Lasso Grafting
Literature #1
2023/1/26
B4 寺⽥周平Contents• Introduction• MainØ LassoGraft Technology®
Ø Application of LG technology
ü Addbody®
ü Mirabody® and other grafted proteins• Summary

2023/1/25

2

Macrocyclic peptides
350

表1

3

医薬品群の比較
低分子医薬品

中分子医薬品

高分子医薬品形態

有機小分子

特殊環状ペプチド

抗体

分子量500 以下

1000〜3000

150,000 以上

標的特異性

低い

高い

きわめて高い

毒性・副作用

多い

少ない

少ない

経口投与

可能

一部可能

不可能

細胞膜透過性

高い

低い〜高い

なし

製造コスト

低い

低い〜普通

高い

免疫原性

低い

低い

普通〜高い

標的結合面積
PPI* 阻害

狭い

普通〜広い

広い

一部可能

可能

可能

生体内安定性

低い〜普通

普通〜高い

きわめて高い*PPI：タンパク質間相互作用．

üSmall molecules and macromolecules may have some problems
for pharmaceutical use.
üMacrocyclic peptides has stability in vivo by virtue of its cyclic
structure and noncanonical amino acid they composed of.
2023/1/25

Miura, T., et al. Seikagaku. 2021 93(3), 349-358.

The platforms for selecting peptide binding drug targets
Phage display•

In vivo translational synthesis
Small library size (~109)
Toxic peptides cannot be handled becauseof the use of E. coli.

OBOC (one-bead-one-compound) display
224•

Fernanda C. Bononi and Leonard G. Luyt

figure shows how the split synthesis
and mix procedure performed in between coupling steps in the synthesis of an OBOC
Chemicallibrary allows for the generation of beads that contain thousands of different compounds with the same length.
The number of possible amino acid sequences increases exponentiallySmalllibrary size(~107) with each step of the library synthesis
Fig. 1 The synthesis of OBOC peptide libraries using the split-mix methodology, where A–E are amino acids. The

The first OBOC library synthesized contained 19 naturalamino acids, excluding cysteine due to the potential of disulfide
bond formation, and the screening was performed by having
acceptor molecules that were coupled to an enzyme (alkaline phosphatase) or fluorescein and added in soluble form to the peptide
library. Positive beads were stained and were easily seen using a
low-power dissecting microscope, against a background of colorless, nonreactive beads. The positive beads were then removed foranalysis and the sequences determined by automated Edman
degradation.
OBOC libraries have the advantage of being able to incorpoacids, unnaturalaminoacids,223-237.
as well as non-peptide
rate
Bononi, F.
C.,D-amino
et al. Peptide
Libraries.
2015,
structures. This is a very useful tool for drug development, since
peptides containing unnatural amino acids are generally more resis-

Ø Simplicity of operation and the size of the library that can becreated are important.
2023/1/25

Zambrano-Mila, M. S. et al, Ther. Innov. Regul. Sci., 54(2), 308-317. (2020)

4

mRNA display

vitro selection, mRNA display, phage display, unnatural amino acids, ribosome display

on and directed evolution
ng of trillions of protein
a single experiment. In
king to alter binding or
n or peptide have a ranget their disposal to isolate
ures of variants. The goal
que advantages of mRNAor to most other directed

techniques have been
Here, we will
only brieﬂy
Procedure
Figure 1. In vitro selection by mRNA display. (a) Schematic of thefor comparison to mRNA
covalent mRNA−protein fusion. The puromycin linkage is shown byuish between
and library
1. screens
A DNA
is transcribed→ mRNA
modifiedwith puromycin →a diamond.
(b) An mRNA selection
for protein
binding. A DNA
ery variant be individuallylibrary
is transcribed, theresultingmRNA modiﬁed withfusion.
puromycin,
TranslatedtogeneratethemRNA−protein
and then translated to generate the mRNA−protein fusion (1). The
or quantitative (e.g., size)
fusions
undergo ananexperiment-speciﬁc
selection step (2) forligandd trait to 2.
survivalof theThe
fusions
undergoexperiment-specificselection
step.

binding or enzyme activity, in which the fusions with the desired
function are immobilized and undesired fusions are removed. Either
hnique predicatedon the transcribed
3. Reverse
into cDNA → Amplified via PCR → Next cycle
before, or sometimes after, the selection step, fusions are reversen a protein variant and its
transcribed into cDNA. The cDNA of selected fusions is ampliﬁed via
n vitro translation (Figure
PCR (3) in order to regenerate the DNA template library for the next
2023/1/25
Newton, M. S., et al. ACS synthetic biology, 9(2), 2019, 181-190.
ﬁed at its 3′-end withselection round.

5Flexible In vitro Translation (FIT) and genetic code reprogrammingüFlexizyme can incorporate non-canonical aminoacids into tRNA.
üThe reconstituted translation system assigns nonnormal amino acid-tRNA complexes to codons,allowing for the synthesis of non-natural peptides.
2023/1/25

Passioura, T., et al. Chem. Commun., 2017, 53(12), 1931-1940.

6

RaPID system

7

RaPID : Random non-standard Peptide Integrated Discovery

e.g.)Fig. 4 Flexible in vitro translation. The canonical codon table for bacterial translation is shown on the left (first base of the codon is shown on the left sideof the table, second base of the codon is indicated along the top and third base of the codon is indicated on the right side of the table). A reconstituted
translation reaction using this genetic code requires many individual components including the 20 proteinogenic amino acids and their cognate
AARSs as well as ribosomes, tRNA and other co-factors. Translation of an appropriate mRNA template in this reaction yields a peptide initiated by formylmethionine and comprised entirely of proteinogenic residues as shown bottom left. Removal of amino acids and AARSs from this reaction ‘‘vacates’’
the corresponding codons, and under these conditions translation of the same template will not occur due to lack of the requisite aminoacylated
tRNAs (middle panel). Addition of pre-aminoacylated tRNAs (right side) reprograms these amino acids (in this case, N-methylated Phe, Ser, Gly and Ala,
and a-N-chloroacetylated Trp) into the corresponding positions of the codon table, and under these conditions translation of the same mRNA templateleads to the synthesis of a peptide containing multiple non-canonical residues.

Random sequenceThis journal is © The Royal Society of Chemistry 2017

Chem. Commun., 2017, 53, 1931--1940 | 1935

ü RaPID system makes it possible to find out target-selective cyclic peptide.
2023/1/26

Passioura, T., et al. Chem. Commun., 2017, 53(12), 1931-1940.

nto SFTI-1
nists of p53
discovered

the target binding site. Another consideration is the effect of the
epitope on the structural and functional integrity of the scaffold.
Collectively, these considerations suggest that grafting onto regionsof the scaffold that display high flexibility or that have high evolutionary variability (for example, loop 6 of cyclotides) is preferred

Peptide grafting approachxt step is totional and/d product.
might be)lorotoxin16,
II50 and thexample, forserine proSFTI-1 andative ability
le above of
ntracellularchosen theral similare inhibitors
ntial therawere chosen
hose of theed peptides
y of natural
es in the defolds availng a greaterab

NATURE CHEMICAL BIOLOGY

8cEnhancedstability
Intracellulardelivery

Oral
activity

Epitope:

Scaffold: kalata B1

E V G W Y

Grafted peptide

Fig. 3 | Applications of molecular grafting. a, Molecular grafting ontoa cyclic peptide scaffold can be used to stabilize a biologically active
peptide sequence. In a recent study, the kalata B1 scaffold was used tostabilize peptide epitopes from myelin oligodendrocyte glycoprotein for thetreatment of multiple sclerosis28. b, Molecular grafting can also be used
to deliver peptides into cells. A peptide sequence from p53 grafted onto
MCoTI-I could inhibit a protein–protein interaction interface of intracellular
Hdm2 or HdmX (human double minute 2 or X proteins)37. c, Grafted
peptides can potentially be delivered via oral administration. Kalata B1
grafted with a bradykinin receptor antagonist had orally delivered activity inan animal model of disease40.

C G E T C V G G T C N T P G C T C E V G W Y C T R N G L P V
Loop 1Loop 2Loop 3Loop 4Loop 5Loop 6

In another studgrafted onto MC
sequence to MC
GFP fluorescenc
sion, and the gr
scriptional respo
Recent efforts to
ing and chemica
cyclic disulfidestability alreadytrating peptides
Cyclic disulfi
deliverable activ
toxin Vc1.1 isinjected, but nocyclized version
ple is the graftinsequence onto S
active peptides flinear antagonis
ity32,40. Similarly,
be orally active
multiple sclerosareas of axonal dcaution that weto induce oral aoral uptake are m
currently an are
now several man
and/or bioavaila

ü Peptide grafting can create new functional molecules thatcombine the functions of peptides and scaffold proteins.
ü Grafting synthetic de novo identified peptides into an
d be grafted
independently folded domain of a natural scaffold would often
etween two
result in the misfolding and inactivation of both entities.
The process oure.com/naturechemicalbiology

erica Inc., part of Springer
Nature. All rights reserved.
2023/1/25

421

Fig. 2 | Molecular grafting of epitopes onto scaffolds. In moleculargrafting, a peptide epitope (which has desired biological activities) is‘grafted’ onto a constrained peptide (which has desired biopharmaceuticalproperties), resulting in a new grafted peptide that has the desired
properties of both chemical inputs. The epitope can be chosen from a
range of sources, such as (i) naturally occurring peptides or fragments ofan interacting protein, (ii) chemical peptide libraries or (iii) recombinant
peptide libraries. The scaffold can be chosen from a panel of cyclic
disulfide-rich peptides, such as those shown in Fig. 1.

Grafted peptides are typically highly stable because they retain the We now describ
Wang, C.of
K.,theet al.
Nat. Chem.cyclic
Biol. 2018,
14(5),and
417-427.
stabilizing structural features
scaffold—abackbonepoints to be cona reinforcing network of disulfide bonds.
and those of othMacrocyclic peptides discovered by RaPID
Peptide name

Binding protein (target)

Original RaPID peptide

Sequence used for grafting

PB1m61

PlxnB1

Ac-wRPRVARWTGQIIYCWRPRVARWTGQIIY

PB1m6A92

PlxnB1

Ac-wRPYIERWTGRLIVC

WRPYIERWTGRLIV

PB1m71

PlxnB1

Ac-wNSNVLSWQTYSWYC(C)NSNVLSWQTYSWY(C)

aMD43

MET

Ac-yRQFNRRTHEVWNLDCYRQFNRRTHEVWNLD

aMD53

MET

Ac-yWYYAWDQTYKAFPCYWYYAWDQTYKAFP

By convention, RaPID peptides are cyclized via the N-chloroacetyl group of the initiator amino acid and thesulfhydryl group of the terminal Cys. The small letter indicates D-amino acids.
PlxnB1; transmembrane receptors for Semaphorins in neural tissues.
MET; hepatocyte growth factor receptor.

ü From previous studies, the above binding peptides were selected.

2023/1/25

1) Matsunaga, Y., et al. Cell Chem. Biol., 2016, 23(11), 1341-1350.
2) Bashiruddin, N. K., et al. PNAS. 2020, 117(49), 31070-31077.
3) Ito, K., et al. Nat. Commun. 2015, 6(1), 6373.

9

Contents• Introduction• MainØ LassoGraft Technology®
Ø Application of LG technology
ü Addbody®
ü Mirabody® and other grafted proteins• Summary

2023/1/25

10

LassoGraft Technology®

11(i) Dimer grafting on uteroglobin (UG)ü RaPID-derived pharmacophore sequences can be readily implanted
into surface-exposed loops on recombinant proteins (LassoGraft
Technology®)ü Binding affinity of the parental peptide was not lost by dimer
grafting.
2023/1/25

Mihara, E., et al. Nat. Commun. 2021, 12, 1543.

Loop grafting on various proteins

12(ii) loop grafting on proteins• β-sandwich fold

• all-α2023/1/26• β-hairpin• β-barrel• α/βMihara, E., et al. Nat. Commun. 2021, 12, 1543.

Loop grafting on various proteins
75

75

P

50

P

50

12.5% (NR)

(e)

P

50

10% (NR)

10% (NR)(f)
PlxnB1-PA

MET-PA

kDa
250
150
100
75

kDa
250
150
100
75

kDa
250

50

50

50

37

37

37

25

25

25

10% (NR)

kDa

M

150
100
75

10% (R)* (R)
10% (NR)

RBP-PA
PlxnB1 MET-MAP -MAPü Lasso-grafted scaffold proteins retains the binding
ability to the proteins on which lasso-grafting
peptide targets.
37

37* (NR)(h)

Ni-NTA

50

50

P

HSA-His2023/1/26

100
75

M: MET, P: PlxnB1, *: grafted protein(NR): non-reducing condition, (R): reducing condition

(g)

75

Pe
p
_in tide
ser natio mens
ite

IgG1-Fc
Protein A

kDa
100

13

PlxnB1
-Fc

MET
-PA

anti-PA

kDa

P

150

M(α)

*
M(β)

100
75

M
P

50
37Mihara, E., et al. Nat. Commun. 2021, 12, 1543.

25
20

Lasso-grafting on Fc domain

at the same graft site. To ﬁrst identify an exogeneous peptidegrafting site that would disrupt gene transduction but not prevent
14
viral packaging, we grafted a 12-residue peptide tag with noreceptor engagement (PA tag,25) into the Cap gene of AAV serc)2 (AAV2) at each of two loops (s1 and s2, see Fig. 2j and
otype
(a)
(b)
PlxnB1
METkine"c constants
Table S2) that analyte
had been reported to tolerate
a peptide insertion26.
-1s-1)
AAV2
kon (µM(PA_s1-Cap
koff (s-1) KD (nM)b)
and PA_s2As expected, both mutant AAV2s
PB1m6A9
PB1m7
aMD4
aMD5
AAV2
Cap
) PB1m6
were (freeefﬁciently
pep"de) produced
3.46 in HEK
0.012 cells3.5at a titercomparable
to thatof on
theUG)
wild-type(Fig. 6a),PB1m6
(gra#ed
0.091virus0.0041
45.8indicating
successful viral assembly and packaging. In contrast, the gene
PB1m6A9(freewaspep"de)7.59as desired
0.0021 by 0.28
transduction
activityaffected
one of the
PB1m6A9(gra#edon
UG)
0.17
0.00148.26
mutations; while the s1 mutant retained activity comparable to
PB1m6A9 (gra#ed on Fc)* 18.0
0.0004that of wild-type
AAV, the s2 mutant completely
lost0.023
its capacity
(c)
(d)
(e)
to infect (Fig.
6a).(freeThisdeleterious 0.038
effect fromthe s2275
modiﬁcation
EGFR
TrkB
α6β1 integrin
PB1m7
pep"de)0.010
on AAV2 PB1m7
infectivity
is likely
the disruptionof binding toN(1)(gra#edon UG) due to0.01580.051 3250
A6-2f
tkD5
IB8
24
AAV2-speciﬁc
sulfate proteoglycan
PB1m7receptor(gra#ed onheparan
Fc)*
0.896
0.055
61.4 and/orN(2)the pan-AAV endocytosis receptor AAVR27. By using thisC(2)PA_s2-CapAAV2 incapable of infecting cells as a capsid scaffold,
C(1)aMD4 (free pep"de)
1.4
0.003
2.4
we tested if
presentationofm6A9
peptidegraftedonaMD4 (gra#ed on UG)
0.044
0.004
91.6the capsid
could mediate
gene ondelivery
intocells.
aMD4 (gra#ed
Fc)*
0.156 PlxnB1-expressing
0.0007
4.5
Fig. 4 FACS analysis of peptide-grafted Fc protein binding to cell surface Unfortunately, when the capsid was solely composed of theaMD5AAV2(free, pep"de)
4.8
0.011 low 2.3
target receptors. HEK293T cells transiently expressing PlxnB1 (a), MET
the virus showedvery
titers (genm6A9_s2-Cap
ü
Peptide-graftedFc
proteinbindingaMD5 (gra#ed on UG)
0.088
0.019
211
(b), EGFR (c), TrkB (d), or α6β1 integrin (e) are incubated with eacherally less than 3% of the wild-type virus), suggesting that it is notto cellsurfacereceptors.
peptide-graftedFc (redhistogram)target
or control Fc(gray histogram), followed fully compatible with capsid assembly and/or
gene packaging.
(determinedusing SPR)We
Fig. 1 Conversion of RaPID-derived cyclic peptides into protein grafts. a Flow-chart of the lasso-graft method. Once a high afﬁnity cyclicby labeling with Alexa Fluor 488-labeled anti-human Fc.
therefore decided to employ a chimeric virus format, where only athe normal infectious ability of AAV by grafting a peptide with nocell surface receptor engagement into a loop in the Cap capsid
protein and then restore function to the AAV-peptide fusion

obtained against a target via the RaPID system in the discovery phase (left), the sequence information is used to formulate binding proteins iphase (right) by inserting the peptide either (i) in between the N- and C-termini of a dimeric protein or (ii) in the middle of an exposed loop oprotein. b Disulﬁde-stabilized homodimeric structure of uteroglobin(PDB ID: 2UTG). The dottedline shows the peptide-grafting site betw(a)(b) Avelumab(c)
control IgG
YW64.3 (anti-Nrp1)
OKT3(anti-CD3) peptides before
C-terminus of molecule 1 (cyan) and the N-terminus of molecule 2 (salmon).(anti-PD-L1)
c Kinetic binding constantsfor RaPID-derived
2023/1/26lasso-grafting. Binding kinetics of UG or Fc proteins lasso-grafted with either PlxnB1-binding peptides (PB1m6, PB1m6A9, and PB1m7) or M
Mihara, E., et al. Nat. Commun. 2021, 12, 1543.
peptides (aMD4 and aMD5) was determined for their respective target molecules using SPR (actual sensorgrams are shown in Fig. S1c–h). V

ü The RaPID peptides for many different targets can be readily grafted
into various protein scaffolds.

Generating multi-specific antibody by Lasso-Grafting

15

The gray histograms show(a) untransfected HEK293T cells(b)(c) the labeling of the same cells with the IgG control.

ü By grafting peptides into the Fc domain, multispecificity antibodies werecreated. <“Addbody®”>ü Using this method, Addbody-mediated heterotypic cell engagement wasachieved.
2023/1/26

Mihara, E., et al. Nat. Commun. 2021, 12, 1543.

Contents• Introduction• MainØ LassoGraft Technology®
Ø Application of LG technology
ü Addbody®
ü Mirabody® and other grafted proteins• Summary

2023/1/25

16

MET targeting therapeutics

17• A tyrosine kinase-type receptor• Dimerizes and phosphorylates its
intracellular domain upon activation by theligand.
• Met-HGF is centrally involved inmorphogenesis during development, wound
repair and organ homoeostasis⭕ rHGF has been shown to have therapeutic
efficiency.
Short half-life
Poor BBB permeability

2023/1/25

海津正賢, & ⾼⽊淳⼀. (2016). HGF-Met シグナル伝達の構造的基盤. ⽇本⾎栓⽌⾎学会誌, 27(1), 77-84.

Agonist activity of peptide grafted Fc by graft position

Articlehttps://doi.org/10.1038/s41551-022-00955-6

a mRNA display
C

C

C

S

S

C

C

Lasso grafting into loops of Fc

Met binders(RaPID system)

C

S

S

T1

T1~
B3

Hinge

T3
T2

aMD4

S

M2
M3

S

T1
T3

T2
T3

0
–10

–9

–8

–7

–6

100

M2

50

M3

0
–10

Concentration (log[M])

–9

–8

–7

M3

B3

B1

Phospho-Met (%)

Phospho-Met (%)

T1

50

Phospho-Met (%)

B3

aMD4dimer

B1
B2

B2

100

T2

M2

aMD5

b

–6

100

B3
50

B1
B2

0
–10

Concentration (log[M])

–9

–8

–7

–6

Concentration (log[M])

50

0
–10

–9

–8

–7

–6

Concentration (log[M])

100

50

M3
M2

0
–10

–9

–8

–7

–6

Concentration (log[M])g. 1 | Met agonists are generated by the insertion of Met-binding
2023/1/25
macrocyclic peptides
into the structural loops of Fc protein. a,harmacophore sequences of Met binders (aMD4 or aMD5; shown in red)

Phospho-Met (%)

aMD5dimer

Phospho-Met (%)

Phospho-Met (%)

c
100

18

ü B3 was the optimal site for aMD4
ü B1 was the optimal site for aMD5.

100

B1

B2

50

B3
0
–10

–9

–8

–7

–6

Concentration (log[M])(Fc(aMD4)) (b) or aMD5-grafted Fc (Fc(aMD5)) (c). EHMES-1 cells were treatedwith Fc variants (coloured) or dimer peptides (grey). Cellular Met activation wasquantified in situ with anti-phospho-Met (Tyr1234/1235) antibody. The results

Sakai, K., et al. Nat. Biomed. Eng. 2022, 1-13.

Peptide grafted Fc has a long blood half-life
Articlehttps://doi.org/10.1038/s41551-022-00955-6b–6

Fc(aMD4)

B2M
T3
M2

Streptavidin chip

180°
M3

Serum conc. (log[M])

WT

–7

d

Fc(aMD4)B3

–8

i.v.
mFcRn−/−
hFcRn+

–9

HGF

–11
–12

0

B3

3
B1
B2

2

Fc(aMD4)

Fc

–10

M

B1

c
i.v.

–8

M

B3

–7

Fc

B2

2

4

Time (h)68Serum conc. (log[M])FcRn

P = 0.043

T2
T3

T2

BiotinFcRn/B2M

P = 0.040

T1

T1

KD to FcRn/B2M(log[M])aFc (t1/2: 46.6 h)–7Fc(aMD4)B3 (t1/2: 49.4 h)–8Minimum conc.
for Met activation

–9

–10

0

100

200

300

Time (h)2023/1/26

4

BrdU+ (%)

46 h, collect liver

MD4)B3PBS

e Fc(aMD4)B3
f
g
BrdU
DAPI
Merge
ü Lasso-grafting
into
most
loops
did
not
affecttheaffinityof Fc to FcRn.
or PBS, s.c.
ü Fc(aMD4)B3 had half-life comparable to that of control Fc. P < 0.0001
liverü The serumHumanizedconcentrationof Fc(aMD4)B3 remained above 1 nM, the minimum
concentration BrdU,
for2 hactivating Met
3
2

1
FcRn:⾎漿中から細胞内に取り込まれたIgG抗体を⾎漿中に戻しリソソームでの分解を防ぐ、リサイクルの働きを持つ。
0

Sakai, K., et al. Nat. Biomed. Eng. 2022, 1-13.

19

Fc(aMD4)B3 acts as an agonist of HGF.
–12

0

2

4

6

–10

8

0

100

BrdU

DAPI

Merge

P < 0.0001
4

BrdU+
RNA-seq

Fc(aMD4)B3

BrdU, 2 h
46 h, collect liver

20g

BrdU+ (%)

Humanized liverf

PBS

Fc(aMD4)B3
or PBS, s.c.

300

Time (h)Time (h)

e

200

3
2

0

h

BrdU

1

PBS

thymidineFc(aMD4)B3

i

Fold enrichment
0

50

100

PBS

Mitotic DNA replication(GO:1902969)

Sterol biosynthetic process(GO:0016126)

Fc(aMD4)B3

Steroid biosynthetic processlog2change3.0
0(GO:0006694)

jNumber of genes
0

50

100

Organic substance metabolic process–3.0(GO:0071704)

Organic cyclic compound metabolic process(GO:1901360)

Cell cycle(GO:0007049)

group). For HGF, somesamplessynthesis
were below the limit
of detection.
e–j, In vivo
ü Fc(aMD4)B3 significantly increased per
replicativeDNAofhepatocytes.
activity of Fc(aMD4)B3 in mice with humanized liver (PXB-mice). e, Schematic.
→ Lasso-grafting generated a Met-activating
Fcsynthesis.
that f,isRepresentative
bioactivein vivo,
with af,g, Replicative DNA
images showing
BrdU staining
in the liver of Fc(aMD4)B3or PBS-treatedmice. Scale bar, 100 µm. Fc.
g, Percentage
markedly improved half-life that is comparable
to
that
of
unmodified
of BrdU+ cells in the liver. Results are presented as mean ± s.e.m. (n = 4 mice per

Fig. 4 | Lasso-grafting Fc yields Met agonists with extended half-life and invivo activity. a, Peptide-inserted loops (T1 to B3) in Fc (grey) are designed awayfrom the binding site to FcRn (orange) and β2-microglobulin (β2M, yellow)(PDB ID: 4N0U). b, KD values of Fc and Fc(aMD4) to FcRn/β2M determined bysurface plasmon resonance. Results are shown as mean ± s.e.m. (duplicatesof2023/1/26
3 concentrations of analytes; unpaired two-tailed t-test). c,d, Fc(aMD4)B3
showed a long serum half-life in mice. Wild-type C57BL/6 mice (c) or mFcRn−/−

Sakai,
K., et h,al.HeatNat.map
Biomed.
group; unpaired two-tailed t-test). h–j, Gene expression
profiles.
of Eng. 2022, 1-13.
DEGs between Fc(aMD4)B3-treated and PBS-treated PXB-mice livers (n = 4 miceArticle
MET agonist
with BBB penetrationsTfR(aMD4)cdTfR(aMD4)Rat
α-mouse TfR
Human
IgG FcFlag

aMD4ds at B3

Fc
(a
sT MD
f R 4)dT (aM
fR D4
(a )
M
D4
Fc
)
(a
sT MD
fR 4
dT (aM )
fR D
(a 4)
M
D4
)

b

Flag-Fc(aMD4)
Fc(aMD4)
Light chain
ReducingdTfR(aMD4)

1.0

0.36 ± 0.04
sTfR(aMD4)

0.5

030.8 ± 12.7
Fc(aMD4)
0

50

100

Concentration (nM)

kDa
245
180
140
100
75
60
50
40
35
30
25
20
15
10

Heavy chain

Relative binding to mTfR

Fc(aMD4)d
Relative phospho-Metahttps://doi.org/10.1038/s41551-022-00955-6

1.0

Fc(aMD4)
sTfR(aMD4)

2.1 ± 0.2
20.2 ± 3.40.50dTfR(aMD4)

39.2 ± 22.3

0

50

100

Concentration (nM)

Non-reducing

400

1.0

300

10

Brain

0.4

rum (nM)

0.8
10
TfR: トランスフェリン受容体。受容体介在性トランスサイトーシスによりBBBを介した脳内ドラッグデリバリーを⾏うことができる。
Sakai, K., et al. Nat. Biomed. Eng. 2022, 1-13.
24 h
0.6

rain (nM)2023/1/26

1.2ain)/(Serum)15

ose per gram brain

P < 0.0001
e
f
g
h
i
P < 0.0001
P = 0.0053
ü Via lasso-grafting,Fabaffinitywaslargelypreserved.
P<
0.0001P=
0.0002
P = 0.0001 P = 0.0011
P < 0.0001 P = 0.0001
P < 0.0001 P = 0.0095
i.v.
ü Via lasso-grafting,
Met activity was reduced. (due to the balky Fab)
WT
200

5

21

i.v.
WT
24 h
Brain
serum

Brain (nM)15

P = 0.0001 P = 0.0011

10

5

PB
Fc
S
(a
M
sT
D4
fR
)
(a
dT MD
4)
fR
(a
M
D4
)

0

j

PBS

g

P < 0.0001

hi

P < 0.0001 P = 0.0002

1.2

0.8
0.6
0.4
0.2
0

P = 0.0053
P < 0.0001 P = 0.0001

P < 0.0001 P = 0.0095

400

1.0

Fc(aMD4)

22

100

300
200
100

10

5

0

0

PB
Fc
(a S
sT MD
4)
fR
(a
dT MD
fR
4)
(a
M
D4
)

P < 0.0001

Serum (nM)f50

Concentration (nM)

Fc
(a
sT MD
4)
fR
(a
dT MD
4)
fR
(a
M
D4
)

e

0

Non-reducing% injected dose per gram brain

Reducing

0% (Brain)/(Serum)

Light chain

Relat

MET agonist with BBB penetrationsTfR(aMD4)

Fc
(a
sT MD
fR
4)
(a
M
dT
D4
fR
)
(a
M
D4
)

35
30
25
20
15
10

Fc(aMD4)

dTfR(aMD4)

Human IgG FcüsTfR(aMD4) showed the highest percentage injected dose per gram brain,
brain-to-serum ratio, as well as the highest brain concentration .
2023/1/26

Sakai, K., et al. Nat. Biomed. Eng. 2022, 1-13.

jsTfR(aMD4)

Fc(asT M D
fR
4
(a
M
dT
D4
fR
(a
M
D4

PB
Fc
(a
sT MD
4
fR
(a
dT MD
fR
4
(a
M
D4

Fc
(a
sT M D
4)
fR
(a
dT MD
4)
fR
(a
M
D4
)

Fc(aMD4)23

dTfR(aMD4)

Human IgG Fc
NeuN

Human IgG Fc

PBS

%

PB
Fc
S
(a
M
sT
D4
fR
)
(a
M
dT
fR D4)(aM
D4)

IHC using Fc(aMD4), sTfR(aMD4), dTfR(aMD4)

Fig. 5 | Lasso-grafting Fc of anti-TfR antibodies yields Met agonists with
BBB penetration. a, Schematic representations of Fc(aMD4), dTfR(aMD4) andsTfR(aMD4). All variants were grafted with aMD4ds at the B3 loop of human
IgG Fc. Rat anti-mouse transferrin receptor Fab was fused with Fc(aMD4). b,
SDS–PAGE of purified variants. c, Lasso-grafting of aMD4 on anti-TfR antibodies
preserved affinity to TfR. The apparent KD values (in nM) of Fc(aMD4),
sTfR(aMD4) and dTfR(aMD4) to mouse TfR (mTfR) were determined by ELISA
and are presented as mean ± s.d. (n = 3 independent experiments). d, Cellular Met
activation by aMD4-grafted anti-TfR antibodies. EHMES-1 cells were treated with
2023/1/26
Fc(aMD4),
sTfR(aMD4) or dTfR(aMD4). Cellular Met activation was quantified
in situ with anti-phospho-Met (Tyr1234/1235) antibody and presented ase–j, BBB penetration of aMD4-grafted anti-TfR antibodies. Schematic (e),brain concentration (f), percentage injected dose per gram of brain (g), serum
concentration (h) and brain-to-serum ratio (i) at 24 h after a single injection ofFc(aMD4), sTfR(aMD4) or dTfR(aMD4) at equimolar amount per kg (12, 20 and26.5 mg kg−1, respectively) via the tail vein. Results are shown as mean ± s.e.m.
(n = 4 mice per group; unpaired two-tailed t-test). j, Representative imagesof immunohistochemical staining of brain sections from mice at 24 h after asingle injection of Fc(aMD4), sTfR(aMD4) or dTfR(aMD4) via the tail vein. Notethe broad staining for sTfR(aMD4) in the brain parenchyma and localization
Sakai,StainingK., et al. Nat.
Biomed. Eng. 2022,
1-13.
around NeuN-positive neuronal cell bodies.
for dTfR(aMD4)
was moreprominent in endothelial cells than in neuronal cell bodies. Scale bars, 50 µm.

üsTfR(aMD4) showed prominent parenchymal staining co-localized with
NeuN, GFAP and Iba1, indicating its distribution to neuron, astrocytes andmicroglia, respectively.

Short summary‘Designer HGF’
Fc(aMD4)B3
Discovery

Receptor
activation

24
Naturalcytokine

Met
Lasso-grafted
cytokine mimic

Concentration agonist (M)Host protein

Met
Hinge

T1

T3

Hepatic Met
activation

Lasso-grafting into Fc

T2
RaPID selection(>1012 diversity)

Anti-TfR(aMD4)
B2
B3

Identification ofsurrogate Met agonists(‘macrocyclic guest peptide’)M2

B1

Identification ofsuitable Fc loop(‘host-protein scaffold’)

Receptor
activation

M3

Potent Met-agonist–Fc constructwith enhanced PK

Naturalcytokine
Lasso-grafted
cytokine mimic

Concentration agonist (M)BBB penetration
Lasso-grafting into anti-TfR

Met-agonist–anti-TfR construct withenhanced PK and BBB penetrationüBinding to FcRn resulted in a longer half-life.
üBBB permeability was improved by incorporating TfR.
→Lasso grafting on Fc fragments enhanced pharmacokinetics.
2023/1/25

Kapurniotu, A., et al. Nat. Biomed. Eng. 2022, 1-3.

Contents• Introduction• MainØ LassoGraft Technology®
Ø Application of LG technology
ü Addbody®
ü Mirabody® and other grafted proteins• Summary

2023/1/25

25

Antibody w/o Fab; Mirabody® acts as a receptor agonist 26
Fc(m7)B2
agonistFc(m6A9)B3
antagonistüMirabody® acted as the agonist toward PlxnB1.
üm7-Fc activates receptor dimerization and subsequent signaling.
üm6A9-Fc acted as antagonist toward PlxnB1 and inhibited Sema4D.
2020). Therefore, it is expected that the binding mechanism the PlxnB1 sema domain b-propeller (Figures 5A and 5B). Eight
üIt
could
potentiallybebiotherapeutics
due
toto thosethein m6,
longwould beessentiallythe sameas that of the original
peptideof the 14 residues in m6A9 wereidentical
and theplasma half-life.
Figure 3. Activation of PxlnB1 signaling by Fc(m7)(A) Binding of various Fc(m7) proteins to cell-surface PlxnB1. FCM histograms and MFI values are shown as in Figure 2B.
(B and C) Cell collapse response upon addition of Fc(m7). Fc(m7) proteins with varying grafting sites, excluding non-binding T3 graft, were added to PlxnB1-expressing cells at a constant concentration (100 nM), and the cellular indices were recorded after normalizing the signal at T = 0 h (B). For the strongest agonist,Fc(m7)B2, concentration dependency was evaluated and is shown as overlaid cell index curves (C, left) and the plots of the maximum collapse response (C, right).
The EC50 value is shown as mean ± SD derived from three independent experiments using the curve fitting by RTCA software.

m6. In fact, the conformation of the PlxnB1-bound m6A9 wasvery similar to that of m6 determined previously (Matsunaga
2023/1/25
et al., 2016), forming an anti-parallel b-sheet like a long tongue

side-chain conformation of Arg7 and Trp8, the two most important residues for PlxnB1 binding in m6, was completely
Sugano-Nakamura.
et al. Nat. Commun. 2021, 12, 1543.
conserved (Figures 5A and 5B). Curiously, theN aminoacid

Lasso-grafting on AAV capsid

27

AAV (adeno-associated virus)• Gene-delivery vehicles
NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-21875-0• Broad tropism (Binds to(a)receptors expressed on the
WT-CapPA_s1-Cap
surface of many cells)
Fluorescence• Capsid engineering has been
(AcGFP)ONS | https://doi.org/10.1038/s41467-021-21875-0
ARTICLE
used to impart tissue specificity.
AAV2

3.2 x 107

Fluorescence(AcGFP)

AAV2 Cap mutation

AAV1 Cap mutation
WT

PA_s2

PA/aMD4_s2

AAV1 Cap mutation(d) PA_s2(e)üPA_s2
By grafting
AAV acquired cell-specificlocalization.
PA/m6A9_s2 on S2 site, lasso-grafted
WT
PA/aMD4_s2
2023/1/26

CHO

4.0 E+05

pe

WT

(c)

(c)

cell type

2.5 x 107

3.2 x 107

PA/m6A9_s2

Expi293

1.7 x 107

PA_s2

PlxnB1-Expi293

titer (vg/µl)

WT

PA_s1-CapAAV2 PA_s2-CapAAV2

PA_s2-CapAAV2

2.5 x 107

AAV2 Cap mutation

cell type

WT-CapAAV2

AAV2

CHO

(b)

(a)

1.7 x 107s1
ARTICL

MET-CHOtiter (vg/µl)s2

Expi293

PlxnB1-Expi293
MOI = 12,500

2.0 E+06

CHO
MET-CHO
Mihara, E., et al. Nat. Commun. 2021, 12, 1543.
MOI = 12,500

PA/aMD4_s2

Luminescence (RLU)

MOI = 12,500

3.0 E+05

2.0 E+06

PlxnB1-Expi293

MOI = 25,000
MOI = 50,000

2.0 E+05

1.0 E+05

0.0 E+00(f)
2.5 E+06

CHO

MOI = 12,500

1.5 E+06

2.5 E+06

MET-CHO

MOI = 25,000
MOI = 50,000

1.0 E+06

0.5 E+06

0.0 E+00

28(g)

PlxnB1-Expi293
203%

MOI = 1,000

2.0 E+06

MOI = 5,000

1.5 E+06
100%
1.0 E+06

0.5 E+06

MET-CHO

3.0 E+06

MET-CHO

3.0 E+06

220%

fraction of capsid subunits are substituted with m6A9_s2CapAAV2. By co-transfecting cells with plasmids coding for
PA_s2-CapAAV2 and m6A9_s2-CapAAV2 at a DNA ratio of 9:1, a597%
chimeric virus incorporating a fraction of m6A9_s2-CapAAV2
subunits per capsid was prepared. The resultant chimeric virus
successfully transduced the AcGFP gene into Expi293F cells that597%
MOI = 1,000
MOI = 5,000

2.0 E+06

1.0 E+06
100%

100%

0.0 E+00(g)

PlxnB1-Expi293

0.5 E+06

0.0 E+00

Luminescence (RLU)(f)

Luminescence (RLU)

Expi293

1.0 E+05

0.0 E+00(e)
4.0 E+05

Lumi

PA_s2

CHO

cell type(d)

Luminescence (RLU)

WT

MET-CHO

Expi293

PA/m6A9_s2

PlxnB1-Expi293

cell type

PA_s2

Lumi

Lasso-grafting on AAV capsid
WT

704%

100%
0.0 E+00

stably expressed human PlxnB1 (Fig. 6b), while no detectable
AcGFP expression was observed with the parent cell line after
incubation with the chimeric virus. We next expanded this analysis to another AAV serotype (AAV1) using MET-binding
aMD4 peptide as the targeting moiety. Unmodiﬁed AAV1 capsid
was capable of transducing both Chinese hamster ovary (CHO)

MOI = 1,000

2.0 E+06

MOI = 5,000

1.5 E+06

100%

1.0 E+06

0.5 E+06

0.0 E+00

220%

MOI = 1,000

Luminescence (RLU)

Luminescence (RLU)

ü Only viruses expressing a peptide that binds specifically to the receptor
could perform gene transfer to cells expressing the receptor protein.
ü Gene transfer efficiency of mutant viruses is higher than that of wild-typeviruses.
203%

MOI = 5,000

2.0 E+06

NATURE COMMUNICATIONS | (2021)12:1543 | https://doi.org/10.1038/s41467-021-21875-0 | www.nature.com/naturecommunications

1.0 E+06

100%

100%

704%

100%

0.0 E+00

MOI:with
Multiplicity
infectionの略で細胞１個あたりのウイルスの数
2023/1/26
expressed human PlxnB1 (Fig. 6b), while no detectablefraction of capsid
subunits are substituted
m6A9_s2- ofstably
CapAAV2. By co-transfecting cells with plasmids coding for AcGFP expression was observed with the parent cell line after
PA_s2-CapAAV2 and m6A9_s2-CapAAV2 at a DNA ratio of 9:1, a incubation with the chimeric virus. We next expanded this ana-

Mihara, E., et al. Nat. Commun. 2021, 12, 1543.

7

Contents• Introduction• MainØ LassoGraft Technology®
Ø Application of LG technology
ü Addbody®
ü Mirabody® and other grafted proteins• Summary

2023/1/25

29

Summary
RaPID systemüRapid screening of macrocyclic peptides
Lasso-GraftingüFunctionalization of macrocyclic peptides in vivoüFunctionalization of host proteinsüGeneration of multispecific antibodies (Addbody®)üImproved bioavailability and BBB permeabilityüGeneration of receptor agonist/antagonist (Mirabody®)

2023/1/25

30