Antibody Fragment–Drug, Centyrin, Peptide Drug and Oligonucleotide 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.
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 (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.
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.
Sheridan, C. Ablynx's nanobody fragments go places antibodies cannot. Nat Biotechnol 35, 1115–1117 (2017).
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.
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.
