. Peptide is conjugated to a radioactive chemotherapeutic drug via an amide linker. This system allows targeting radiation to tumor and induce selective killing of cancer cells. Clinical trials showed that 177Lu-Dotatate improves progression-free survival and complete/partial shrinkage of tumor in somatostatin-receptor positive patients.

Another example of PDCs that use SSTR2-targeting peptide for solid tumor penetration is PEN-221. Its octreotide peptide is conjugated to a cytotoxic payload DM1 maytansine with a cleavable linker. It is currently investigated in a Phase 2a trial in patients with small-cell lung cancer to evaluate its efficacy, safety, and pharmacokinetics.

Phage display technology can be utilized to select peptides that have a high affinity to tumor-specific antigens. The bicyclic peptide of the PDC BT1718 (also named as Bicycle Drug Conjugate) has been discovered via phage display selection. When three cysteines at fixed positions of the peptide made to covalently bond with a reagent such as TBMB, a bicycle-like structure emerges. This peptide has a strong affinity to MT1-MMP (human matrix metalloprotease 14) that is highly expressed in multiple cancers including triple-negative breast, non-small cell lung, and soft tissue sarcoma. The cytotoxic payload of BT1718 is DM1. After successful anti-tumoral effect at in vitro and in vivo lung tumor xenograft mouse models, it is now tested in Phase I trial in patients with advanced solid tumors and Phase II trial in patients with non-small cell lung cancer.

Oligonucleotide Conjugates

Oligonucleotides (ON) are highly anionic (i.e. negatively charged) molecules with potential therapeutic applications. They can be designed as single-stranded antisense ON or double-stranded siRNAs for silencing of genes characterizing progression of a particular disease. However, the anionic nature of ONs hinders their internalization into cells [their site of action]. Moreover, there is a huge need to improve their stability and targeting. Lastly, achieving the cytosolic delivery of the ON (rather than being trafficked to the lysosome) is a critical barrier for the use of ON as therapeutics. In order to circumvent these barriers, conjugation of ONs to polymers, peptides, proteins and antibodies has been suggested. For example, conjugation of VEGFR2 siRNA to cyclic RGD, a tripeptide, improved its in vitro and in vivo performance. The conjugate knocked down the target gene, decreased angiogenesis and tumor growth in a mouse model.

Despite being few, there are preclinical studies regarding antibody-ON conjugates (AOC) reported in the literature. Some of them compared non-covalently and covalently conjugated versions of siRNA and antibody in terms of cellular uptake, nuclear translocation, and gene silencing. Although conjugation method did not matter when it comes to cellular uptake, non-covalent conjugations appeared superior than the covalent ones for the other two outputs. Inferiority of covalent conjugation stayed same for both cleavable and non-cleavable bonds. It can be inferred that covalent bonding leads to poorer intracellular trafficking at least for some receptors. However, another study reported contrasting results. They targeted CD19 receptor (ALL biomarker) with an antibody conjugated to an antisense ON with (covalent) disulfide bond. This AOC knocked down ALL fusion protein effectively both in vitro and in vivo, also doubled the survival time of human ALL. This story suggests that not all receptors are created equal at least when targeting them with AOC and endosomal escape of AOC is receptor-specific.

References

Sheridan, C. Ablynx's nanobody fragments go places antibodies cannot. Nat Biotechnol 35, 1115–1117 (2017).

List T, Neri D. Immunocytokines: a review of molecules in clinical development for cancer therapy. Clin Pharmacol. 2013;5(Supplement 1):29-45

Kim, Ji-Sun et al. Critical Issues in the Development of Immunotoxins for Anticancer Therapy. Journal of Pharmaceutical Sciences, Volume 109, Issue 1, 104 - 115

https://sesenbio.com/wp-content/uploads/2019/01/BLADDR_Congress_2018_Posterl.pdf

https://www.fda.gov/news-events/press-announcements/fda-approves-new-kind-treatment-hairy-cell-leukemia

http://www.philogen.com/en/products/pipeline/oncology/daromun_26.html

https://www.arobiotx.com/research-development

S. D. Goldberg et al. Engineering a targeted delivery platform using Centyrins, Protein Engineering, Design and Selection, Volume 29, Issue 12, 28 December 2016, Pages 563–572

C. Shi et al. Bioanalytical workflow for novel scaffold protein–drug conjugates: quantitation of total Centyrin protein, conjugated Centyrin and free payload for Centyrin–drug conjugate in plasma and tissue samples using liquid chromatography–tandem mass spectrometry, Bioanalysis 2018 10:20, 1651-1665

He, R.; Finan, B.; Mayer, J.P.; DiMarchi, R.D. Peptide Conjugates with Small Molecules Designed to Enhance Efficacy and Safety. Molecules 2019, 24, 1855.

Wang Y., Cheetham A.G., Angacian G., Su H., Xie L., Cui H., Peptide–drug conjugates as effective prodrug strategies for targeted delivery. Advanced Drug Delivery Reviews 2017, 110–111,112-126.

https://www.tarvedatx.com/pipeline

https://www.bicycletherapeutics.com/wp-content/uploads/22_AACR-BT1718_-01-04-2017-Abstract-No-1167.pdf

https://clinicaltrials.gov/ct2/show/NCT03486730

M. Gooding et al. (2016) Oligonucleotide conjugates – Candidates for gene silencing therapeutics, European Journal of Pharmaceutics and Biopharmaceutics, 107, 321-340.

Winkler J. (2013) Oligonucleotide conjugates for therapeutic applications. Therapeutic Delivery,4(7):791-809.

I. Dovgan et al. (2013) Antibody−Oligonucleotide Conjugates as Therapeutic, Imaging, and Detection Agents, Bioconjugate Chemistry 2019 30 (10), 2483-2501.

Podcast: How to Create the Ultimate Bispecific

What does it take to create the perfect bispecific?

That's what we asked Volker Schellenberger, President and Chief Technology Officer at Amunix Pharmaceuticals in a very first episode of the Life Sciences podcast.

As co-founder of Amunix, Schellenberger is the lead inventor of the company’s XTEN as well as XPAT platforms of protease-activated T-cell engagers. In conversation with Conference Producer Eden Turner, he discussed the key steps to producing the ultimate bispecific, including the most important considerations for designing a platform and notable trends in antibody engineering techniques.

Listen to the full podcast episode below or click here to listen on Soundcloud.

Antibody Therapeutic Modalities Report 2020

Global research study into the development, targets, challenges and technologies of different therapeutics. October 2020

Antibody Therapeutic Modalities Report 2020

Within antibody engineering and therapeutics, there are now a number of different modalities the industry is working on, including monoclonal antibodies (mAbs), protein therapeutics, bispecifics, immuno-oncology checkpoints, antibody-drug conjugates (ADCs) and CAR-T/cellular therapies. At the same time, work on these modalities is increasingly exploring new non-cancer targets.

Key Insights

17% of respondents working on immuno-oncology checkpoints have therapies in Phase III or IV - the highest of any modality.
36% of respondents are working on therapeutics targeting auto-immune diseases - the highest target after cancer.
55% of respondents say that new targets/discovery approaches is the biggest challenge they are facing.
62% of respondents working with mAbs see computational approaches/Machine learning/Artificial Intelligence as the most promising technology for enabling development.

Which of the following types of therapeutic
is your company currently working with?

Unsurprisingly, mAbs were the most common (79%) type of therapeutic that respondents are working on, followed by 57% of companies developing protein therapeutics and 55% bispecifics.

Over the course of this report, we explore what phase of development companies are in for each modality, what they are targeting, the challenges that still need to be overcome and the enabling technologies and platforms being used.

Demographics

Respondent organizations

Respondent job function

Regions respondents are based

What phase(s) of development are the therapeutics you are working with currently in?

In this section we look at the development phase that different modalities are in. As this chart shows, the majority of all respondents are currently in discovery/pre- clinical, with fewer and fewer in each stage - just 3% of respondents are working on therapeutics in Phase IV.

Looking more closely at the stage of development of different modalities, it is a similar story for most. However, protein therapeutics appear to be slightly more advanced amongst respondents, with 36% in Phase II-IV trials, versus just 21% of those working with bispecifics.

Immuno-oncology checkpoints has the highest proportion of therapies in Phase III and IV, with 17% of respondents working on them at that stage of development. This is contrast to just 6% of those working on CAR-T/cellular therapies.

What are the targets of the therapeutics you are working on?

When asked about what respondents are targeting with antibody therapeutics, unsurprisingly cancer comes out top by far with 73% of respondents. Auto-immune (36%) and infectious (27%) diseases are the next most common targets.

What are the targets of the therapeutics you are working on? - BY MODALITY

When comparing targets by different modalities, some interesting differences emerge. For example there is over 20% difference between modalities for cancer, auto-immune diseases, rare diseases and infectious diseases.

What are the biggest challenges you are
facing with the therapeutics you are working on?

There are still a huge number of challenges that need to be overcome within antibody engineering and therapeutics, particularly around novel modalities. New targets/discovery approaches (55%) was the biggest challenge most respondents are facing, closely followed by transitioning from discovery to the clinic (44%).

What are the biggest challenges you are facing with the
therapeutics you are working on? - BY MODALITY

Each different therapeutic modality has a unique set of challenges. New targets/discovery approaches is by far the biggest challenge for those working on immuno-oncology checkpoints (70%), whilst delivery is a challenge for 49% of those working on CAR-T/Cellular therapies, but just 19% of mAbs.

Which enabling technologies or platforms do you believe will have the most success for development of different therapeutics? (Click to expand any of the charts)

In general, machine learning/Artificial Intelligence and novel screening are the technologies respondents hold most hope for the development of the different therapeutics. As these charts show, unsurprisingly, different technologies are seen as more promising for some modalities than others.

Antibody Therapeutic Development in Response to COVID-19

By Silvia Hnatova

INTRODUCTION

COVID-19 is a virus of zoonotic origin with 96% homology with is the closest relative in bat, Rhinolophus affinis (Andersen et al., 2020). It has a transmission rate higher than SARS and MERS, with R0 estimate 3.02 (Majumder and Mandl, 2020). Case to fatality ratio estimate is 1.38% although the totalnumbe]r of patients is likely to be underestimated (Verity et al., 2020).

Multiple traditional and off-the-shelf approaches are being clinically tested and validated for COVID-19 treatment: antibody-based therapies, existing drugs re-purposed for COVID-19, mRNA therapies and convalescent plasma from COVID-19 survivors. Here, we will evaluate the antibody therapies for COVID-19 currently in clinical trials and preclinical development.

ANTIBODY THERAPEUTIC DEVELOPMENT IN RESPONSE TO COVID-19

The structural basis of COVID-19 bears 80% identity with SARS-CoV (Yan et al., 2020). The virus is an enveloped, single-stranded RNA virus
(Kuiken et al., 2003). The receptor-binding domain of COVID-19 (RBD) binds to angiotensin-converting enzyme (ACE2) upon entering human cells (Yan et al., 2020). Spikes, the main structural proteins, of COVID-19 are formed of S protein trimers that bear similarity with HIV glycoprotein 160 and Ebola virus glycoprotein (Weissenhorn et al., 1999). The vast majority of novel therapeutics strategies are focused on targeting the RBD domain and S proteins of COVID-19. Another class of antibody therapeutics re-purposes antibodies previously approved for use in autoimmune diseases.

Clinical trials repurposing antibodies approved for autoimmune diseases

A major therapeutics strategy for COVID-19 is re-purposing existing drugs. Critical COVID-19 patients display cytokine storm, due to the abnormal release of interleukin 6 (IL-6), tumour necrosis factors alpha (TNF-alpha) and IL-12 (Xu et al., 2020). The cytokine storm is suspected to contribute to the eventual respiratory failure leading to death. Antibodies against factors involved in cytokine storm are being repurposed for COVID-19. These therapeutics have an added advantage: they are approved for use in autoimmune conditions and their
safety profiles are known.

Regeneron Pharmaceuticals together with Sanofi are re-purposing their drug sarilumab (Kevzara ®), for use in COVID-19. Currently, there is a global trial program enrolling patients in multiple countries, and a phase II/phase III clinical trial. Sarilumab, IL-6 receptor antagonist, is currently licensed for use in rheumatoid arthritis patients.

Tocilizumab (Actemra) is IL-6 inhibitor owned by Roche. Ten clinical trials using tocilizumab, a rheumatoid arthritis drug, in COVID-19 patients are being carried out by numerous hospitals across the world: Spain, Italy, Switzerland, US and China. Hubei Xinhua Hospital and Wuhan hospitals estimate completion date is 20th June 2020. In a retrospective study of tocilizumab, 90.5% patients displayed absorption of lung lesions on CT scans (Zhang et al., 2020). Peking University First Hospital is carrying Tocilizumab combined with favapiravir is also in clinical trials for COVID-19 patients, expected to be completed in May 2020.

Papa Giovani XXIII Hospital together with support from Ergomed plc are reporting positive results from observational case-control studies using siltuximab, a therapeutic for neoplastic diseases. One-third of 21 patients reported improvement in clinical state in previous case studies.

I-Mab Biopharma is developing TJM2 antibody to neutralize GM-CSF in COVID-19 patients. TJM2 neutralization of GM-CSF resulted in a reduction of myeloid and T-cell infiltration in the nervous system and prevention of cytokine release syndrome in human xenografts (Sterner et al., 2019). I-Mab’s Company’s Investigational New Drug application has been approved by FDA, enabling clinical trials using TJM2.

Gimsilumab, a monoclonal antibody against GM-CSF, is in clinical trials by Roivant Sciences. Phase 1 of gimsilumab has already been carried out using patients with lung disease. The company is now awaiting clearance for the Phase II clinical trials going ahead. Humanigen is also planning a Phase III trial with lenzilumab or Humaneered ® anti-GM-CSF monoclonal antibody. Phase I and II trials have previously been carried out. The current Phase III trial is for use in COVID-19 patients.

Antibodies ready to enter clinical testing

Regeneron Pharmaceuticals announced the discovery of hundreds of fully human antibodies against COVID-19, using the VelocImmune mice and blood from recovered COVID-19 patients. Regeneron previously used a multi-antibody approach for Ebola treatment, currently under review by the FDA. Regeneron is using VelociMab technology to scale up the manufacturing of two selected antibodies that will target the spike protein to neutralize the virus. The aim is to develop prophylactic doses by later this year. Clinical trials are yet to be announced. Regeneron partnered with Health & Human Services’ Biomedical Advanced Research and Defense Authority, to support scaling up and meet demand as soon as the antibody is validated.

Distributed Bio uses a similar strategy to Regeneron, by optimizing anti-SARS protective antibodies for COVID-19 using the DBio’s Tumbler technology. The antibodies block the ability of COVID-19 to bind ACE2 receptor, its main point of infection in human cells (Yan et al., 2020). Besides, Distributed Bio developed fully human antibodies against COVID-19 using the SuperHuman 2.0 technology. Clinical trials with human participants are yet to be announced.

GSK bought Vir Biotechnology and together they explore VIR-7831 and VIR-7832, monoclonal antibodies developed originally against SARS, for use in COVID-19. Clinical trials are yet to be announced. Vir has also secured rights to Xencor’s Xtend Fc technology aimed at extending the half-life of antibodies that could be used for COVID-19.

Vanderbilt University Medical Center is using convalescent plasma from COVID-19 survivors to isolate monoclonal antibodies. Clinical trials have not yet been announced, whilst Vanderbilt is undertaking clinical trials examining nonantibody approaches remdesivir, hydroxychloroquinone and DAS191 in COVID-19.

Antibodies in pre-clinical development

Due to homology between COVID-19 and SARSCoV, monoclonal antibodies against SARS-CoV that bind with COVID-19 may be re-purposed for use in COVID-19 patients (Zhai et al., 2020). However, not all anti-SARS antibodies are successful in binding COVID-19 receptor-binding domain (RBD, Tian et al., 2020).

CR3022 is an example of an antibody that was developed against SARS-CoV (ter Meulen et al., 2006). CR3022 was discovered in the phage library derived from the blood of convalescent SARS patients. CR3022 recognises and binds to SARS-CoV (ter Meulen et al., 2006). CR3022 was found to potentially bind COVID-19, via recognising an epitope that does not overlap with ACE2-binding site for COVID-19 RBD (Tian et al., 2020). Crystal structure of CR3022 was described by The Scripps Research Institute in the US and it described that CR3022 binds a highly conserved epitope on both SARS and COVID-19 (Yuan et al., 2020). The knowledge of the conserved epitope vulnerable to binding by antibodies may enable the design of antibodies that could serve for COVID-19 treatment, by providing in vivo protection. However, CR3022 does not neutralize COVID-19 in vitro, possibly due to its lower affinity to COVID-19 than SARS (Yuan et al., 2020). The Wilson lab at Scripps research previously pioneered antibodies for influenza and HIV treatment. It is currently doing more research for antibody-oriented vaccine, inspired by CR3022 design. Antibody design previously used to target SARS and MERS is now being re-purposed for COVID-19. Previously, inhibitors of spike heptad repeat 1 and 2 were found to be efficient for SARS-H2RP and MERS-H2RP (Xia et al., 2020). In vitro assay using ACE2-expressing 293T cells demonstrated inhibition of COVID-19 by SARSCoV-H2RP inhibitor and pan-CoV fusion inhibitor EK1 epitopes of COVID-19 (Xia et al., 2020). The pan-CoV fusion inhibitor targeting HR1 domains of HCoV S proteins was shown to inhibit infection of five coronaviruses, including COVID-19, in mice (Xia et al., 2020). The antibody was delivered as an intranasal suspension to mice, showing potential both as a treatment and as a prophylactic measure (Xia et al., 2020). The authors suggest that this drug, EK1C4 can be used as treatment and prevention in COVID-19 patients. The group is located at Fudan University in China.

Junshi Bioscience together with Institute of Microbiology of the Chinese Academy of Sciences are developing neutralizing antibodies for COVID-19, after preliminary in vitro and in vivo studies. However, no studies have been published yet.

Mabpharm Limited is developing ACE-MAB fusion protein to target the spike protein of COVID-19. If successful, the company could be ready for a quick scaling up using its cGMP cell line.

AbCellera and Lilly are developing antibodies by screening immune cells from COVID-19 survivors, identifying 500 fully human antibodies. If successful, Lilly would be responsible for scaling up the drug. Another company using the blood of COVID-19 survivors in Korea, Celltrion Group. Besides, Celltrion is hoping to develop a ‘super antibody’, not only against COVID-19, but also against other similar viruses. The company has completed initial screening processes and is planning clinical trials, with candidate screening to be completed by mid-April.

Wang et al. (2020) also identified a monoclonal antibody against COVID-19, 47D1, using SARS-S hybridomas derived from immunized mice. However, the mechanism of action of the antibody is yet unknown, unlike the RBD or spike protein-binding antibodies. Harbour Biomed (Shanghai) together with Mount Sinai Health System would be responsible for scaling up. Last Wrapp et al. (2020) identified singledomain antibody, that was capable of neutralizing COVID-19 S pseudotyped viruses.

Additional anti-COVID-19 antibodies currently in preclinical development are shown in the table on the following page.

Additional anti-COVID-19 antibodies currently in preclinical development

Company	Antibody Type	Target
Amgen, Adaptie Biotechnologies	Virus-neutralizing
Aqualung Therapeutics	Monoclonal	ALT-100
Beijing Degengrei Biotechnology Co.	Monoclonal	BDB-1
Bioduro LLC	Human synthetic antibodies	Targeting both ACE2 and spike protein
Carterra Inc., Coronavirus Immunotherapy Consortium
Dyadic International Inc	Monoclonal antibodies
Emergent Biosolutions	Antibodies from convalescent plasma
Eusa Pharma	Monoclonal	IL-6
Flanders Institute for Biotechnology	Single-domain
Gigagen	Polyclonal	Recombinant anti-coronavirus 19 hyperimmune gammaglobin
GT Biopharma, Cytovance Biologics	Tri-specific recombinant fusion protein conjugate	Anti-CD16, anti-CDC33, IL-15
Harbour Biomed, Mount Sinai Health System	Monoclonal
Immunoprecise Antibodies	Monoclonal
Inflarx		Anti-C5a
Kiniksa Pharmaceuticals	Monoclonal	Anti GM CSF (mavrilimumab)
Mateon Therapeutics	Monoclonal	OT-101
Medicago
Nascent Biotech	Monoclonal	Vimentin
Ostrich Pharma USA
Proteona	Broadly neutralizing
Sab Biotherapeutics, US Department of Defense	Polyclonal
Tiziana Life Sciences	Monoclonal	IL-6
Xavier Saelens	Single-domain

Conclusion and Future Perspectives

Hundreds of researchers are racing to find the treatment for COVID-19. They are using different approaches: monoclonal antibodies against COVID-19, convalescent plasma and repurposing existing drugs. The industry is working in tandem with researchers to facilitate rapid scaling up and to resolve any logistics issues. Fortunately, antibodies previously developed for SARS patients can be trialled in a record time, and FDA is facilitating the process. Monoclonal antibodies have shown promising results and so have antibody approaches using convalescent plasma from COVID-19 survivors. Approaches inhibiting the cytokine storm associated with respiratory failure in COVID-19 patients are also optimistic. Re-purposing existing drugs with a known safety profile would naturally be faster, safer and more efficient.

There are currently 366 clinical trials from all over the world and partnerships between the industry and academia have been formed at a rapid pace. Next few months will shed light on the next promising therapeutics for COVID-19.

About the Author

Silvia Hnatova is a Ph.D. candidate in biochemistry at the University of Cambridge, working on Alzheimer's disease. Her research experience includes cell and molecular biology, biochemistry and genetics, and her main domain of interest is bioinformatics and neurodegenerative diseases.

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