Recent Advances in the Development of Novel Protein Scaffolds Based Therapeutics

International Journal of Biological Macromolecules 102 (2017) 630–641

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

journal homepage: www.elsevier.com/locate/ijbiomac

Review

Recent advances in the development of novel protein scaffolds based therapeutics

a,∗,1 a,1 a,b,1 a a

Asim Azhar , Ejaj Ahmad , Qamar Zia , Mohd. Ahmar Rauf , Mohammad Owais ,

c,∗

Ghulam Md Ashraf

Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh, UP, India

Gagan College of Management and Technology, Aligarh, UP, India

King Fahd Medical Research Center, King Abdulaziz University, P.O. Box 80216, Jeddah 21589, Saudi Arabia

a r t i c l e i n f o a b s t r a c t

Article history: Antibodies occupy a central position when it comes to binding proteins with desired antigenic speciﬁci-

Received 7 December 2016

ties. During the past decade, a plethora of recombinant or humanized versions of antibodies have entered

Received in revised form 10 April 2017

clinical settings with outstanding accomplishments. Yet, they suffer from several drawbacks such as high

Accepted 11 April 2017

molecular weight, limited tissue penetration, instability, high production cost, requirement for large

Available online 13 April 2017

doses and potential cytotoxicity. As a result, new generation of receptor proteins has been developed,

that are derived from small and robust immunoglobulin (Ig) or non-immunoglobulin based “scaffolds”.

Keywords:

Combinatorial protein engineering has tremendous scope in the development of these protein scaffolds

Immunoglobulin based protein scaffolds

with immunoglobulin like speciﬁcity and/or prescribed binding functions. The advancement made in

Non-immunoglobulin based protein

scaffolds this regard can boast of developing various validated Ig based and non-Ig protein scaffolds with desirable

Adnectin therapeutic potential. The newly emerging technology has profound scope in translational biology and

Anticalin offer matching replacement for existing immunotherapeutic agents. Only few data from early clinical

Avimer studies are available yet, but many more are likely to come in the near future. Here, we provide a glimpse

BiTE

of recent clinical advances in the ﬁeld of existing protein scaffolds.

DARPins

Contents

1. Introduction ...... 631

2. Non-immunoglobulin based protein scaffolds ...... 631

2.1. Lipocalin...... 631

2.2. Designed ankyrin repeat proteins (DARPins)...... 631

2.3. Adnectin...... 634

2.4. Avimers ...... 634

3. Immunoglobulin like scaffolds ...... 634

3.1. Anticalins...... 635

3.2. Cystine knot miniproteins (Knottin)...... 636

3.3. Bispeciﬁc design ...... 636

3.3.1. Bispeciﬁc t cell engager (BiTE) ...... 636

3.3.2. Dual afﬁnity re-targeting (DART) ...... 637

3.3.3. Tetravalent tANDem AntiBodies ...... 638

∗

Corresponding authors.

E-mail addresses: [email protected] (A. Azhar), [email protected], [email protected] (G.M. Ashraf).

Authors contributed equally.

http://dx.doi.org/10.1016/j.ijbiomac.2017.04.045

A. Azhar et al. / International Journal of Biological Macromolecules 102 (2017) 630–641 631

4. Conclusion ...... 638

Conﬂict of interest...... 638

Acknowledgements ...... 638

References ...... 638

1. Introduction proteins include low toxicity, enhanced physiological circulation

and strong retention at the target site as well as cost-efﬁcient

Biomolecules that can speciﬁcally connect to targets play crit- production process through chemical synthesis or recombinant

ical roles as therapeutic and diagnostic agents in medicine, and bacterial methods.

as tools in basic and applied research. The ﬁeld has been domi- The ideal protein scaffold possesses a ﬁrm protein folding com-

nated by monoclonal antibodies, which are the gold standard with ponent which spatially brings together several exposed loops,

regard to antigen-speciﬁc protein therapeutics [1,2]. In addition, forming an extended network that ensures tight binding to the bio-

new antibody-based formats are presently undergoing preclini- logical target. The scaffold may retain a conventional Ig backbone or

cal and clinical assessment [3–6]. As an alternative, other proteins derived from a completely unrelated protein [11,12]. The scaffold

have been tested as backbones for afﬁnity molecules by convert- are generally used as a substitute for biological factor, neverthe-

ing them to library of protein binders following randomization of less they have great therapeutic potential as well [17]. The protein

amino acids at the binding surface. Among these formats are dia- scaffolds are effective for molecular targeting to neutralize a tar-

bodies, multivalent, multispeciﬁc and multimodular antibodies, as get protein, drug delivery to speciﬁc cells, such as tumor cells, and

well as heavy (H) chain-based antibodies derived from camel or tumor imaging through attachment of a ﬂuorophore to the protein

shark [7,8]. Regardless of their usefulness and widespread applica- scaffold. During the development of scaffolds, the utmost important

bility, they suffer from several drawbacks; including high molecular criteria are to select the validated biological targets to minimize the

weight, limited tissue penetration, instability, high production potential safety risks, and increase the likelihood of achieving the

cost, requirement for large doses, potential immunogenicity proof concept as future therapeutics in human.

and complex intellectual property issues [7,9]. Moreover, frag- In the present review, we have provided a comprehensive and

ment crystallizable region (Fc)-mediated complement-dependent elaborative discussion on various types of novel protein scaffolds

cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity and their applications in medicinal chemistry. Table 1 lists vari-

(ADCC) can give rise to adverse reactions. Therefore, the concept ous novel protein scaffolds with their properties and applications.

of a universal binding module was extended from antibodies to Table 2 summarizes molecular target of various novel protein scaf-

alternative protein frameworks referred to as scaffolds. folds.

In recent years, protein based scaffolds have been widely intro-

duced as a possible substitute for protein based biomacromolecules 2. Non-immunoglobulin based protein scaffolds

[10–12]. Literally, scaffold is deﬁned as a mechanical construction

which supports machines and workers during various activities 2.1. Lipocalin

concerning the building, repair or cleaning of an apparatus or struc-

ture [13]. However, the term ‘protein scaffold’ is mostly used either Lipocalins are a large family of secretory proteins consisting

in the context of intracellular signal transduction or to deﬁne enti- of 100–300 amino acid residues (Fig. 1a). They facilitate trans-

ties for protein engineering [14]. In signal transduction, scaffolds portation of small lipophilic molecules such as steroids, bilins,

can bind to multiple members of a signalling pathway, prevent- retinoids, lipids, odorants, and various secondary metabolites. The

ing their inactivation and degradation and help them to localize lipocalin displays a conserved exon/intron arrangement [18], share

to speciﬁc cellular compartments as central watchdogs of various one or three conserved sequence motifs, called structurally con-

molecular signalling pathways. Nonetheless, scaffolds have a dif- served regions (SCRs) [19], and a common tertiary structural design

ferent meaning in the area of protein engineering. They represent a [20–22]. Lipocalins contain an eight stranded anti-parallel ␤-barrel

new generation of universal binding frameworks and can be viewed structure with a repeated +1 topology, enclosing an internal ligand

as the building blocks for proteins with intended functions. With binding site [22,23]. The lipocalins are highly conserved during the

respect to scaffolds as entities for protein engineering, which will process of evolution, with new members being discovered every

be reviewed here, there are two basic options for the structural year [24].

basis of artiﬁcial binding sites: i) binding can be mediated by one Recently, human tear lipocalin and neutrophil-gelatinase asso-

or more loops on a rigid protein structure which imitate the anti- ciated lipocalin had been developed, that contain randomization

body paratope, or ii) by surface-exposed side chains of secondary of 16 accessible positions within the four exposed loops [25].

structure elements. Both loop and rigid structure-mediated bind- These non-natural lipocalin-based structures, described as anti-

ing are suitable for the generation of high afﬁnity binders. Peptides calins, have been commercially developed by Pieris Pharmaceutical

with known afﬁnity against a target can be inserted into a scaffold (Freising, Germany) [26].

protein in order to combine characteristics of both the peptide and

the scaffold protein, such as speciﬁcity, stability, and improvement 2.2. Designed ankyrin repeat proteins (DARPins)

of tissue penetration [15,16].

The scaffold proteins embody a novel class of proteins that com- Ankyrin repeat (AR) proteins are present in abundant range of

plement the immunoglobulins (Igs) and antibody derivatives in living being belonging to bacterial, archeal and eukaryotic kingdom.

the development of new agents for medical imaging and therapy. They are involved predominantly in key protein–protein inter-

Currently, a number of powerful techniques such as phage display actions; such as those occurring in transcription, regulation, and

method, cell and ribosomal display are available for combinatorial transport etc [27,28].

engineering and establishment of large molecular datasets, from DARPins are fully engineered scaffolds resembling natural and

which binders with high affinity and specificity for a given target human AR proteins, typically exhibiting high affinity and highly

protein can be isolated. Although high affinity and specificity rep- specific target binding (Fig. 1b). These DARPins are composed of

resent key features, other desired properties of engineered afﬁnity stacked ARs [29]; with each AR being fabricated of 33 amino acids,

632 A. Azhar et al. / International Journal of Biological Macromolecules 102 (2017) 630–641

Table 1

List of novel protein scaffolds.

Name of pro- Mol. Weight Major Functions Used for disease Developer Date of FDA approval References

tein/antibody/hormones (in kDa)

Designed ankyrin 14–21 Binds to VEGF-␣ Age related Macular Molecular partners, Ongoing Phase III [43]

repeat proteins disease (AMD) Allergan clinical trial

or Diabetic macular

edema (DME)

Adnectin 10 Binds to VEGFR2 Hypercholesterolemia Bristol-Myers Phase I completed [59]

Squib

Avimers 9–18 Inhibitor of IL-6 Crohn’s disease Amgen, Inc. Phase I clinical trial [60]

Anticalins 20–22 Binds VEGF-␣, IL-4Ra, AMD, Severe Asthma, Pieris Various products in [74–79]

Hepcidin Anemia Phase I and II clinical

trials

Knottin 3–6 Protease inhibitors Chronic pain Medimmune Preclinical trial [99–103]

Bispeciﬁc design 55 Recruit T- cells Refractory acute Amgen Inc. FDA approved in 2014 [114]

Bispeciﬁc T cell lymphoblastic

engager (BiTE) leukemia (ALL)

Dual afﬁnity 50 T-cell mediated HIV-1, several types of MacroGenics Inc Various stages of [124–128,132]

re-targeting (DART) clearance cancer clinical trials

Tetravalent tandem 110 Activates NK and DC Speciﬁcally targets Afﬁmed Phase II clinical trials [134,141]

antibodies cells cancer cell therapeutics

FDA, Food and Drug Administration; EMA, European Medical Agency; NK, Natural Killer cells; DC, Dendritic cells.

Fig. 1. Some of the proteins scaffolds under active development. (a) Lipocalins (b) DARPins (c) Adnectins (d) Avimers (e) Anticalins and (f) Knottins. Illustrations were

generated using Discovery Studio Visualiser 4.0.

␤ ␣

that form a -turn followed by two anti-parallel -helices and a exception of Cys (to eliminate disulﬁde formation), Pro and Gly

␤

loop binding to the -turn of the next AR [30,31]. The number of (residues located in the helix).

repeats vary in different proteins with up to 29 consecutive repeats The most advanced DARPin program (Abicipar pegol, pre-

can be found in a single protein [32]. However, AR domain, usually viously AGN-150998 or MP0112) corresponds to development

consist of four to six repeats, having molecular mass of about 14 of a highly potent VEGF (vascular endothelial growth factor)-␣

−

18 kDa. These repeats stack onto each other, leading to a right- inhibitor (IC50 < 10 pM). Currently, MP0112 is being investigated

handed solenoid structure with a continuous hydrophobic core and in three different clinical trials. The ﬁrst two trials are safety and

a large solvent accessible surface [33,34]. efﬁcacy studies of Abicipar pegol in patients with exudative age-

Most DARPins show high thermodynamic stability against heat related macular degeneration (AMD) to establish comparability

as well as chemical denaturants [35,36]. They generally do not between Japanese and non-Japanese patients [Clinical trial num-

aggregate even at high concentration [37]. They can be expressed at ber NCT02181517 and NCT02186119]. The third study is to test

very high yield in soluble form in the cytoplasm of E. coli (typically the safety and efﬁcacy of abicipar in patients with diabetic macular

100–200 mg soluble puriﬁed protein per liter culture) [38]. Using edema (DME) [Clinical trial number NCT02181504]. These parallel

ribosome display method, DARPins have been shown to bind with open-label non-controlled trials have shown that MP0112 is safe

target proteins in picomolar range. and well tolerated when given as a single intravitreal injection of

Synthetic DARPin libraries have been designed [30], to identify 0.04–1.0 mg [41]. The most frequent adverse event associated with

speciﬁc binders and can be further evolved by process such as ribo- it was a transient, sterile inﬂammatory response [41,42]. A phase

some display method [39]. Using an iterative process of sequence III safety and efﬁcacy study of Abicipar Pegol [Clinical trial num-

and structural analyses, a consensus framework for these scaffolds ber NCT02462928] in patients with neovascular AMD is currently

has been constructed [40]. The critical surface residues that might undergoing [43].

potentially interact with the target were then randomized with the

A. Azhar et al. / International Journal of Biological Macromolecules 102 (2017) 630–641 633 References [41–43] [56–58] [59] [10,60] [73–75] [12] [76] [77–79] [80] [81] [88] [111–114] [115] [123–125] [126,127] [128] [131] [132] [134–136] [140,141] AG

Squibb

Partners

and

Inc

GmbH GmbH

Research

AG AG AG AG AG AG

Amgen Bristol-Myers Amgen Developed Allergan Adnexus Pieris Pieris Pieris Pieris Pieris NA Pieris Amgen MacroGenics MacroGenics MacroGenics MacroGenics MacroGenics Afﬁmed Afﬁmed Molecular Bristol-Myers

FDA

after

the

completed

by accelerated

trial program

trial

trial trial

trial, trial trial trial trial trial trial trial

the III I I I II IIa I/II I I I I I II

Approved Phase Phase approval Phase 2014 (BMS-844203) NCT00353756 Status Phase Phase Discontinued Phase Preclinical Preclinical Precilinical Precilinical Phase Phase Phase Pre-Clinical Phase NA NCT02321592 Phase Phase Phase under

cancer types

diseases disorders and

acute (GBM) IL-6 B-cell-CLL

and

(FID) iron cancer disease

(B-ALL) (AML) and

tumor lymphocytic

glioblastoma

asthma

non-Hodgkins lymphoma DME myeloid

tumors and

Solid Colorectal lymphoma Autoimmune Immuno-Oncology HIV B-cell Primary Indication AMD, Neutralizes AMD Multiple Non-Hodgkin’s lymphoma, Hodgkin’s deﬁciency Severe B-precursor Relapsed/refractory acute Hypercholesterolaemia Anaemia Lung (chronic Autoimmune B-cell multiforme lymphoblastic leukemia leukemia) gastrointestinal patients functional non-Hodgkin lymphoma leukemia

cells

of kill

cells

killing

T to levels

target

Action cells

bridging activation cell

the

inhibitors; T

actions

activity

T T-cell T

body’s cells by of

IL-23

CD19 pathway cholesterol CD137 T

malignant IL-13 PCSK9

B-cell

inhibitor inhibitor detection the

disease

inhibitors

recruitment

B-cell

the of cells

antagonist

␣ ␣

recruitment and

HER2-positive

antigen

and free

cell

GPA33-expressing

cell

cells endogenous antagonist CD123-expressing towards cells ED-B Mechanism VEGF- Inhibition Lowered Crohn’s VEGF- Inhibits Promote Neutralization c-Met Speciﬁc IL-17 Target CD3 Redirect Redirected Redirected Inhibit Redirected T NK killing cell–killing hepcidin IL-4 depletion cells VEGFR-2 EpCAM clustering of tumor without and with CD137-positive

edema.

Format DARPins Adnectin Adnectin Avimer Anticalin Anticalin Anticalin Anticalin Anticalin Anticalin BiTe DART DART DART DART DART TandAb TandAb BiTe Anticalin macular

cell

Diabetic

alpha

Bispeciﬁc

CD3

2 molecule

scaffolds.

epithelial GPA33 DMD,

CD79B CD3

)

CD3 CD3 CD16 + +

Env+

IL-23 ␣ ␣ ␣

+ + +

and and receptor

protein

Targets VEGF- VEGFR-2 PCSK9 IL-6 VEGF- IL-4 4-1BB/HER2 Hepcidin c-Met Lipocalin IL-17, CD19xCD3 CD3 CD123 CD3 CD19 CD32B HIV-1 CD19 CD30 adhesion (EpCAM) (IL-4R novel

degeneration;

various

macular

N9B

(MT-103) (PRS-050) (MT110)

® and

target TM

(PRS-190)

N7E

2 Age-related

Molecule MP0112 CT-322 BMS-962476 AMG-220 Angiocal PRS-060 PRS-343 PRS-080 PRS-110 N7A, DigiCal BLINCYTO Solitomab MGD006 MGD007 MGD011 MGD010 MGD014 AFM11 AFM13 Molecular Table AME,

634 A. Azhar et al. / International Journal of Biological Macromolecules 102 (2017) 630–641

Molecular Partners AG (Zurich, Switzerland) initiated a ﬁrst-in- lowered cholesterol and free PCSK9 levels. In genomic transgenic

human study to investigate the safety, tolerability and blood levels mice, BMS-962476 potently reduced free human PCSK9 (ED50

of MP0250, a second DARPin candidate, in patients with cancer, in ∼0.01 mg/kg) followed by ∼2-fold increases in total PCSK9 before

July 2014 [Clinical trial number NCT01042678]. return to baseline [59].

DARPins have also been developed to target Human telomere Adnectins, with speciﬁcity for epidermal growth factor recep-

quadruplex with afﬁnities of the selected binders ranging from 3 tor (EGFR) or interleukin 23 (IL-23) has been generated using the

to 100 nM. The meticulously designed DARPins were able to differ- mRNA display technique [60]. A bispeciﬁc adnectin was designed

entiate the human telomere quadruplex with the telomeric duplex to inhibit the cross-talk between EGFR and insulin-like growth fac-

and other quadruplexes [44]. Novel DARPins (D1.73, D5.15, D6.11, tor receptor-1 (IGFR-1) in cancer cells [61]. To this end, individual

D8.1, D8.4, and D9.2) have been developed with speciﬁcity for cas- adnectins with afﬁnities of 10 nM for EGFR and 1 nM for IGFR-1

pases 1, 5, 6, and 8 [45]. The DARPin G3 is a low molecular weight were optimized for blocking either EGFR or IGFR-1, arranged in tan-

protein (14–15 kDa) with picomolar afﬁnity (91 pmol/L) for HER2 dem by a short linker and PEGylated. The combination of adnectins

[46–48]. It has a short half-life in mice ( < 3 min), lacks biological CT-322 and AT580-Peg40 (an IGFR-1inhibitor) revealed an 83%

activity and binds to HER2 in the presence of trastuzumab and per- reduction in tumor growth, a nearly 5 times lower vessel density,

tuzumab in vitro [46,48]. Radiolabelled DARPin G3 was found to be less necrotic areas and less appearance of angiogenesis [62].

helpful in selective imaging of HER2-positive tumors, and identiﬁes

a suitable format for clinical application [29].

2.4. Avimers

Recently, a novel class of ﬂuorogen-activators, termed FADAs

(ﬂuorogen-activating designed ankyrin repeat proteins), has been

The avimer consists of a single domain multimeric artiﬁcial pro-

generated by combined selections with ribosome display and yeast

tein (Fig. 1d), and its structure was specially designed with the aim

surface display [49]. The FADAs based biosensor enhance the ﬂu-

of increasing binding afﬁnity through single-domain multimeriza-

orescence of malachite green (MG) dyes by a factor of more than

tion (the avidity effect), known as the ‘avidity multimer’ or avimer

11000 and can be used for imaging of proteins on the cell surface

[63]. It is not structurally related to antibodies and classiﬁed as

as well as in the cytosol [50].

a type of Ab mimetic. Avimers consists of two or more peptide

sequences of 30–35 residues long and connected through linker

2.3. Adnectin

peptides. The antibody surrogates have high ligand afﬁnity, low

immunogenicity and the capacity for high expression in bacteria

Adnectin (a trademark of Adnexus, a Bristol-Myers Squibb R&D

[64]. It was ﬁrst discovered and developed by Avidia (now Amgen,

Company) (Fig. 1c) is a class of new family of targeted therapeu-

Mountain View, USA) [65].

tics based on the 10th ﬁbronectin type III domain ( Fn3) [51,52]. It

The scaffold is based on the genetic oligomerization of small

shares structural homology with antibody variable domains, a beta-

protein domains called A-domains existing in various cell-surface

sheet sandwich fold (94 residues) of two anti-parallel beta sheets

receptors such as the low-density lipoprotein (LDL) receptor. Sev-

with diversiﬁed solvent-accessible loops at each pole of the domain

eral libraries of assorted A-domains were generated by exon

[51]. The 15–21 amino acid long loops were randomized, displayed

shufﬂing. The A-domains share only the universally conserved

in both phage and yeast and translated into a scaffold that exhibits

residues of the A-domain. Finally, upon sequential panning and

drug-like properties. However, they differ from antibodies in pri-

screening of multiple A-domains (a process called as ‘domain walk-

mary sequence, and have a simple single-domain structure lacking

ing’) with each domain recognizing a different epitope, the multiple

disulﬁde bonds or free cysteines. As a consequence, Fn3 retains

A-domains were linked by genetic fusion to form a high afﬁn-

its high thermostability [51,53]. Adnectins have been designed to

ity avimer. Remarkably, although A-domains contain six cysteines

bind with high afﬁnity and speciﬁcity to therapeutically relevant

that form three disulﬁde bonds, avimers containing up to eight A-

targets [51].

domains with a total of 48 cysteines (24 disulﬁde bridges) can be

The ﬁrst Adnectin tested in a clinical trial, CT-322 (PEGylated

produced in the cytoplasm of E. coli and then properly folded upon

Adnectin ), aimed to target VEGFR (VEGF Receptor)-2 [54,55]. The

air oxidation to give yields >1 g/L in high cell-density fermentation.

VEGFR-2-binding ﬁbronectin moiety was expressed produced in

Such yields have been observed with several avimers to different

E. coli to increase the serum half-life of adenectin by introducing a

ligands [64]. In addition, avimers are highly stable after weeks of

40 kDa branched PEG molecule attached to it [56]. Preclinical and

incubation at elevated temperature in buffer and for several days

phase I studies on CT-322 demonstrated that it was well tolerated

in serum.

and produced pharmacological effects expected from the inhibi-

AMG-220 (previously known as C-326), an avimer that neu-

tion of the VEGFR-2 pathway [55]. The parental adnectin binds

tralizes the pro-inﬂammatory cytokine interleukin (IL)-6, has been

strongly to VEGFR-2 with high binding afﬁnity (Kd = 11 nM) and

originally developed by Avidia (acquired by Amgen in 2006). AMG-

potently inhibits VEGFR-2 signalling, migration, and tube formation

220 possess the binding afﬁnity in the range of picomolar, and

in human umbilical vascular endothelial cells. It also inhibits the

sub-picomolar potency in an IL-6 stimulated TF-1 leukemia cell

growth of a broad range of human tumor xenograft in the murine

proliferation assay [64,66]. The efﬁcacy of AMG-220 has been suc-

models [57]. A open-label run-in/phase 2 study to assess its efﬁcacy

cessfully evaluated in patients suffering from Crohn’s disease [10].

and safety in recurrent glioblastoma, was terminated prior to reach-

Unfortunately, the compound was halted by the manufacturer [60].

ing the planned enrollment for all treatment groups because data

from the completed CT-322 monotherapy treatment arm revealed

3. Immunoglobulin like scaffolds

insufﬁcient efﬁcacy. Despite biological activity and a tolerable side

effect proﬁle, CT-322 failed to meet the prespeciﬁed threshold for

Antibody fragment derivatives such as single chain variable

efﬁcacy in recurrent glioblastoma [58].

domain fragments (scFv) and diabodies were developed in the 80 s

Proprotein convertase subtilisin kexin-9 (PCSK9) is an impor-

and extensively evaluated as a potential research tools for imaging

tant pharmacological target for decreasing low-density lipoprotein

and therapeutic applications [67]. However, single-domain Ig folds

(LDL) in cardiovascular disease. A PCSK9-binding adnectins (BMS-

had been discovered recently either by genetic engineering or as

962476) has been identiﬁed through selection from highly diverse

part of naturally occurring proteins in certain living organisms like

libraries using mRNA display. In hypercholesterolemic, overex-

camelids and sharks [68].

pressing human PCSK9 transgenic mice, BMS-962476 rapidly

A. Azhar et al. / International Journal of Biological Macromolecules 102 (2017) 630–641 635

3.1. Anticalins ceptor and an established marker for tumor-speciﬁc inﬁltrating T

lymphocytes (TILs), and is therefore, an attractive target for cancer

Anticalins are artiﬁcial proteins that bind to both large sized immunotherapy. The mode of action of this CD137/HER2 bispeciﬁc

antigens as well as small molecules with equal propensity (Fig. 1e). is to promote CD137 clustering by bridging CD137-positive T cells

Although not related in primary sequence, anticalins are struc- with HER2-positive tumor cells, and to thereby provide a potent

turally similar to antibodies as they too normally stabilized by co-stimulatory signal to tumor antigen-speciﬁc T cells [76].

disulﬁde bridges which make them a type of antibody mimetic. PRS-080, an anticalin against hepcidin, targets the anemia and

They are derived from human lipocalins (discussed previously), functional iron deﬁciency (FID) [77]. During chronic inﬂammation,

which are a family of naturally binding proteins and are being hepcidin levels in the body are elevated. Hepcidin traps iron in

used in place of monoclonal antibodies. However, they are about body stores and thereby leads to functional iron deﬁciency and

eight times smaller in size and contain only one non-glycosylated impaired erythropoiesis. In turn, neutralization of hepcidin by PRS-

polypeptide of about 180 amino acids with molecular mass 20 kDa. 080 restores iron utilization and erythropoiesis. In 2015, Pieris

Anticalins have better tissue penetration than antibodies and are has completed dosing in a Phase 1b trial (Clinical Trial number

◦

stable at temperatures up to 70 C. Unlike antibodies, they can be − NCT02340572) in anaemic patients with chronic kidney disease

produced in bacterial cells like E. coli in large amounts [69]. The undergoing hemodialysis. In this trial, PRS-080 showed a favorable

anticalin technology is exclusively commercialized by Pieris Phar- safety proﬁle [78]. Within one hour of PRS-080 administration, a

maceuticals in Freising, Germany [26]. marked decrease in plasma hepcidin was seen, followed by eleva-

Anticalin contains a rigidly conserved eight antiparallel ␤-barrel tions of both serum iron concentration and transferrin saturation

backbone with four loops variable region, (that can be targeted [78]. Moreover, the durations of serum iron elevation and trans-

for randomization), which together form a pliable cup-like binding ferrin saturation increased in a dose-dependent manner. Pieris will

pocket and an attached ␣-helix [69–71]. Conformational deviations conduct a multi-dose trial (Phase 2a study), in the same patient

are primarily located in the four loops reaching in the ligand bind- population which is scheduled for completion in the second half of

ing site [9]. Mutagenesis of amino acids at the binding site allows for 2017 [79].

changing the afﬁnity and selectivity. Recently, researchers reported Anticalin (PRS-110) based on the Lcn1/Tlc (tear lipocalin) scaf-

an efﬁcient display of anticalin libraries on E. coli that enabling fold has been developed against hepatocyte growth factor receptor

FACS and hence have an attractive future alternative for the gen- (HGFR; c-Met proto-oncogene) [12] and was shown to act as

eration of new speciﬁc anticalins [72]. The monovalent anticalins a highly potent and speciﬁc Met antagonist with both ligand-

have potential advantage over bivalent antibodies as they cease to dependent and ligand-independent activity [80]. Three different

cause unwanted side effects because of receptor cross-linking and extra domain-B (ED-B) speciﬁc anticalins, namely N7A, N7E and

pose an agonistic effect. N9B, based on the human Lipocalin 2 (Lcn2) scaffold, were devel-

Anticalins are now efﬁciently produced in both E. coli and yeast oped by replacing 20 amino acids in four structurally variable loops

cells, and found to be soluble and stable [25]. Several anticalins [81]. These anticalins were found to speciﬁcally detect ED-B in pri-

have been selected and produced against various medically rele- mary glioblastoma multiforme (GBM; WHO IV) but not in tumors of

vant targets, including cytotoxic T-lymphocyte antigen 4 (CTLA-4, lower histopathological grade or in tumor-free brain [82]. Another

CD152), VEGF-␣ and c-Met oncogene, having afﬁnity in nanomo- anticalin has been recognized which inhibits the interaction of

lar to picomolar range [25,71]. An anticalin directed against VEGF CTLA4 (CD152), (a transmembrane cell surface receptor expressed

(PRS-050; Pieris AG) and exhibiting encouraging binding and afﬁn- by T-cells), with both B7.1 (CD80) and B7.2 (CD86) expressed on

ity proﬁle, has been developed [73]. The binding afﬁnity of anticalin tumor cells [71].

molecule to VEGF was found to be in the range of nanomolar con- The ﬂuorescein-speciﬁc anticalin (FluA) was developed, after

centration. During phase-I study; PRS-050 was shown to be safe genetic fusion to an Ig Fv fragment that recognized the Fn

and well-tolerated with a complete lack of immunogenicity and no extra-domain-A as angiogenesis marker, to pre-targeted payload

trace of anti-drug antibodies (ADAs) in humans. Anticalins were delivery in a mouse tumor xenograft model [83]. Furthermore, the

further linked to a 40 kDa PEG moiety to enhance their half-life in afﬁnity-improved digoxigenin-binding anticalin (DigiCal) showed

blood circulation [73]. Furthermore, the binding showed powerful promising results in a rat study as antidote for the treatment of dig-

antagonistic effects both in vitro and in animal models, including italis intoxication [84]. Recently, a new bilin-binding protein (BBP)

an age-related rabbit model of macular degeneration (AMD), mouse derived anticalin was selected via ribosome display to recognize

xenograft models of HCT116, and U87-MG tumor cells [25,74]. Tox- the sex hormone estradiol [85].

icological studies in rats and cynomolgus monkeys suggest that Chemical coupling of ﬂuorescent dyes and radionuclides to

these animals are well tolerated [74]. This anticalin, now known anticalins either via Lysine residue exposed on the protein sur-

as Angiocal , is expected to enter clinical Phase II trial [75]. face or via genetically introduced Cysteine residues has also been

Another example is an anticalin (PRS-060) speciﬁc for the IL-4 demonstrated [69,86]. In fact, radio-conjugated anticalins have

receptor alpha (IL-4R␣), thereby inhibiting the actions of IL-4 and been used successfully as bio-imaging reagents [69,86]. Schlehu-

IL-13, and is currently in preclinical development to treat severe bera and Skerra (2001) reported the construction of a functional

asthma [12]. IL-4R␣ has a key role in chronic inﬂammation through fusion protein from two independent anticalins, a so-called duo-

interaction with both IL-4 and IL-13 in the lungs. The high thermal calin, containing an anticalin with ﬂuorescein speciﬁcity on the one

stability of anticalins motivated researchers to locally administer it hand and an anticalin with digoxigenin speciﬁcity on the other [87].

through alternative routes, potentially leading to improved patient It was demonstrated that both domains of the fusion protein have

compliance as well as directed local administration to the inﬂamed essentially kept their ligand-binding properties with respect to the

tissue. The small size and biophysical stability of PRS-060 enables separately produced anticalins. Moreover, these fused, bispeciﬁc

direct delivery to the lungs, such as through the use of an inhaler anticalins or duocalins had the potential to mediate signal trigger-

[12]. ing by cross-linking of cellular receptors. The bispeciﬁc duocalins

The third anticalin-based drug candidate, PRS-343, is a bispe- can also be exploited to target cells of the immune system to tumors

ciﬁc protein targeting the immune receptor CD137 and the tumor by cross-linking tumor cells with immune effector cells. A success-

target HER2. PRS-343 is the result of a genetic fusion of a variant of ful example is the reduction of the immune suppression exerted by

the HER2-targeting antibody trastuzumab with an anticalin speciﬁc the transmembrane CTLA4 by an anticalin [16].

for CD137. 4-1BB (CD137) is a potent costimulatory immunore-

636 A. Azhar et al. / International Journal of Biological Macromolecules 102 (2017) 630–641

linear (also known as acyclic) knottin peptides in that their N- and

The PRS-190 bispeciﬁc anticalin, or Duocalin , is a ﬁrst-in-class

C-termini are not connected through a peptide bond.

drug candidate that is designed to speciﬁcally bind two clinically

The engineered EETI-II peptide derived binders with an RGD

validated targets in the autoimmune space. The PRS-190 is a highly

motif grafted onto one of the loops (Min-23) was shown to fold in

speciﬁc and potent bispeciﬁc antagonist targeting IL-17 and IL-

◦

a native-like manner and possess high stability (T around 100 C)

23, two key members of the Th17 family of cytokines, involved m

[99,100]. It has been successfully used in targeting tumor vascula-

in autoimmunity and hyperinﬂammation. This approach creates a

ture in the murine xenograft. The truncated C-terminal fragment of

synergistic therapeutic effect while enabling a more efﬁcient devel-

the AgRP as scaffold for peptide grafting of the RGD motif, adopt-

opment pathway than that for a drug cocktail approach [88].

ing a very similar approach as described for the EETI-II scaffold

[101]. Tumor targeting was conﬁrmed and inhibition of platelet

aggregation was also demonstrated [102,103].

3.2. Cystine knot miniproteins (Knottin) Knottin binders with different speciﬁcities have also been gener-

ated employing peptide grafting procedure that involves insertion

Cystine knot miniproteins (also called Knottin) represent a large of known sequences having high afﬁnity [104]. Still, it remains to

family of structurally related peptides (30–50 amino acids long) be seen how generally applicable cystine knot miniproteins can be

(Fig. 1f) with diverse amino acid sequences, showing high potency used for de novo isolation of binders to any protein target. Other

and selectivity [89]. They share a common structural motif, char- cystine-stabilized scaffolds, such as the human kringle domain,

acterized by a core of antiparallel ␤-strands stabilized by at least might provide an alternative for the creation of large repertoires

three disulﬁde bonds. In a characteristic cystine-knot motif, the of molecules, allowing for selection of binders against different

ﬁrst and fourth and the second and ﬁfth cysteine residues form targets.

disulﬁde bonds; the disulﬁde bond formed between the third and

sixth cysteine residues passes through the other two disulﬁdes,

creating a macrocyclic knot [90]. This disulﬁde-constrained core 3.3. Bispeciﬁc design

◦

provides amazing thermal (above 95 C), chemical (guanidine, urea

and acidic pH) and proteolytic stability to the scaffold [91]. 3.3.1. Bispeciﬁc t cell engager (BiTE)

The cystine knot miniprotein offers an attractive platform in BiTE was ﬁrst developed by Micromet Inc Germany (now

the development of peptide-based therapeutics. The disulﬁde- Amgen) to overcome the challenges associated with the quantity,

constrained structural core can be functionalized by decoration quality and stability of the bispeciﬁc Ab compared to the mono-

with bioactive-loop residues. Methods have been developed to clonal Ab of early CD19xCD3 formats [105]. BiTE is a 55 kDa single

generate miniproteins with pre-arranged binding characteristics polypeptide molecule containing only 1 N- and 1 C-terminus, in

against a range of different target molecules [92]. Cystine knot pro- which four variable domains of heavy and light chains are linked

teins have been isolated from many different sources and are used together and provide antigen-binding speciﬁcities for two antigens,

for various applications including molecular imaging and therapeu- like pearls on a string of polypeptide linkers (Fig. 2). One of the vari-

tic [93]. FDA approved its use for the treatment of severe chronic able domain is designed against any antigen of interest (surface

pain [4]. marker) and the other recognises and binds to the CD3 component

Despite the vast number of naturally occurring knottin peptides, of the T cell receptor (TCR) complex, present on the surface of T

+ +

only a few have been developed and validated as molecular scaf- cells (mainly CD4 and CD8 effector and memory T cells) result-

folds for protein engineering. These include trypsin inhibitor from ing in T cell activation [106]. By using just one CD3-speciﬁc binding

the seeds of the squirting cucumber Ecballium elaterium (Ecbal- arm, such bispeciﬁc antibodies can mono-valently bind to all T cells,

lium elaterium trypsin inhibitor [EETI]) [95], or from the fruit but do so only with rather low afﬁnity [39], which will not trigger

of Momordica cochinchinensis (Momordica cochinchinensis trypsin T-cell signalling by CD3, unless the BiTE antibody is presented to

inhibitor [MCoTI]) [96], a peptide insecticide from the African plant the T cell in a multivalent fashion by a target cell [107] . The BiTE

Oldenlandia afﬁnis (kalata B1) [97], and a Agouti-related protein antibody construct is designed to selectively target the endogenous

(AgRP), a human neuropeptide that binds to the melanocortin T cells towards malignant cells. These BiTE -activated T cells can

receptor and regulates appetite [98]. EETI and MCoTI have simi- then engage with malignant cells leading to subsequent expansion

lar tertiary structures despite their diverse amino acid sequences. of local cytotoxic T-cell (CTL) pool in targeting additional malignant

MCoTI and kalata B1 are both cyclotides, while EETI and AgRP are cells [39,108].

Fig. 2. Immunoglobulin based scaffolds with bispeciﬁc design: (a) The structure of BiTEs. The two linkers outside the chain are about 15 amino acids, and are sufﬁciently long

and ﬂexible to make light chains and heavy chains form the right conformation; (b) The structure of dual-afﬁnity re-targeting proteins (DARTs). The linker between heavy

and light chains is as short as about ﬁve amino acids. Because of the short linker between the two domains of scFv, the two domains of the same scFv cannot pair and are

forced to homodimerize with its homologous partner in another scFv. The adding of another cysteine residue at the end of VHA and VHB is helpful for the stability of this

kind of bispeciﬁc antibody by forming a disulﬁde linkage; (c) The structure of Tandem diabodies (TandAbs). The three linkers of TandAb are all short linkers; it is hard to pair

within the chain while easy to dimerize between two chains and generate.

A. Azhar et al. / International Journal of Biological Macromolecules 102 (2017) 630–641 637

Key features of BiTE based antibodies that contrast them from VH of the ﬁrst variable region linked to the VL of the second binder,

other bispeciﬁc antibody constructs include: (i) a high potency of and the VH of the second variable region linked to the VL of the ﬁrst

redirected lysis with EC50 values ranging from 0.1 to 50 pmol/L that may or may not be connected via disulphide linkage (Fig. 2)

(2–1000 pg/mL), (ii) target cell-dependent activation of endoge- [120]. While in the diabody format, the two polypeptide chains

nous T cells thereby avoiding the need for ex vivo expansion or associate non-covalently, DART format may provide additional sta-

manipulation [107,109], and (iii) support of serial lysis by acti- bilization through a C-terminal disulﬁde bridge. This introduction

vated T cells, i.e., activity at low E:T ratios. BiTE antibodies do of interchain disulﬁde bonds at their C-termini makes them more

not rely on the generation of speciﬁc T-cell clones, on the activ- structurally compact, forming stable cell-to-cell contacts between

ity of co-stimulatory molecules, or on normal antigen presentation target and effector cells and increasing potency than BiTEs. DARTs

[108]. BiTE antibodies are typically secreted as recombinant, non- can be produced easily without refolding in large quantities, reveal

glycosylated proteins by eukaryotic cell lines. exceptional stability in both buffer and human serum, are not prone

The BiTE technology presents a very promising mode of action. to aggregation during storage and showed high potency in both

However, its low circulating half-life (2–3 h) requires incessant in vitro and in vivo studies.

intravenous infusion in order to maintain steady-state serum levels Johnson and co-workers linked an Fv speciﬁc for human CD16

[108]. BiTE antibodies have so far been constructed to more than 10 (Fc␥RIII) on effector cells to an Fv speciﬁc for mouse or human

different target antigens, including CD19, EpCAM, Her2/neu, EGFR, CD32 B (Fc␥RIIB), a normal B-cell and tumor target antigen and

CD66e (or CEA, CEACAM5), CD33, EphA2, and MCSP (or HMW-MAA) demonstrated extremely potent, dose-dependent cytotoxicity in

[105,106]. re-targeting human PBMC (peripheral blood mononuclear cells)

The most clinically advanced BiTE is blinatumomab (previously against B-lymphoma cell lines as well as in mediating autologous

known as AMG103 and MT103) targeting CD19 and CD3 [110]. B-cell depletion in culture [11]. DART molecules constructed by

CD19 is a tumor marker expressed in high number over the sur- pairing an Fv region of mAb directed against CD32 B with an Fv

face of B cells. Phase I and II clinical trials with the CD19xCD3 BiTE region from a mAb directed against CD79B, the ␤-chain of the

blinatumomab have demonstrated promising results in relapsed B- invariant signal-transducing dimer of the B cell receptor complex

cell non-Hodgkins lymphoma (phase I trial) [111] and B-precursor was shown to trigger the CD32B-mediated inhibitory signalling

acute lymphoblastic leukemia (B-ALL) (phase II trial) [112]. The pathway in activated B cells, resulting in inhibition of B cell pro-

clinical responses in non-Hodgkin’s lymphoma (NHL) patients were liferation and Ig secretion [121].

obtained at a serum level (15 mg/m /day of blinatumomab) that Moore et al. (2011) developed DART to eradicate B-cell lym-

was 10,000-fold below the levels reported for the monoclonal Ab phoma through co-engagement of the B cell–speciﬁc antigen CD19

rituximab [113]. This high potency may be related to the high lytic and the TCR/CD3 complex on effector T cells [122]. The DART

potential of CTLs that are activated by engagement of only a few CD3 molecules were found to be more potent in directing B-cell lysis

subunits and can rapidly induce cell lysis as well as selective expan- as compared to single-chain, bispeciﬁc antibody. The enhanced

+ +

sion of CD8 and CD4 effector memory T cells. In December 2014, activity with the CD19xCD3 DART molecules was observed on all

Blinatumomab (trade name or BLINCYTO ) was approved by the CD19-expressing target B-cells, evaluated using resting and pre-

US Food and Drug Administration under the accelerated approval stimulated human PBMCs or puriﬁed effector T-cell populations

program [114]. [122].

Another BiTE antibody, named Solitomab (MT110) is bispe- MGD006 is a DART that simultaneously binds the cell surface

ciﬁc for CD3 and epithelial cell adhesion molecule (EpCAM) [115]. protein CD123 as well as CD3. CD123, the Interleukin-3 receptor

It is currently being tested in a phase I/II trial with lung, col- alpha chain, has been reported to be over-expressed on cancer

orectal and gastrointestinal cancer patients (Clinical trial number cells in a wide range of hematological malignancies including acute

NCT00635596). EpCAM (CD326) is frequently expressed on human myeloid leukemia (AML) and myelodysplastic syndrome (MDS).

adeno- and some squamous cell carcinoma, and also on cancer stem It does not contain an Fc region and, therefore, has a rather

cells [116]. To assess the therapeutic window of an EpCAM/CD3- short serum half-life. The binding to CD3 helps in the recruit-

bispeciﬁc single-chain antibody construct of the bispeciﬁc T-cell ment of T cells to cancers and is currently in phase I trials in

engager (BiTE) class, a murine surrogate of MT110 (muS110) patients with hematological cancer [123]. A phase I dose-escalation

was constructed from single-chain antibodies speciﬁc for murine study is currently being done to investigate the safety, pharma-

EpCAM and CD3 antigens [117]. MuS110 was well tolerated and cokinetics and preliminary activity of MDG006 in patients with

showed signiﬁcant antitumor activity at a concentration as low as relapsed/refractory acute myeloid leukemia (AML) (Clinical Trial

5 ␮g/kg bw (body weight), in both syngeneic 4T1 orthotopic breast number NCT02152956) [124]. This currently enrolling study rep-

cancer and CT-26 lung cancer mouse models. Moreover, muS110 resents the ﬁrst clinical use of DART molecules in patients with

exhibited similar in vitro characteristics and in vivo antitumor activ- cancer [125].

ity as MT110 [117]. Another CD3-binding DART (MGD007) (GPA33 x CD3 DART),

Utilizing the same technology, melanoma (with MCSP spe- that targets gpA33-positive cells for recognition and elimination

ciﬁc BiTEs) and acute myeloid leukemia (with CD33 speciﬁc by co-engagement of CD3-expressing T lymphocytes. GPA33 is a

BiTEs) can be targeted [9]. Research in this area is currently 43 kDa membrane-bound glycoprotein with expression restricted

ongoing. Another avenue for novel anti-cancer therapies involves to the surface of normal human colon and small bowel epithelial

re-engineering some of the currently used conventional antibodies cells. In contrast to MGD006, this DART is fused to an Fc region

like trastuzumab (targeting HER2/neu), cetuximab and panitu- and, therefore, has a prolonged serum half-life. MGD007, with

mumab (both targeting the EGF receptor), using the BiTE approach enhanced pharmacokinetic (PK) properties, is being evaluated in a

[118]. BiTEs against CD66e and EphA2 has been developed as well Phase I trial in patients with colorectal cancer (Clinical Trial number

[105]. NCT02248805) [126]. MGD007 mediates redirected T cell killing

of GPA33-expressing CRC (colon cancer) cell lines in vitro and

3.3.2. Dual afﬁnity re-targeting (DART) regression of transplantable CRC cell line xenografts in preclinical

Dual-Afﬁnity Re-Targeting (DART) proteins are bispeciﬁc, tumor models. Preclinical toxicology studies in cynomolgus mon-

diabody-like entities that can bind two distinct cell-surface keys demonstrated that MGD007 could be administered safely with

molecules simultaneously. First developed by the biotechnology a pharmacokinetic proﬁle supportive of repeat dosing on a weekly

company, MacroGenics Inc (USA) [119], DART antibodies have the basis [127].

638 A. Azhar et al. / International Journal of Biological Macromolecules 102 (2017) 630–641

MGD011 (also known as JNJ-64052781), a humanized induced speciﬁc lysis of CD19-positive cell lines (Raji, Daudi, and

CD19xCD3 DART in MP3 format, is being evaluated in a Phase I BJAB) in the presence of T cells [137]. Thus, UC-MSCs releasing

trial in patients with B-cell hematological malignancies (Clinical TandAb could be an efﬁcient strategy for the treatment of B cell

Trial number NCT02454270). CD19, a lymphocyte-speciﬁc marker lymphoma.

expressed from early B-lymphocyte development through mature One of the TandAb, AFM13 is directed against CD30 and CD16A

memory B cells, is highly represented in B-cell malignancies. (or Fc␥-R-IIIa) in order to selectively activate natural killer (NK),

MGD011 was well tolerated with prolonged pharmacokinetics dendritic cells, and macrophages [138]. This TandAb has two bind-

properties, consistent with that of a human IgG1 in monkeys. ing sites for CD16A and two for CD30, the antigen identifying

MGD011-mediated killing of CD19 target cells was accompanied Hodgkin lymphoma cells. The binding and cytotoxicity of the

by target-dependent T-cell expansion and activation, cytokine TandAb were compared with antibodies with identical anti-CD30

release and upregulation of perforin and granzyme B, consistent domains: (i) a native IgG, (ii) an IgG optimized for binding to Fc

with the redirected T-cell mediated killing mechanism of action receptors, and (iii) a bivalent bispeciﬁc CD30/CD16A diabody. Due

endowed in its design [128]. to its CD16A-bivalency and reduced koff, the TandAb was retained

DART have also been pursued to attenuate autoimmune dis- longer on the surface of NK cells than the IgGs or the diabody. This

orders: MGD010, a CD32BxCD79 B DART in MP3 format, that contributed to the higher potency and efﬁcacy of the TandAb rela-

simultaneously bind Fc␥R-IIb (CD32B) and the B-cell receptor com- tive to those of the other anti-CD30 antibodies. Moreover, TandAb

ponent, CD79B, on B cells; it is being tested in a Phase I trial in cytotoxicity was found to be independent of the CD16A allotype,

normal volunteers (Clinical Trial number NCT02376036) [129,130]. whereas the anti-CD30 IgGs were substantially less cytotoxic when

CD32 B is a checkpoint molecule expressed on B lymphocytes that, NK cells with low afﬁnity CD16A allotype were employed [139]. The

when co-ligated with CD79B, a component of the B-cell antigen therapeutic potential of AFM13 has been explored through clin-

receptor complex, delivers a co-inhibitory signal that dampens ical Phase I, showing good safety and tolerability proﬁles in the

B-cell activation. Earlier studies established that CD32BxCD79 B treatment of Hodgkin lymphoma [140]. A phase II study is under-

DARTs engage CD32 B in a BCR-dependent manner and inhibit way for this novel tetravalent bsAb AFM13 as the ﬁrst-in-class

B-cell activation without B-cell depletion. Favourable pharmacody- NK cell-speciﬁc agent for cellular immunotherapy targeting CD30

namics properties and lack of safety signals, including no cytokine malignancies (GHSG-AFM13, NCT02321592) [141].

release nor B-cell depletion, were observed in non-human primate

dose-escalated study with MGD010 [131].

4. Conclusion

HIVxCD3 DART with HIV arms derived from the non-

neutralizing mAbs A32 and 7B2 combined with an anti-CD3 binding

The fast growing scientiﬁc endeavours focussing therapeutic

arm (hXR32) was able to recognize HIV-1 Env-expressing cell lines

proteins has led to the development of novel classes of engineered

and to elicit redirected T cell killing activity, even when cell-surface

proteins. With the help of technologies such as phage display

Env-expression appeared low [132]. HIVxCD3 DARTs derived from

method, researchers are now able to screen a number of potential

PGT121, PGT145, A32, and 7B2, but not VRC01 or 10E8 antibodies,

candidates for therapeutic use. In addition, the clinical evaluation

mediated potent CTL-dependent killing of quiescent primary CD4 T

of several alternative scaffolds as antibody mimetics is ongoing.

cells infected with diverse HIV isolates 108].

These new scaffolds might be able to ﬁll an important void, since the

potential of mAbs to bind to grooves or catalytic pockets is limited. A

3.3.3. Tetravalent tANDem AntiBodies

variety of the scaffolds like BiTEs and DARTs have shown promising

TandAbs (Tetravalent tandem Antibodies) are tetravalent, sin-

results, and many of them have entered clinical settings. However,

gle chain bifunctional proteins that recognize a speciﬁc biological

only a selected few scaffold based therapeutics have reached the

target and utilizing their second functionality, bind with high afﬁn-

market. Hence, further in-depth research works are needed to con-

ity receptors on immune cells to eliminate the target cell. These

solidate these promising protein scaffolds. Efﬁcient induction of

molecules assemble by dimerization of a protein that contains four

apoptosis of tumor cells is a further prospect of these new agents.

variable H- and L-chains in an orientation that prevents “wrong”

Coupling of the new scaffold-based entities to cytotoxic payloads

intramolecular pairing (Fig. 2). TandAbs have four binding sites,

is another perspective. Additionally, dosage-schedule and duration

and they are larger than BiTEs but do not carry Fc domains. Non-

of treatment are also avenues for further research.

adjacent conformation (VHA-VLB–VHB-VLA; A and B being the

targeted antigens) of both binding unit connected by a 12 amino

Conﬂict of interest

acid linker results in larger size (110 kDa) that greatly improves its

serum half-lives [133].

The authors conﬁrm that this article content has no conﬂicts of

AFM11 (CD19xCD3), a T-cell recruiting molecule is in phase I

interest.

clinical development for indications such as NHL (non-Hodgkin

lymphoma), B-cell-CLL (chronic lymphocytic leukemia) and other

hematological malignancies [134]. AFM11 has 2 binding sites for Acknowledgements

CD3 and 2 for CD19, an antigen that is expressed from early B

cell development through differentiation into plasma cells. AFM11 The authors are grateful to Interdisciplinary Biotechnology Unit,

exhibits potent cytotoxic activity in vitro, with (EC50) in the low- Aligarh Muslim University, Aligarh for the facilities. Asim Azhar is

and subpicomolar range. T cells displayed a sevenfold increase in thankful to University Grant Commission for providing the fellow-

cytotoxic activity after the addition of CD19xCD3 TandAb [135]. ship in terms of UGC-DSKPDF. Ghulam Md Ashraf is grateful to King

In a NOD/SCID xenograft model, AFM11 induced dose-dependent Fahd Medical Research Center (KFMRC) and Deanship of Scientiﬁc

growth inhibition of Raji tumors in vivo, and radiolabeled TandAb Research (DSR), King Abdulaziz University (Jeddah, Saudi Arabia)

exhibited excellent localization to tumor but not to normal tissue. for the facilities.

In a human ex vivo B-cell-CLL study, AFM11 exhibited substantial

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