BioDrugs (2018) 32:233–243 https://doi.org/10.1007/s40259-018-0278-1 REVIEW ARTICLE 1 2 Christine Rothe Arne Skerra Published online: 10 May 2018 The Author(s) 2018 Abstract Anticalin proteins are an emerging class of clinical- (HER2), thus offering a novel treatment option in immuno- stage biopharmaceuticals with high potential as an alternative oncology. to antibodies. Anticalin molecules are generated by combina- torial design from natural lipocalins, which are abundant plasma proteins in humans, and reveal a simple, compact fold Key Points dominated by a central b-barrel, supporting four structurally variable loops that form a binding site. Reshaping of this loop Anticalin therapeutics offer a promising alternative region results in Anticalin proteins that can recognize and to antibodies, which currently dominate tightly bind a wide range of medically relevant targets, from biopharmaceutical drug development but have small molecules to peptides and proteins, as validated by X-ray technical limitations. structural analysis. Their robust format allows for modiﬁcation Anticalin proteins are derived from natural human in several ways, both as fusion proteins and by chemical con- lipocalins and provide several beneﬁts such as small jugation, for example, to tune plasma half-life. Antagonistic size, robust and compact fold, pronounced target Anticalin therapeutics have been developed for systemic speciﬁcity, and the potential to construct fusion administration (e.g., PRS-080: anti-hepcidin) or pulmonary proteins with high formatting ﬂexibility. delivery (e.g. PRS-060/AZD1402: anti-interleukin [IL]-4-Ra). Moreover, Anticalin proteins allow molecular formatting as bi- Anticalin-based biopharmaceuticals have and even multispeciﬁc fusion proteins, especially in combina- demonstrated safety and tolerability in early clinical tion with antibodies that provide a second speciﬁcity. For studies and offer both new treatment options in example, PRS-343, which has recently entered clinical-stage immuno-oncology and innovative routes of delivery development, combines an agonistic Anticalin targeting the such as inhalation. costimulatory receptor 4-1BB with an antibody directed against the cancer antigen human epidermal growth factor receptor 2 1 Reprogramming Natural Lipocalins & Christine Rothe firstname.lastname@example.org for Medically Relevant Target-Binding Activities & Arne Skerra email@example.com The lipocalins constitute a protein family with many rep- Pieris Pharmaceuticals GmbH, Lise-Meitner-Straße 30, resentatives in diverse phyla of life, including mammals, 85354 Freising, Germany insects, plants and even bacteria . Their members typi- ¨ ¨ Lehrstuhl fur Biologische Chemie, Technische Universitat cally represent secretory monomeric proteins that share a Munchen, Emil-Erlenmeyer-Forum 5, 85354 common fold dominated by a b-barrel, which is Freising (Weihenstephan), Germany 234 C. Rothe, A. Skerra complemented by an a-helix that leans against its side. At Employing targeted random mutagenesis of the binding one end, the b-barrel is closed by three densely packed site, encompassing the region of the four structurally loops and a hydrophobic core of predominantly variable loops, in combination with phage display selection hydrophobic side chains; at the other end, it is open to and enzyme-linked immunosorbent assay (ELISA) solvent. There, four loops form a binding pocket of vari- screening enabled successful identiﬁcation of BBP variants able size and shape . with novel speciﬁcities against several small molecules, The role of natural lipocalins mostly lies in the com- including ﬂuorescein and digitalis. These studies provided plexation of small molecules for various physiological proof of concept that the lipocalin scaffold is suitable for purposes . For example, retinol-binding protein (RBP) generation of novel ligand speciﬁcities while maintaining a transports the poorly soluble and chemically sensitive stable protein fold (for review, see Skerra  and Schle- vitamin A from its storage site in the liver through the huber and Skerra ). bloodstream to various tissues. Similarly, apolipoprotein With the goal of making Anticalin technology amenable D—in plasma associated with high-density lipoprotein to broader application in medical therapy and in vivo (HDL) particles—is involved in the transport of proges- diagnostics, we subsequently recruited human lipocalin terone and arachidonic acid. Other lipocalins have scav- scaffolds, thereby reducing the risk of immunogenicity in enger functions, such as the neutrophil gelatinase- human patients upon repeated dosing. Based on systematic associated lipocalin [NGAL], which tightly binds certain biochemical and structural characterization of all known bacterial siderophores and thus restricts iron supply to human lipocalins [3, 13], two members appeared particu- invading microbes . In insects and other invertebrates, larly suitable for the generation of human Anticalin lipocalins often serve for coloration by complexing pig- libraries (Fig. 1): tear lipocalin (Tlc) [14, 15], also known ments, for example the bilin-binding protein (BBP) of the as lipocalin 1 (Lcn1), and the neutrophil-gelatinase asso- European butterﬂy Pieris brassicae . Interestingly, ciated lipocalin (NGAL), also known as lipocalin 2 (Lcn2) blood-sucking ticks are a rich source of lipocalins that or siderocalin [3, 4]. interfere with the host’s innate immune system, for Both human lipocalins were successfully employed for example by blocking complement  or by scavenging the preparation of advanced phage display libraries using histamine  and/or releasing nitric oxide . procedures similar to those previously developed for the Of note, the ligand-binding activities of natural lipoca- BBP. Furthermore, trinucleotide-based DNA synthesis was lins are strictly conserved in each organism. While their applied to allow better control of amino acid composition predominant speciﬁcities for small molecules may be seen (and avoid stop codons) at the randomized positions as complementary to the biological role of immunoglobu- [16, 17]. Using this methodology, a series of Anticalin lins, which mainly recognize protein antigens, the ﬁxed proteins has been developed over recent years, providing genetic status of lipocalins distinguishes these proteins smart high-afﬁnity protein reagents directed against vari- from the somatically diversiﬁed antibody repertoire that is ous targets of biomedical relevance, both for basic research subject to continuous generation by the immune system . and for therapeutic application. In the human body, 12–15 different lipocalin isotypes with For example, the ﬁrst NGAL-derived Anticalin protein distinct ligand speciﬁcities or physiological functions have was selected against human cytotoxic T-lymphocyte- been characterized , many of them abundant plasma proteins (much like the immunoglobulins). Initial application of protein engineering to the lipoca- lins was motivated by the discovery of some structural similarity between their binding sites and those of anti- bodies (immunoglobulins), which comprise four variable loops in the former and six complementarity-determining regions (CDRs; ‘‘hypervariable loops’’) in the latter [2, 9]. Based on this notion and utilizing the methodology of combinatorial genetic engineering that emerged during the 1990s, we explored the possibility of reshaping the binding pockets of natural lipocalins to create novel ligand-binding functions [10, 11], leading to so-called ‘‘Anticalin’’ pro- Fig. 1 Structural plasticity of the loop-based binding sites of natural teins. Early attempts started with the BBP , for which a human lipocalins: tear lipocalin (Tlc; Lcn1) and neutrophil-gelatinase high-resolution X-ray crystallographic analysis was avail- associated lipocalin (NGAL; Lcn2), two validated scaffolds for the able at the time . selection of Anticalin proteins (PDB IDs 1XKI and 1L6M, respec- tively; graphics prepared with PyMOL) Anticalin Proteins as Therapeutic Agents in Human Diseases 235 associated antigen 4 (CTLA-4, cluster of differentiation Further to these scientiﬁcally motivated studies, several [CD]-152) , the prototypic checkpoint receptor for T disease-related Anticalin programs were launched with the cell activation . After afﬁnity maturation, the ﬁnal goal of medical applications, predominantly in the areas candidate showed high afﬁnity towards the ectodomain of of oncology and inﬂammation (Table 1). Anticalin-based the human membrane protein (K = 240 pM) as well as biopharmaceuticals have shown promising results in vari- pronounced cross-reactivity towards murine CTLA-4. In a ous preclinical programs as well as early clinical studies for mouse model of Leishmania infection, treatment with the several indications. So far, ﬁve Anticalin therapeutic can- PEGylated Anticalin protein led to signiﬁcant lowering of didates have reached the clinical development stage and parasite burden in the liver and spleen, which is indicative will be discussed in the following sections. of an enhanced cellular immune response due to efﬁcient checkpoint receptor blockade. After systematic optimization of the NGAL random 2 Anti-VEGF-A Angiocal : The First Anticalin library design [16, 20], Anticalin proteins were selected Protein to Enter Clinical Study against a range of targets, e.g., lanthanide chelate com- plexes, including medically relevant radioisotopes , the The ﬁrst Tlc-derived Anticalin protein with prospects for extra domain B (ED-B) of oncofetal ﬁbronectin [16, 22], therapeutic application was selected against human vas- prostate-speciﬁc membrane antigen (PSMA) , vascular cular endothelial growth factor (VEGF)-A, a key factor of endothelial growth factor (VEGF) receptor 3 (VEGFR-3) tumor angiogenesis and ocular diseases . This Anti- , the tumor cell membrane-exposed form of heat shock calin protein (PRS-050) tightly binds VEGF-A, with a very protein 70 kDa (Hsp70) , and the Alzheimer amyloid low K value of *20 pM, and effectively prevents b-peptide (Ab). receptor binding and activation . Intravitreal adminis- In parallel, the toolbox for selection and screening of tration of the Anticalin protein in a rabbit model suppressed lipocalin variants with novel ligand speciﬁcities was VEGF-induced blood–retinal barrier breakdown. To enable expanded . Beyond phagemid panning and colony- prolonged systemic neutralization of VEGF-A in vivo, the screening procedures, which were instrumental in the early plasma half-life of the engineered lipocalin was extended days of Anticalin technology, automated high-throughput by site-directed PEGylation. PEGylated PRS-050 efﬁ- microculture screening in combination with ELISA have ciently blocked VEGF-mediated vascular permeability and facilitated the quick identiﬁcation of lead candidates growth of tumor xenografts in nude mice, which was exhibiting the desired target-binding proﬁle. More recently, accompanied by reductions in microvessel density. a technique was developed for the display of engineered PEGylated PRS-050 (‘‘Angiocal’’) was subjected to a lipocalins in an active state on the surface of live Escher- ﬁrst-in-human, dose-escalation phase I study in patients ichia coli cells via fusion with a bacterial autotransporter with advanced solid tumors [30, 31]. PRS-050 was well- protein . The bacterial Anticalin libraries generated in tolerated when administered as a 2-h infusion at doses up to this way resemble a naı¨ve immune B cell repertoire with 10 mg/kg. No signs of toxicity or immunogenicity were clonal membrane-anchored antibodies, thus allowing the observed, based on the absence of anti-drug antibody use of ﬂuorescence-activated cell sorting (FACS) for the (ADA) in 24 patients (except for one patient ), rapid and automated enrichment of bacterial cells that including samples from six patients who had received express mutant lipocalins with binding activity towards a biweekly dosing. ﬂuorescently labeled antigen . Table 1 Anticalin programs in various indications that have passed early clinical development Program Target Indication Partner Status PRS-050 VEGF-A Solid tumor – Phase I completed DS-9001a PCSK9 Dyslipidemia (Daiichi Sankyo) Phase I completed PRS-080 Hepcidin Anemia ASKA Pharmaceutical Phase Ia/Ib completed Phase IIa launched PRS-060 IL4-Ra Asthma AstraZeneca Phase Ia launched PRS-343 4-1BB/HER2 bispeciﬁc Immuno-oncology – Phase Ia launched HER2 human epidermal growth factor receptor 2, IL interleukin, PCSK9 proprotein convertase subtilisin/kexin type 9, VEGF vascular endothelial growth factor Daiichi Sankyo discontinued development for strategic reasons and returned this Anticalin program to Pieris 236 C. Rothe, A. Skerra Free VEGF-A was undetectable after PRS-050 dosing at 4 A Hepcidin-Targeting Anticalin Fusion Protein levels of C0.5 mg/kg and remained so over the 3-week for the Treatment of Anemia observation period in most patients, thus suggesting target saturation. Notably, signiﬁcant reductions in circulating PRS-080 is another Anticalin protein with prolonged cir- matrix metalloproteinase 2 (MMP-2) levels indicated an culation, this time via site-speciﬁc PEGylation, which tar- anti-angiogenic effect. Pharmacokinetic/pharmacodynamic gets hepcidin (hepatic bactericidal protein) alias liver- (PK/PD) modelling, based on a measured terminal half-life expressed antimicrobial peptide 1 (LEAP1). Hepcidin of 6 days, recommended a biweekly dosing regimen for a plays a major role in the regulation of iron metabolism phase II study, comparable to dosing schedules common , particularly in patients with functional iron deﬁciency for antibodies . (FID) anemia . FID is a major cause of anemia of chronic disease (ACD), which particularly develops in patients with infections, inﬂammatory diseases, cancer, or 3 An Anti-PCSK9 Anticalin Fusion Protein chronic kidney disease (CKD). Routine use of erythropoi- for the Treatment of Metabolic Diseases etin-stimulating agents (ESAs) and high-dose intravenous iron supplementation corrects the condition in most An Anticalin protein that targets proprotein convertase patients; however, ESAs may result in a number of adverse subtilisin/kexin type 9 (PCSK9) showed convincing pre- clinical outcomes, and concerns about the long-term safety clinical activity in the treatment of dyslipidemia . of intravenous iron administration have been raised PCSK9 binds to the low-density lipoprotein receptor (LDL- [38, 39]. R), inducing its internalization and degradation. An anti- Hepcidin binds to ferroportin, the only iron transporter PCSK9 antibody blocking the binding of PCSK9 to LDL-R on the surface of absorptive enterocytes, macrophages, was demonstrated to lower plasma levels of LDL choles- hepatocytes, and placental cells, and causes its internal- terol (LDL-C) and, consequently, led to fewer cardiovas- ization and degradation , thereby blocking iron export cular events in patients with atherosclerotic cardiovascular from the body’s depositories and reducing its availability disease receiving standard statin therapy . for erythropoiesis . Consequently, antagonizing hep- A corresponding Anticalin fusion protein with an albu- cidin has the potential to improve iron availability and min-binding domain (ABD), DS-9001a, also blocks erythropoiesis while avoiding overload with exogenous PCSK9 binding to LDL-R and thus inhibits PCSK9-me- iron and, thus, may allow lower dosing of ESAs . diated LDL-R degradation. In cynomolgus monkeys, a Based on ﬁndings from PK/PD modelling, the hepcidin- single administration of DS-9001a signiﬁcantly reduced targeting Anticalin protein was conjugated with a 30-kDa serum LDL-C levels for about 3 weeks. Furthermore, PEG polymer to achieve a suitable plasma half-life. Con- administration of DS-9001a had synergistic effects with sidering both the in vivo synthesis rate of the target and the conventional statin application in murine in vivo models. therapeutically allowable upper threshold concentration of The genetic fusion of the anti-PCSK9 Anticalin protein hepcidin in the bloodstream, this pharmacokinetic opti- to an ABD  signiﬁcantly increased its inherent plasma mization proved particularly important for such an Anti- half-life, as shown in rats, allowing convenient dosing in calin protein with scavenger function. In fact, when the humans. The beneﬁcial features of the small lipocalin Anticalin protein was coupled with PEG chains of different scaffold were maintained, such as lower cost of goods by lengths (20, 30, and 40 kDa), a depot-like accumulation of production in a microbial expression system, which is of the hepcidin–Anticalin complex was observed in cases of particular interest for drugs intended for chronic treatment. branched 40-kDa PEG, whereas a low steady state con- Furthermore, the small size and high solubility of the centration of the complex was achieved when using the Anticalin-based biopharmaceutical offers the possibility of shorter and linear 30-kDa polymer. Hence, the PRS-080 improving treatment convenience by applying a high drug biopharmaceutical development program demonstrates the concentration in a low injection volume. beneﬁt of tunable PK. DS-9001a was investigated in a single ascending-dose Data from a phase I study in healthy volunteers indi- phase I study in healthy volunteers. Doses up to 450 mg cated that a single intravenous infusion of PRS-080 up to a were injected subcutaneously in a randomized, placebo- dose of 16 mg/kg body weight was safe and well tolerated. controlled setting; hence, a favorable safety and tolerability This beneﬁcial safety proﬁle was conﬁrmed in a subsequent proﬁle was demonstrated for DS-9001a, whereas in silico single-administration, ascending-dose phase Ib study (up to screening indicated low immunogenic potential . 8 mg/kg body weight) in patients with end-stage CKD Importantly, PK/PD analysis revealed a dose-dependent requiring hemodialysis. Notably, administration of this decrease of free PCSK9 as well as LDL-C levels. Anticalin protein resulted in a signiﬁcant decrease in free Anticalin Proteins as Therapeutic Agents in Human Diseases 237 hepcidin concentration within 1 h after infusion, followed mutein that also antagonizes IL-4Ra and was in clinical by robust mobilization of iron, with dose-proportional testing via the inhalation route. Notably, PRS-060/ increases in both level and duration of serum iron con- AZD1402 has demonstrated proof of concept as well as centration, as well as subsequent transferrin saturation . feasibility for pulmonary delivery in a disease model of Based on the encouraging data from this clinical trial, a humanized mice that only express the human version of the phase IIa study was initiated to evaluate the safety and PK/ receptor and its respective cytokines. PRS-060/AZD1402 PD of repeated PRS-080 administration in anemic CKD can be nebulized in high yield and without aggregation under hemodialysis (NCT03325621, ClinicalTrials.gov). using approved devices , thus enabling inhaled delivery directly to the lungs. This Anticalin drug candidate is currently subject to clinical testing in a ﬁrst-in-human 5 An IL-4-Ra-Targeting Anticalin Protein phase I study in healthy volunteers (NCT03384290, in Respiratory Disease ClinicalTrials.gov). Asthma is one of the most common respiratory diseases, with 5–10% of patients having moderate to severe condi- 6 Anticalin Fusion Proteins as Novel Multispeciﬁc tions, which often remains uncontrolled despite high-dose Agents in Immuno-Oncology inhaled corticosteroids (ICS) in combination with a long- acting beta-2 agonist (LABA)—as a second controller— Multispeciﬁc Anticalin-based fusion proteins can be used and/or systemic corticosteroids . A large proportion of to pursue innovative therapeutic strategies in immuno-on- patients with asthma have a disorder in the T helper type 2 cology, particularly by addressing the ‘‘immunological (T 2) pathway where the cytokines interleukin (IL)-5, IL-4, synapse’’ that can form upon contact between an immune and IL-13 play an important role. Antibodies such as cell and a cancer cell at their interface. This may enhance mepolizumab, reslizumab, or benralizumab, which target activation of tumor-speciﬁc T cells near the tumor site, IL-5 or its receptor IL-5Ra, are already approved for use in thereby avoiding some of the toxicities usually observed patients with severe eosinophilic asthma . with peripheral T-cell activation in healthy tissues. In Notably, the cytokines IL-4 and IL-13 both signal via the recent years, immuno-oncological approaches have sig- IL-4 receptor a (IL-4Ra) subunit, which renders IL-4Ra a niﬁcantly changed cancer therapy, and several corre- cornerstone of intervention. Indeed, clinical trials with bio- sponding biopharmaceuticals are currently being evaluated logics targeting IL-13 alone, including lebrikizumab and for the treatment of a broad range of human tumors. tralokinumab, did not yield convincing results . Only Monoclonal antibodies targeting inhibitory checkpoint dupilumab, which simultaneously targets both IL-4 and molecules such as CTLA-4 and programmed cell death IL-13 by blocking IL-4Ra, showed promising effects . protein 1 (PD-1) were the ﬁrst to show promising thera- Dupilumab has recently completed phase III studies in sev- peutic outcomes with durable response or cure in different ere asthma, and approval for this indication is expected in the cancer types, demonstrating immunotherapy as a viable next 12–18 months. However, a general drawback may be approach [48–50]. Despite these encouraging results, many that all these antibodies are administered systemically via the patients have shown only minimal beneﬁt, thus providing intravenous or subcutaneous route. strong motivation for alternative treatment strategies, par- Inhaled biologics, on the other hand, could offer several ticularly in the light of a growing number of known co- potential advantages. Beside much better convenience for stimulatory receptors, such as 4-1BB, CD27, CD40, Ox40, patients, local delivery to the lung might require a signif- or GITR . icantly lower dose, which could result both in a cost of Among those, 4-1BB (CD137) is a member of the tumor goods advantage and an expanded patient population. necrosis factor receptor super-family (TNFRS) and offers a Lower systemic target engagement, with the potential for compelling therapeutic target as it plays a central role in better tolerability, could offer further beneﬁts. A potent the regulation of the immune response. 4-1BB is mainly ? ? IL4-Ra-targeting Anticalin protein, PRS-060 alias expressed on activated CD8 and CD4 T cells, activated AZD1402, is currently being developed jointly by Pieris B cells, and natural killer (NK) cells, whereas its ligand, and AstraZeneca as an inhalable biologic for the treatment 4-1BBL, is constitutively expressed on antigen-presenting of moderate to severe asthma that is not well controlled by cells (APCs) [52, 53]. Data from in vivo murine models standard of care.  and human ex vivo assays  as well as results from In a cell-based functional assay, PRS-060/AZD1402 adoptive T-cell therapy  have demonstrated the beneﬁt demonstrated in vitro potency comparable to that of of 4-1BB co-stimulation for the elimination of tumors. dupilumab. Moreover, this Anticalin protein showed sig- Activation of 4-1BB requires higher-order cell surface niﬁcantly higher in vitro potency than pitrakinra, an IL-4 receptor clustering, which occurs under physiological 238 C. Rothe, A. Skerra conditions by binding of the trimeric 4-1BBL on APCs. Of All four formats of the bispeciﬁc fusion protein bound to note, soluble trimeric 4-1BBL cannot trigger activation of 4-1BB and HER2, respectively, with nearly identical 4-1BB. This sophisticated mode of action may explain why afﬁnities compared to the parental building blocks and the success of monoclonal antibodies targeting 4-1BB that were capable of binding both targets simultaneously . are currently in clinical development remains limited . Furthermore, all constructs showed comparable beneﬁcial Biotherapeutics addressing this costimulatory pathway biophysical properties and PK proﬁles in animals. Notably, must effectively activate the 4-1BB receptor, but their however, HER2-dependent agonistic engagement of 4-1BB activation should be restricted to the tumor and its in ex vivo T cell activation assays was found to depend on microenvironment (TME) to reduce systemic effects and the geometry of the bispeciﬁc protein. The genetic fusion unwanted toxicity. PRS-343 is an Anticalin–antibody of the Anticalin protein to the C-terminus of the heavy fusion protein that targets both 4-1BB and the well-known chain of modiﬁed trastuzumab (PRS-343) appeared to be breast cancer antigen human epidermal growth factor more potent than the other three formats. receptor 2 (HER2) at the same time. PRS-343 was designed This indicates that the geometry of the bispeciﬁc con- to activate 4-1BB on T cells by clustering the receptor at struct and particularly the distance between the two distinct the tumor site but not in the periphery, thus enhancing the binding sites are key for optimal tumor-localized activation T-cell receptor-mediated activity and effecting tumor of co-stimulatory receptors . Furthermore, this destruction (Fig. 2). demonstrates the value of Anticalin technology for the The two building blocks of PRS-343 are an Anticalin generation of innovative bi- and multispeciﬁc biopharma- protein selected against 4-1BB, which binds in a ligand- ceuticals. Several ex vivo assays and murine in vivo tumor independent manner, and an Fc-silenced version of the models have conﬁrmed the local activation of T cells in a HER2-targeting antibody trastuzumab [57, 58]. The for- tumor-speciﬁc manner and the lack of systemic 4-1BB matting ﬂexibility offered by the Anticalin technology activation, therefore conferring a manageable risk proﬁle. enabled the generation of various Anticalin–antibody Thus, PRS-343 has the potential to offer a therapeutic fusion formats as depicted in Fig. 3. The Anticalin protein alternative for patients with HER2-positive malignancies, was genetically fused to either the N- or the C-terminus of including breast, bladder, and gastric tumors. PRS-343 has the antibody heavy or light chain, thereby resulting in entered clinical development as a ﬁrst-in-class bispeciﬁc different geometries of the fusion protein, wherein the Anticalin–antibody fusion protein functioning as a tumor- antibody and Anticalin binding sites cover a range of dis- targeted immune-costimulatory 4-1BB agonist. The tances concerning the T-cell target on the one hand and the recently launched phase I trial is designed to determine the tumor antigen on the other. safety, tolerability, and potential anti-cancer activity of Fig. 2 The concept of costimulatory T cell engagement. Following cell receptor-mediated activity and leading to tumor destruction. As T binding of the Anticalin–antibody fusion protein to the tumor cell and cells in the periphery should not get activated, toxic side effects are interaction with a T cell in its vicinity, the clustering of the expected to be manageable. HER2 human epidermal growth factor costimulatory tumor necrosis factor receptor (TNFR) 4-1BB provides receptor 2 a local co-activation signal to the latter, thus further enhancing its T Anticalin Proteins as Therapeutic Agents in Human Diseases 239 Fig. 3 Anticalin technology offers a broad molecular formatting ﬂexibility as the distance between antibody and Anticalin binding sites varies between types of fusion proteins. Testing various geometries provides the possibility of matching the distance of the antibody/ Anticalin binding sites with the distance between the binding partners in the immunological synapse, here shown for a member of the costimulatory tumor necrosis factor receptor superfamily (TNFRS) and its respective ligand (TNFRSL) PRS-343 in patients with advanced or metastatic HER2- beneﬁcial biochemical or biophysical properties such as positive solid tumors for which standard treatment options solubility and folding stability. A crucial advantage of are unavailable, or no longer effective or tolerated, or Anticalin proteins for medical applications is their low where the patient has refused standard therapy immunogenic potential, as they are derived from soluble (NCT03330561, ClinicalTrials.gov). human lipocalins that are abundant in plasma or other body The concept of tumor-localized activation of co-stimu- ﬂuids and only deviate by a limited number of amino acid latory checkpoint proteins can easily be exploited further exchanges from their natural counterparts. by (a) combining the 4-1BB agonistic Anticalin protein As outlined above, several Anticalin therapeutics have with other tumor-targeting agents or (b) combining Anti- entered early clinical development and have so far been calin proteins directed against other co-stimulatory targets well tolerated with no signs of overt immunogenicity after with tumor-targeting Anticalin proteins or antibodies. One repeated administration. However, generally all therapeu- such example is PRS-342, a genetic fusion of the 4-1BB- tic proteins carry a risk of inducing an immune response, targeting Anticalin with a glypican 3 (GPC-3)-targeting including even fully human proteins such as insulin or Anticalin protein and a silenced Ig Fc moiety. GPC-3 is an coagulation factors  and humanized or so-called human oncofetal antigen with almost no expression in normal antibodies . Notably, Anticalin proteins differ in fewer adult tissue but increased expression in multiple cancers, positions (typically around 20 mutated residues) from the including hepatocellular carcinoma, Merkel cell carci- endogenous lipocalin scaffold compared with the set of noma, and melanoma [60, 61]. grafted foreign CDR residues (approximately 60) in a typical humanized antibody . Causes for immunogenicity are diverse and not yet fully 7 Prospects for Anticalin Technology understood. Potential T or B cell epitopes within the amino acid sequence or posttranslational modiﬁcations at the Anticalin technology offers a mature toolset for the selec- primary structural level, physical aggregates or sub-visible tion of modiﬁed human lipocalins with speciﬁcities for particles may elicit ADA, and even extrinsic factors such prescribed targets of interest, ranging from small molecules as the administration route or patient characteristics play a over peptides to proteins . The afﬁnities of the resulting role [65, 66]. Nevertheless, there is a gap between the Anticalin proteins are competitive for or even better (par- prediction of immunogenicity and the clinical outcome ticularly for low-molecular-weight ligands) than those ; in fact, even regulatory authorities concede that drug accessible by antibody technology and range from typically immunogenicity can only be fully assessed after late-stage low nanomolar—after initial selection from a naı¨ve random clinical testing and/or market approval . library—to mid-picomolar, usually after afﬁnity maturation In silico analysis tools to predict potential T cell epi- via partial mutagenesis and selection under conditions of topes, although known to overestimate immunogenicity increasing stringency . Apart from ligand afﬁnity and risk, combined with in vitro immunogenicity assays are speciﬁcity, Anticalin proteins can be tailored for other commonly used to rank protein candidates during drug 240 C. Rothe, A. Skerra development. In the context of Anticalin technology, half-life. Beyond that, Anticalin proteins can even be bioinformatics tools to screen for potential T cell epitopes fused to entire antibodies, thereby adding another target and posttranslational modiﬁcation sites in the Anticalin speciﬁcity to existing therapeutic antibodies and thus sequence are routinely applied and complemented by creating novel modes of action, as exempliﬁed by PRS- in vitro immune cell assays as well as biophysical char- 343. Further combinations of Anticalin-based fusion pro- acterization to select Anticalin candidates with a low pre- teins are conceivable, addressing diverse tumor antigens dicted risk for immunogenicity. Indeed, several Anticalin and co-stimulatory or inhibitory checkpoint targets, thus therapeutics that have entered early clinical development offering novel treatment options. The formatting ﬂexibil- were so far well tolerated. ity of Anticalin proteins enables adaption of the geometry Anticalin proteins are small biomolecules with a robust of the binding sites in such bi- and multispeciﬁc bio- structure, which makes them an ideal scaffold for devel- pharmaceuticals to optimally ﬁt the respective target opment as inhaled drugs for the treatment of respiratory combination in immuno-oncology. In addition, the ability diseases , among other applications. By using well- to develop Anticalin therapeutics as inhaled biologics established half-life extension methods (e.g., PEGylation should provide considerable beneﬁts for patients with or fusion with an ABD), the inherently short in vivo cir- respiratory diseases. culation of Anticalin proteins can be prolonged to meet In conclusion, Anticalin proteins offer differentiation pharmacological requirements and even extended to a half- potential compared with standard-of-care therapies, life approaching that of antibodies, if necessary . including antibodies, in several therapeutic areas and have The molecular architecture of Anticalin proteins, com- shown promising results in both preclinical and early prising a single polypeptide chain that folds into a clinical development in various indications. The nature of stable eight-stranded b-barrel with exposed N- and C-ter- lipocalins as endogenous human plasma proteins provides a mini, neither of which are part of the binding site, makes unique beneﬁt and makes Anticalin proteins attractive, also them ideal building blocks to generate bi- and even mul- in comparison with other potential protein drugs based on tispeciﬁc fusion proteins, thus offering novel therapeutic non-immunoglobulin scaffolds (such as Adnectins, Afﬁ- modalities. In this manner, Anticalin proteins can be bodies, or DARPins; for a recent review see Gebauer and genetically fused with other proteins, including Anticalin Skerra ). Importantly, multiple Anticalin-based bio- proteins of the same or different speciﬁcity , thereby pharmaceuticals have proven safety and tolerability in generating multivalent, multiparatopic and multispeciﬁc early clinical development. In summary, Anticalin proteins proteins (Fig. 4). provide a novel class of protein drugs that may comple- For example, the possibility of fusing one or more ment and even surpass conventional antibodies in many areas. Anticalin proteins to the Ig Fc portion was exploited to implement Fc effector functions or to increase plasma Fig. 4 Formatting opportunities for Anticalin proteins. Anticalin therapeutics can be developed as stand-alone small biologics but can also be used as building blocks to generate multispeciﬁc fusion proteins. Multispeciﬁc Anticalin-based biologics are accessible by mutually fusing several Anticalin proteins or by fusing Anticalin proteins to antibodies or an Ig Fc part. mAb monoclonal antibody Anticalin Proteins as Therapeutic Agents in Human Diseases 241 Compliance with Ethical Standards 14. Breustedt DA, Chatwell L, Skerra A. A new crystal form of human tear lipocalin reveals high ﬂexibility in the loop region Funding No sources of funding were used to conduct this study or and induced ﬁt in the ligand cavity. Acta Crystallogr D Biol prepare this manuscript. Open access publication of this article was Crystallogr. 2009;65:1118–25. 15. Breustedt DA, Korndo¨rfer IP, Redl B, Skerra A. The 1.8-A funded by Pieris Pharmaceuticals GmbH. crystal structure of human tear lipocalin reveals an extended branched cavity with capacity for multiple ligands. J Biol Chem. Conﬂicts of interest Arne Skerra is founder and shareholder of Pieris 2005;280:484–93. Pharmaceuticals. Christine Rothe is an employee of and holds own- 16. Gebauer M, Schiefner A, Matschiner G, Skerra A. Combinatorial ership interest in Pieris Pharmaceuticals. The authors have no other design of an Anticalin directed against the extra-domain B for the conﬂicts of interest that are directly relevant to the content of this speciﬁc targeting of oncofetal ﬁbronectin. J Mol Biol. study. 2013;425:780–802. 17. Gebauer M, Skerra A. Anticalins: small engineered binding Open Access This article is distributed under the terms of the proteins based on the lipocalin scaffold. Methods Enzymol. Creative Commons Attribution-NonCommercial 4.0 International 2012;503:157–88. License (http://creativecommons.org/licenses/by-nc/4.0/), which per- 18. Scho¨nfeld D, Matschiner G, Chatwell L, Trentmann S, Gille H, mits any noncommercial use, distribution, and reproduction in any Hu¨lsmeyer M, et al. An engineered lipocalin speciﬁc for CTLA-4 medium, provided you give appropriate credit to the original reveals a combining site with structural and conformational fea- author(s) and the source, provide a link to the Creative Commons tures similar to antibodies. 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Published: May 10, 2018