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We report a method to generate bifunctional antibodies by grafting full-length proteins into constant region loops of a full-length antibody or an antigen-binding fragment (Fab). The fusion proteins retain the antigen binding activity of the parent antibody but have an additional activity associated with the protein insert. The engineered antibodies have excellent in-vitro activity, physiochemical properties, and stability. Among these, a Her2×CD3 bispecific antibody (BsAb) was constructed by inserting an anti-Her2 ScFv into an anti-CD3 Fab. This bispecific antibody efficiently induces targeted cell lysis in the presence of effector cells at as low as sub-picomolar concentrations in vitro. Moreover, the Her2×CD3 BsAb shows potent in-vivo antitumor activity in mouse Her2++ and Her2+ xenograft models. These results demonstrate that insertion of a full-length protein into non-CDR loops of antibodies provides a feasible approach to generate multi-functional antibodies for therapeutic applications.

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Article Engineering Bifunctional Antibodies with

Constant Region Fusion Architectures

Juanjuan Du, Yu Cao, Yan Liu, Ying Wang, yong Zhang, Guangsen Fu, Yuhan

Zhang, Lucy Lu, Xiaozhou Luo, Chan Hyuk Kim, Peter G Schultz, and Feng Wang

J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b09641 • Publication Date (Web): 29 Nov 2017

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Engineering Bifunctional Antibodies with Constant Region Fusion

Architectures

Juanjuan Du†‡, Yu Cao‡, Yan Liu†, Ying Wang†, Yong Zhang†, Guangsen Fu†, Yuhan Zhang†,

Lucy Lu†, Xiaozhou Luo‡, Chan Hyuk Kim†, Peter G. Schultz†‡, Feng Wang†

† California Institute for Biomedical Research, 11119 N. Torrey Pines Road, La Jolla, CA 92037, United States

‡ Department of Chemistry, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, United

States

KEYWORDS Antibody fusion, protein engineering, bispecific antibodies, cancer immunotherapy

ABSTRACT: We report a method to generate bifunctional antibodies by grafting full-length proteins into constant region

loops of a full-length antibody or an antigen-binding fragment (Fab). The fusion proteins retain the antigen binding activ-

ity of the parent antibody but have an additional activity associated with the protein insert. The engineered antibodies

have excellent in-vitro activity, physiochemical properties, and stability. Among these, a Her2×CD3 bispecific antibody

(BsAb) was constructed by inserting an anti-Her2 ScFv into an anti-CD3 Fab. This bispecific antibody efficiently induces

targeted cell lysis in the presence of effector cells at as low as sub-picomolar concentrations in vitro. Moreover, the Her2

×CD3 BsAb shows potent in-vivo antitumor activity in mouse Her2

and Her2

xenograft models. These results demon-

strate that insertion of a full-length protein into non-CDR loops of antibodies provides a feasible approach to generate

multi-functional antibodies for therapeutic applications.

Introduction

Monoclonal antibodies are increasingly important ther-

apeutics due to their high affinity and specificity, long

circulating half-life, and low immunogenicity.

1-3

A new

generation of engineered antibodies, including bispecific

antibodies (BsAb) and antibody conjugates, have given

rise to antibodies with dual functions.

1-6

Since the pio-

neering work of Dr. Nisonoff,

a plethora of methods to

constructs bispecific antibodies have emerged in the past

fifty years, including Ig-fusion,

quadromas,

diabodies,

tandem ScFvs,

11,12

DART,

knobs-into-holes,

DVD-Ig,

etc.

16,17

With over 30 different bifunctional antibodies in

clinical trial, it has become increasingly evident that each

format has its own limitation. The BsAb format is best

chosen to match the specific mechanism of action. Ex-

panding the format arsenal thus facilitates the BsAb de-

velopment. Therefore, exploring more and novel BsAb

formats remains on the cusp of coming years.

Previously, we developed a strategy to generate anti-

body agonists and antagonists by fusing biologically ac-

tive proteins and peptides into antibody hypervariable

loops based on the X-ray crystal structure of the bovine

antibody BLV1H12 which has an ultralong heavy chain

complementarity-determining region 3 (CDR3H). The

hypervariable loop folds into a novel structural motif,

consisting of a solvent-exposed antiparallel β-strand stalk

which terminates in a disulfide-crosslinked knob domain

to afford a unique "stalk-knob" structure. On the basis of

this novel structure, we have grafted a number of cyto-

kines, growth factors, and conformationally restricted

peptides into the CDR loops of bovine and human immu-

noglobulins to generate functional antibody-CDR fu-

sions.

19-27

The immunoglobulin scaffold affords long se-

rum half-life and ease of recombinant protein production,

while the fused polypeptide endows the antibody with

new agonist, antagonist or inhibitory activities.

Figure 1. The topologies of CDR3 fusion in variable region

and non-CDR loop fusion in constant region. Diagrams show

the protein insertion sites and the strand nomenclature for

the β-sheets of IgG variable domains and constant domains.

Similar to the antibody variable domain, the constant

domains also have the characteristic immunoglobulin fold

Constant Domain

C''

CDR1

CDR2

Variable Domain

Loop

Disulfide Bond

β Strands Consist-

ing Two β Sheets

CDR3

Linker

Insert

Protein

Insert

Protein

Insert Protein

Linker

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(Figure 1).

This fold consists of two β sheets, formed

from antiparallel β strands that surround a central hydro-

phobic core. Based on the structural similarities between

the antibody variable and constant domains, we reasoned

that our CDR loop fusion strategy could be extended to

the non-CDR loops of the latter domain. In contrast to a

typical CDR loop fusion, constant region loop fusions are

expected to retain the binding affinity and specificity of

the parent scaffold, resulting in a bifunctional antibody

with the additional activity of the grafted polypeptide. In

this work, we show that non-CDR loops are able to ac-

commodate a variety of biologically active protein inserts.

Results

Full-length erythropoietin can be grafted in the

non-CDR loops of an anti-Her2 antibody. We initially

chose to fuse human erythropoietin (EPO) into an anti-

Her2 antibody (Herceptin) to explore different non-CDR

loops as fusion sites (Figure 2a). Previously, we have fused

EPO into the CDR3H loop of Herceptin to generate a sta-

ble antibody fusion protein (EPO-Herceptin-CDR3H).

The fusion protein retained the biological activity of EPO

and gained a long serum half-life.

22,27

Fusing EPO into

non-CDR loops in the constant domain of Herceptin al-

lows a direct comparison of the ability of variable and

constant domains to accommodate loop fusions. To avoid

interference with Fc receptor binding, we grafted EPO

into loops between the β strands D and E either in the

CH1 (replacing S182 and G183 at the splice site) or CL (re-

placing K169 at the splice site) regions (Figure 2b). To

spatially separate the EPO insert and Herceptin back-

bone, an anti-parallel coiled-coil "stalk" (14 amino acids in

each chain) was used as a rigid linker to connect EPO and

Herceptin (Figure 2b). GGSG and GGGGS adapters were

placed at each end of the coiled-coil sequences to afford

flexibility (Table S1). A similar linker strategy was previ-

ously used successfully to fuse EPO, Granulocyte-colony

stimulating factor (GCSF) and Exendin-4 into the CDR

loops of human and bovine antibodies.

1-3,22-25,29

The dis-

tance between the N- and C-termini of EPO (~15.8 Å,

PDB# 1BUY) is close to the axial distance between the two

coiled-coil chains in the stalk (~11 Å, Figure S1). Thus, fu-

sion of the N- and C-termini of EPO with the coiled-

coil"stalk"should not interfere with folding of either the

antibody or EPO. Moreover, because the receptor-binding

surface of EPO is on the opposite face to the fusion site,

the fusion protein should retain EPO receptor (EpoR)

binding. The two fusion constructs (hereafter referred to

as EPO-Herceptin-CL and EPO-Herceptin-CH1) were ex-

pressed in Free-Style HEK293 cells by transient transfec-

tion and purified using protein G chromatography. The

unoptimized expression yields of EPO-Herceptin-CL and

EPO-Herceptin-CH1 after purification are 13 mg/L and 5

mg/L, respectively.

Figure 2. Design, generation, and characterization of EPO-Herceptin constant domain fusions. (a) Model of the EPO-Herceptin

constant domain fusion. (b) Map of the key elements of EPO-Herceptin fusions: numbers indicate the fusion sites in the Kabat

numbering scheme. (c) Dose-dependent TF-1 proliferation stimulated by EPO-Herceptin-CL, EPO-Herceptin-CH1 and EPO-

Herceptin-CDR3H (positive control). (d) Binding of EPO-Herceptin-CL, EPO-Herceptin-CH1 and Herceptin (positive control)

against immobilized Her2-Fc antigen measured by ELISA.

The proteins were then analyzed with SDS-PAGE (Fig-

ure S2). Under non-reducing conditions, Herceptin has an

apparent molecular weight of ~160 kDa, higher than the

calculated mass (145 kDa) due to glycosylation. EPO-

Herceptin-CH1 (Lane 2) and EPO-Herceptin-CL (Lane 3)

migrate at ~200 kDa, higher than the theoretical mass of

189 kDa, again due to glycosylation. Under reducing con-

ditions, the light chain of EPO-Herceptin-CL migrates at

~55 kDa, higher than the theoretical molecular weight of

45.6 kDa due to glycosylation. The heavy chain of EPO-

Herceptin-CL migrates at ~50 kDa, consistent with the

expected molecular weight of 49.1 kDa. Similarly, EPO-

Herceptin-CH1 shows a heavy chain of ~80 kDa and a

light chain of ~ 23 kDa, consistent with the theoretical

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molecular weights of 71.3 kDa plus glycans and 23.5 kDa,

respectively. In addition, after treatment with peptide-N-

glycosidase to remove N-glycosylation and dithiothreitol

(DTT), the molecular weights of the heavy chains and

light chains of Herceptin, EPO-Herceptin-CL and EPO-

Herceptin-CH1 were measured by mass spectrometry. As

shown in Figure S3 and Figure S4, the mass spectrum of

EPO-Herceptin-CH1 heavy chain shows multiple peaks

with two major peaks of 71138 Da (22020 Da higher than

the Herceptin chain, consistent with the expected molec-

ular weight increase of 22016 Da) and 72086 Da (multiple

peaks, due to O-glycosylation). The molecular weights of

the light chains of Herceptin and EPO-Herceptin-CH1 are

the same (23968 Da) and consistent with the theoretical

mass (23972 Da). Similarly, compared with Herceptin

(Figure S3), EPO-Herceptin-CL shows the same heavy

chain molecular weight (Figure S5), and a molecular

weight gain of 22185 Da (non-glycosylated form, theoreti-

cal molecular increase: 22184 Da) in light chain, indicating

one EPO is fused in each light chain. Gel filtration anal-

yses in PBS (pH 7.4) indicated that EPO-Herceptin-CL

and EPO-Herceptin-CH1 both have apparent molecular

weights of ~200 kDa, as expected. Additionally, aggrega-

tion was determined by size exclusion chromatography,

in which EPO-Herceptin-CL (8.2 mg/mL) or EPO-

Herceptin-CH1 (10.5 mg/mL) was loaded. Integrated UV

absorbance peaks at 280 nm indicate that less than 5%

protein forms aggregation in both samples from protein G

purification (Figure S6).

EPO-Herceptin non-CDR loop fusions retain the

activities of both the insert and the scaffold. We next

examined the activity of EPO-Herceptin-CL and EPO-

Herceptin-CH1 using an EPO-dependent TF-1 prolifera-

tion assay. EPO-Herceptin CDR3H loop fusion (EPO-

Herceptin-CDR3H) is used as a positive control to com-

pare non-CDR fusion and CDR fusion.

1-6,22,27

TF-1 cells are

human bone marrow erythroblasts, which have a strong

growth dependency on EPO. Both EPO-Herceptin-CL and

EPO-Herceptin-CH1 stimulate TF-1 cell proliferation in a

dose-dependent manner (Figure 2c). The EC

is 0.16±0.01

nM for EPO-Herceptin-CL, 0.30±0.02 nM for EPO-

Herceptin-CH1 and 0.21±0.02 nM for EPO-Herceptin-

CDR3H, similar to the EC50 of recombinant EPO

(0.13±0.02 nM). These observations indicate that the fu-

sion of EPO into non-CDR loops of constant domains

does not significantly affect EPO activity, similar to fusion

into CDR3H loop.

18,22

To test whether non-CDR loop fusion affects the anti-

gen binding affinity of the antibody variable domain, we

carried out an ELISA against immobilized Her2 extracel-

lular domain fused with Fc (Her2-Fc) (Figure 2d). Both

EPO-Herceptin-CH1 and EPO-Herceptin-CL bind to

Her2-Fc in a dose-dependent manner, with EC

2.4±0.2 nM and 1.2±0.1 nM, respectively, which are com-

parable to that of Herceptin (1.9±0.1 nM). In addition,

flow cytometry analysis shows that both EPO-Herceptin-

CL and EPO-Herceptin-CH1 bind to Her2+ SK-BR-3 cells

with similar affinities (EC

= 7.5±4.3 nM and 11.0±2.3 nM,

respectively) to that of the parental antibody Herceptin

(EC

= 15.3±4.0 nM) (Figure S7). These results demon-

strate that the antibody binding affinity is not significant-

ly affected by the non-CDR loop fusion. Collectively, these

observations demonstrate grafting of a functional protein

in non-CDR loops in constant region can generate fusion

proteins retaining both the biological activity of the insert

and the antigen-binding affinity of the scaffold antibody.

Her2×CD3 bispecific antibodies can be generated

by fusing an anti-Her2 ScFv into the non-CDR loops

of an anti-CD3 antibody. Next we determined whether

an antibody single-chain variable fragment (ScFv) could

be grafted into the loops of constant domains. This strat-

egy would enable us to generate BsAbs targeting cancer

cells using for example, an antibody fragment specific for

tumor associated antigens, and a parent antibody scaffold

specific for CD3 on T cells. As shown in Figure 3a, a Her-

ceptin ScFv (hereafter referred to as Her2ScFv) was in-

serted into the loop connecting β strands D and E (to re-

place K169) in the CL domain of a humanized anti-CD3

antibody (SP34). We opted to fuse Her2ScFv into SP34-

Fab instead of fusing SP34 ScFv into Herceptin-Fab, be-

cause the properties of Her2ScFv are well documented.

19-

27,30,31

We chose the CL domain as the insertion site due to

higher expression yield of CL domain fusion in the case of

EPO-Herceptin fusion. In contrast to EPO, whose N- and

C-termini are located in close proximity to each other, the

N- and C-termini of Her2ScFv are separated by approxi-

mately 34 Å (Figure S8). To ensure a correct folding of the

fusion protein, a long flexible linker (GGGGS)3 was used

between the stalk and the N-terminus of the Her2ScFv

(Figure 3a). Additionally, it was previously demonstrated

that an extra disulfide bond at the termini or in the mid-

dle of coiled coils increases the stability of synthetic

coiled-coil structures.

28,32-34

Therefore, in our design, a

disulfide bond was introduced at the site where the

coiled-coil stalk connects to SP34 CL domain to enhance

stability (Table S1). The Her2×CD3 BsAb (referred to as

Her2ScFv-SP34-CL) was expressed in HEK293 FreeStyle

system by co-transfection with plasmids encoding the

fused light chain and the heavy chain (Fab) of SP34. After

purification with protein G and size exclusion chromatog-

raphy, we obtained Her2ScFv-SP34-CL with a yield of 12

mg/L. Gel filtration analysis of Her2ScFv-SP34-CL (2

mg/ml) in PBS showed that around 83% species purified

from protein G chromatography corresponds to the mon-

omer (Figure S9). The isolated fraction was then re-

analyzed by analytical size-exclusion chromatography,

which showed a single peak with molecular weight corre-

sponding to the monomeric Her2ScFv-SP34-CL (Figure

S9). This indicates that the purified monomer is stable in

solution without formation of observable dimers or oli-

gomers.

Figure S10 shows the SDS-PAGE of SP34 Fab (Lane 1)

and Her2ScFv-SP34-CL (Lane 2). Compared with SP34,

which migrates at ~45 kDa under non-reducing condition,

Her2ScFv-SP34-CL has an apparent molecular weight of

~75 kDa on SDS-PAGE. The 30 kDa molecular weight in-

crease is consistent with the molecular weight of the ScFv

insert (29 kDa). Under reducing condition, the heavy

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chains of SP34 Fab and Her2ScFv-SP34-CL migrate at the

same position (~23 kDa). The light chain of Her2ScFv-

SP34-CL migrates at ~54 kDa, approximately ~30 kDa

higher than the light chain of SP34 Fab. Additionally, af-

ter reducing with DTT, two peaks with molecular weights

of 24528 Da and 54252 Da were observed in the mass

spectra, consistent with the theoretical masses of 24532

Da (heavy chain) and 54277 Da (light chain) (Figure S11).

It is worth noting that ScFv fusion proteins, including

the Bi-specific T-cell engager (BiTE) developed by Mi-

cromet/Amgen, tend to form non-specific oligo-

mers/aggregations. A large portion of these oligomers are

in equilibrium with monomers in solution and therefore

difficult to separate, which cause serious manufacture and

toxicity issues.

22,27,35

Dynamic light scattering (DLS) was

used to evaluate the aggregation in concentrated

Her2ScFv-SP34-CL. As shown in Figure S12, a single frac-

tion with hydrodynamic radius of ~10 nm was observed at

10 mg/ml protein concentration, indicating that no aggre-

gation was formed. To determine the thermostability of

Her2ScFv-SP34-CL, the melting temperature (T

) was

measured using the Protein Thermal Shift™ Dye Kit

(ThermoFisher Scientific). The melting curve indicates

two T

s at 70 and 79 °C, likely corresponding to the un-

folding of ScFv and Fab domains, respectively (Figure

S13).

Her2ScFv-SP34-CL fusion protein retains the bind-

ing affinities of the anti-Her2 ScFv insert and the an-

ti-CD3 Fab fragment. The binding affinity of Her2ScFv-

SP34-CL to CD3 was measured by flow cytometry on

CD3+ Jurkat cells (Figure 3b). SP34 Fab was used as a pos-

itive control. Similar to SP34 Fab, Her2ScFv-SP34-CL

binds to Jurkat cells in a dose-dependent manner.

Her2ScFv-SP34-CL has an EC

of 8.7±1.0 nM, slightly

higher than the EC

of SP34 Fab (3.8±0.4 nM). The bind-

ing affinity of Her2ScFv-SP34-CL to Her2 was next meas-

ured by ELISA on immobilized Her2-Fc (Figure 3c). Com-

pared with Herceptin Fab (EC

= 0.9±0.1 nM), Her2ScFv-

SP34-CL binds to Her2-Fc with an EC

of 6.3±0.7 nM.

ScFv fragments reportedly have lower binding affinities

than the corresponding Fab. Nevertheless, the EC

s to

CD3 and Her2 still remain in the nanomolar range.

Figure 3. Design, generation, and characterization of the

Her2×CD3 bispecific antibody (a) Key elements of Her2ScFv-

SP34-CL fusion proteins. Numbers indicate the insertion site

in the CL domain (Kabat numbering scheme). (b) The bind-

ing of SP34 Fab and Her2ScFv-SP34-CL to CD3

Jurkat cells

determined by flow cytometry. (c) The binding of Herceptin

and Her2ScFv-SP34-CL to immobilized Her2-Fc antigen by

ELISA.

Her2ScFv-SP34-CL can effectively recruit CD3

cells

to target Her2

cancer cells in vitro. Next we deter-

mined whether Her2ScFv-SP34-CL could mediate the

formation of an immunological synapse between Her2

target cells and CD3

T cells. To visualize the association

of Her2

cells and CD3

cells mediated by Her2ScFv-SP34-

CL, live Her2

SK-BR-3 cells and CD3

Jurkat cells were

stained with calcein AM and cell tracker Orange, respec-

tively. After co-culturing with or without 100 nM

Her2ScFv-SP34-CL for 12 hours, cells were extensively

washed to remove the unbound Jurkat cells. The cells

treated with Her2ScFv-SP34-CL showed significantly

more Jurkat cells bound to the SK-BR-3 cells than that in

the absence of BsAb (Figure 4a). This result demonstrates

that Her2ScFv-SP34-CL mediates cell-cell interaction be-

tween CD3

cells and Her2

cells.

To measure the activity of Her2ScFv-SP34-CL to selec-

tively direct T cells to kill Her2 expressing cancer cells, we

performed a cytotoxicity assay using cells expressing dif-

ferent levels of Her2. Peripheral blood mononuclear cells

(PBMCs) from healthy donors were purified with Ficoll

and incubated with the target cells in the presence or ab-

sence of Her2ScFv-SP34-CL. LDH release from the lysed

cells was used to evaluate in-vitro cytotoxicity. As shown

in Figure 4b, Her2ScFv-SP34-CL demonstrates excellent

cytotoxicity against Her2-expressing cells. On Her2

MDA-MB-435/Her2 (with stable Her2 expression) and SK-

BR-3 cells, the EC

s of Her2ScFv-SP34-CL are 1.0±0.2 pM

and 0.32±0.04 pM, respectively (Table S2). The sub-

picomolar potency suggests that Her2ScFv-SP34-CL effi-

ciently recruit T cells to lyse Her2 positive cells. On Her2

MDA-MB-231 cells, which have 100-fold lower Her2 ex-

pression compared to Her2

SK-BR-3 cells,

Her2ScFv-

SP34-CL has an EC

of 5.6±0.5 pM. The maximum cyto-

toxicity on Her2

MDA-MB-231 (22%) is lower than that

on Her2

cells (51% on SK-BR-3 and 47% on MDA-MB-

435/Her2). It was previously reported that the surface

antigen expression level positively correlates to maximum

cytotoxicity and negatively correlates to EC50, which is

consistent with our observations.

37,38

No significant cyto-

toxicity was observed on Her2 negative MDA-MB-468

cells. The correlation between EC50s and surface Her2

expression levels suggests that the T cell killing mediated

by Her2ScFv-SP34-CL is highly selective to Her2 express-

ing cells.

Non-specific T-cell activation might result in potential

off-target toxicity. However, no detectable cytotoxicity on

Her2

MDA-MB-468 cells in the T cell killing assay was

observed. To further validate this observation, non-

specific T cell activation was evaluated. As shown in Fig-

ure 4c, T cells activated with anti-CD3 and anti-CD28

antibodies showed a significant upregulation of the early

T-cell activation marker CD69 compared with the non-

treated group. After cells were treated with 200 pM

Her2ScFv-SP34-CL, a dose over 200-fold higher than the

EC50 on Her23+ cells, no significant CD69 up-regulation

was observed. These results demonstrate that Her2ScFv-

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SP34-CL does not cause significant non-specific T cell activation.

Figure 4. T cell recruitment, cytotoxicity and non-specific T cell activation mediated by Her2ScFv-SP34-CL (a) Fluorescence

microscope images of the interaction between SK-BR-3 (green) cells and Jurkat cells (red) in the presence of PBS or Her2ScFv-

SP34-CL. Scale bar = 100 µm. (b) Dose-dependent cytotoxicity on MDA-MB-468 (Her2

), MDA-MB-231 (Her2

), MDA-MB-

435/Her2 (Her2

), and SK-BR-3 (Her2

) cells in the presence of Her2ScFv-SP34-CL and human peripheral blood mononuclear

cells (PBMCs) from healthy donors. (c) Flow cytometry analysis of T-cell activation marker CD69 on human peripheral blood

mononuclear cells (PBMCs) after 20-hour treatment of 200 pm Her2ScFv-SP34-CL (labeled as "Her2ScFv-SP34-CL"). Untreated

PBMCs purified from healthy donors were used as the negative control (labeled as "no treatment"). Human PBMCs activated

with plate-bound anti-CD3 antibody (clone OKT3, eBioscience) and 2 µg/mL of soluble anti-CD28 antibody (clone CD28.2, eBio-

science) were used as the positive control (labeled as "OKT3+aCD28").

The serum stability and pharmacokinetics of

Her2ScFv-SP34-CL. The serum stability of Her2ScFv-

SP34-CL was then assessed to determine whether

Her2ScFv-SP34-CL is stable against proteolytic activities.

Her2ScFv-SP34-CL was incubated in mouse serum for up

to 96 hours. To quantify the concentration of the active

fraction of Her2ScFv-SP34-CL, a sandwich ELISA was es-

tablished with immobilized Her2-Fc as the antigen and

anti-human kappa chain as the secondary antibody. The

sandwich ELISA detects two separate epitopes on the pa-

rental Fab and the insert, respectively. Thus, the sandwich

ELISA results can quantify the amount of the fusion pro-

tein. As shown in Figure 5a, after 96 hours, ~65% of origi-

nal Her2ScFv-SP34-CL was present, demonstrating that

Her2ScFv-SP34-CL is relatively stable in serum.

The pharmacokinetic (PK) properties of Her2ScFv-

SP34-CL were assessed in mice by intravenously injection.

The concentration of the fusion protein was determined

by the ELISA described above. Her2ScFv-SP34-CL showed

a characteristic two-phase pharmacokinetic behavior

(Figure 5b). The half-lives of the distribution and elimina-

tion phases were determined with the WinNonlin® phar-

macokinetic software package. The half-life of the elimi-

nation phase is 9.8±1.9 hour for Her2ScFv-SP34-CL, signif-

icantly longer than ~1.3 hour for a typical Fab.

In con-

trast, Blinatumomab, the anti-CD19/CD3 bispecific T cell

engager (BiTE), has a reported short terminal phase half-

life of only ~2 hours even in human.

With a molecular

weight of approximately 78 kDa, Her2ScFv-SP34-CL is

above the first-pass renal clearance limit, offering a

pharmacokinetic advantage over smaller BiTE format.

Her2ScFv-SP34-CL recruits T cells to suppress tu-

mor growth in vivo. To evaluate the in vivo efficacy of

Her2ScFv-SP34-CL, xenograft models were established by

subcutaneous implantation of 5×10

Her2

(MDA-MB-

453) or 2.5×10

Her2

(MDA-MB-435) cells in female NSG

mice.

One day later, 2×10

fresh human PBMC were in-

jected into the intraperitoneal space. Upon formation of a

palpable tumor, mice were intravenously infused with

2×10

activated human T cells. Her2ScFv-SP34-CL was

then intravenously administered daily at a dose of 1

mg/kg for 10 days. Mice treated with PBMCs and PBS

were used as negative controls. The tumor growth and

body weight change were monitored for up to 50 days. As

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shown in Figure 5c and 5d, shortly after treatment was

initiated, tumor shrinkage was observed in the Her2ScFv-

SP34-CL group, whereas the vehicle group showed rapid

tumor outgrowth. In the Her2

MDA-MB-453 model, the

tumor size decreased and then stabilized in the treatment

group within the 50-day period of this study; and no ap-

parent relapse was observed for at least 30 days after

treatment was stopped. In the Her2

MDA-MB-435 mod-

el, similarly, tumor growth was significantly suppressed

by Her2ScFv-SP34-CL. During these studies, no overt

body weight loss was observed in mice in any group (Fig-

ure S14), indicating Her2ScFv-SP34-CL has no obvious

toxicity to mice in this dosing paradigm. These observa-

tions demonstrate that BsAbs generated by non-CDR loop

grafting represent a potential effective treatment for

Her2

cancers with all types of Her2 overexpression levels.

Figure 5. Serum stability, pharmacokinetics, and in vivo

efficacy of Her2ScFv-SP34-CL (a) The stability of Her2ScFv-

SP34-CL in mouse serum. The Her2ScFv-SP34-CL concentra-

tion is determined by sandwich ELISA with Her2-Fc as cap-

ture antigen and HRP-anti-human kappa 2

Ab. (b) Phar-

macokinetics of Her2ScFv-SP34-CL in mice by i.v. dosing

(n=3). The Her2ScFv-SP34-CL concentration is determined

by sandwich ELISA with Her2-Fc as capture antigen and

HRP-anti-human kappa 2

Ab. (c) Tumor regression medi-

ated by 1 mg/kg Her2ScFv-SP34-CL in MDA-MB-453 (Her2

)

xenograft in NSG mice (n=5). (d) Tumor regression mediated

by 1 mg/kg Her2ScFv-SP34-CL in MDA-MB-435 (Her2

) xen-

ograft in NSG mice (n=5).

Discussion

In our previous work, we reported successful pro-

tein/peptide grafting into CDR3 and CDR2 in antibody

variable regions.

19-27

Herein, we extend this insertion

strategy to the loops in the constant regions, which struc-

turally also belong to the immunoglobulin scaffold. We

demonstrate that these conserved loops are also capable

of accepting large functional protein insertions. In general

CDR loop fusions reduce or destroy the binding proper-

ties of the parent antibody. In contrast, the non-CDR

loops are spatially separated from the antigen-binding

site, and as a result, protein/peptide grafting into these

loops does not significantly impact the binding affinity to

the cognate antigen. Therefore, unlike the mono-

functional antibody CDR-fusions, non-CDR loop fusions

yield antibody fusions for applications such as the genera-

tion of immunocytokines

and immune cell recruiting

bispecific antibodies.

Current published Ig fusion strategies mainly include

N- or C- terminal fusions and hinge insertions. As N-

termini are close to the antigen-binding site, large-size N-

terminal fusion in antibodies often decreases the binding

affinity of the antibody.

C-terminal fusion, comparative-

ly, does not affect the activity of the parental antibody.

Therefore, it is often chosen for immunocytokines and

immunotoxins. However, C-terminal fusion has reported-

ly shorter serum half-life, likely due to interference with

neonatal Fc receptor (FcRn) binding and/or destabilized

Fc dimerization.

In addition to terminal fusions, inser-

tion of peptides/proteins into the hinge region has been

reported. However, the inserted proteins have reduced

FcγR-binding as well as shorter half-lives.

In comparison,

non-CDR fusion site is distant from the antigen binding

site or the Fc domain, minimizing the influence on anti-

gen binding or PK properties. Additionally, as the insert

protein is stemmed out from the antibody scaffold by a

rigid stalk, allowing retained biological functions of the

insert protein. As we demonstrated in our studies, neither

EPO insert nor the anti-Her2 ScFv insert had significant

decrease in its biological activity.

To demonstrate the potential utility of this strategy, we

generated a Her2×CD3 bispecific antibody (Her2ScFv-

SP34-CL) capable of recruiting T cells to Her2+ cancer

cells with high in vitro and in vivo cytotoxity.

The use of bispecific antibodies (bsAbs) to retarget the

immune system to treat cancer has been highly effective

but still faces several challenges.

44,45

Catumaxomab, the

first clinically approved bispecific antibody, was con-

structed from rat-mouse antibody quadromas and has

significant immunogenicity in humans.

Bispecific T cell

engager (BiTE)

and dual-affinity re-targeting (DART)

bispecific constructs have shown excellent efficacy. How-

ever, they suffer from short half-lives and can have poor

physical properties. More recent attempts to generate

heterodimeric IgG-like bispecific antibodies have afforded

long circulation times and high potencies. However,

complete removal of unwanted homodimeric species to

generate highly purified heterodimers remains a manu-

facturing challenge.

Tumor penetration, effective immune synapse for-

mation, and stability are critical properties of T-cell re-

cruiting bispecific antibodies. The optimal size of a bsAb

is a trade-off between serum half-life and tumor penetra-

tion.

48,49

It has been suggested that proteins with molecu-

lar weight between 65 kDa and 110 kDa are most suitable

for penetrating solid tumors.

Thus the small size of

BiTE and DART (~ 50 kDa) afford excellent efficacy, but

suffer from fast clearance. On the other hand, IgG-like

bispecific antibodies have long serum half-lives, but less

efficiently penetrate through the dense extracellular ma-

trix (ECM) of solid tumors.

The scFv-Fab non-CDR loop

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fusion, with molecular weight around ~ 78 kDa, nicely fall

within the optimal range for tumor penetration, and have

a terminal half-life of ~ 10 hours 0Her2ScFv-SP34-CL0 in

mice, significantly longer than for BiTEs.

Efficient immunological synapse formational also af-

fects the ability of immune cells to kill cancer cells. Re-

ducing the distance between the T-cell-binding site and

tumor antigen binding sites on bsAbs normally results in

higher potency.

Indeed, the excellent in vitro and in vivo

efficacies of BiTE and DART are due in part to the short

distance between T-cell- and cancer-cell binding sites.

Similarly, the distance between the CD3-binding site on

the parent SP34 antibody and the Her2-binding site on

the Herceptin scFv insert is small, and the in vitro cyto-

toxicity of Her2ScFv-SP34-CL reaches EC50 values of 10

-12

-13

Stability is another important requirement for bispecif-

ic antibodies. Blinatumomab, for example, has a narrow

therapeutic window, due to non-specific T cell activation

likely caused by bsAb aggregations.

Although small

amounts of Her2ScFv-SP34-CL oligomers are present after

protein G chromatography of the expressed fusion pro-

tein, no further aggregation is observed after the mono-

mer is purified by size exclusion chromatography, and no

aggregates can be detected by dynamic light scattering.

Indeed, the physiochemical properties of Her2ScFv-SP34-

CL upon expression and purification are similar to those

of a typical Fab. More importantly, no nonspecific in vitro

cytotoxicity or T cell auto-activation is observed for this

construct.

The non-CDR loop fusion can be constructed on either

Fab scaffolds or full-length IgG scaffolds, affording either

monovalent or bivalent binding modes. It is also likely

that one can generate multi-valent binding formats that

can include binding to multiple tumor antigens, check-

point proteins or serum albumins. Additionally, unlike

other full-length IgG fusion strategies (such as N- and C-

terminal fusion),

8,43

there are multiple options for the

non-CDR loop fusion sites to that allow one to retain full

antigen-binding and Fc mediated functions. We are cur-

rently exploring the extension of this strategy to trispecif-

ic antibody fusions.

Conclusions

In summary, we demonstrate that full-length proteins

and single chain variable fragments (scFv) can be inserted

into the non-CDR loops of an IgG or a Fab. The antibody

fusion proteins retain the antigen binding activity of the

scaffold antibody as well as full activities of new function-

alities introduced by the insert. The non-CDR loop fusion

proteins have excellent in vitro activity, physiochemical

properties, and thermal and serum stabilities. The

Her2×CD3 bispecific antibodies generated by this ap-

proach can induce targeted cell lysis in the presence of

effector cells at sub-picomolar concentrations in breast

cancer cells. No T cell activation was observed in the ab-

sence of target cells even at high concentration of

Her2ScFv-SP34-CL. Furthermore, Her2ScFv-SP34-CL

showed potent in vivo antitumor activity in mouse Her2

and Her2

xenograft models. Future work includes evalu-

ation of candidate constructs for developability, as well as

potential immunogenicity and toxicity in non-human

primates.

Methods

Cloning of EPO-Herceptin-CL and EPO-Herceptin-CH1

IgG Fusion Protein. The genes encoding Herceptin Fab heavy

chain and light chain were synthesized by Genscript (NJ, USA),

and amplified by polymerase chain reactions (PCR). The mam-

malian expression vector of Herceptin full-length IgG heavy

chain was generated by in-frame ligation of amplified Herceptin

Fab heavy chain (VH and CH1) to pFuse-hIgG1-Fc backbone

vector (InvivoGen, CA). Gene encoding antibody Herceptin light

chain was amplified and cloned into the pFuse vector without

hIgG1 Fc fragment. The gene encoding EPO was synthesized by

Genscript (NJ, USA), and amplified by PCRs. Coiled-coil stalk

was added to both ends of the EPO insert sequence. The se-

quence of the ascending stalk peptide with linkers at each end is:

H2N-GGSGAKLAALKAKLAALKGGGGS-COOH; the sequence of

the descending peptide with linkers at each end is: H2N-

GGGGSELAALEAELAALEAGGSG-COOH. The EPO-Herceptin-

CL light chain was created by replacing the K169 in the CL re-

gion of Herceptin light chain by EPO with a coiled-coil stalk.

EPO-Herceptin-CH1 heavy chain was created by replacing the

S182 and G183 in the CH1 region of Herceptin heavy chain by

EPO with a coiled-coil stalk. The genes encoding the EPO-

Herceptin-CH1 heavy chain and EPO-Herceptin-CL light chain

were obtained by overlap extention PCRs. And the vectors of the

EPO-Herceptin-CH1 heavy chain and the EPO-Herceptin-CL

light chain were generated by in-frame ligation of the amplified

PCR products to pFuse-hIgG1-Fc backbone vector. The resulting

mammalian expression vectors were confirmed by DNA se-

quencing.

Cloning of Her2ScFv-SP34-CL Fab Fusion Protein. The

genes encoding SP34 Fab heavy chain and light chain were syn-

thesized by Genscript (NJ, USA), and amplified by PCRs. The

mammalian expression vectors of SP34 Fab heavy and light

chains were generated by in-frame ligation of amplified SP34 Fab

heavy chain (VH and CH1) or light chain (VL and CL) to pFuse-

hIgG1-Fc backbone vector (InvivoGen, CA) without the Fc frag-

ment. Gene encoding Her2ScFv with the coiled-coil linkers was

synthesized as gBlock gene fragment by IDT, Inc (IA, USA), and

amplified by PCRs. The sequence of the ascending stalk peptide

with linkers at each end is: H2N-

GGSGCAKLAALKAKLAALKGGGGS-COOH; the sequence of the

descending peptide with linkers at each end is: H2N-

GGGGSELAALEAELAALEACGGSG-COOH. Subsequently,

Her2ScFv-SP34-CL light chain was created by replacing the K169

(for Her2ScFv-SP34-CL) in the CL region of SP34 light chain by

Her2ScFv with a coiled-coil stalk. The gene encoding the

Her2ScFv-SP34-CL Fab light chain was obtained by overlap ex-

tention PCR. And the vector of Her2ScFv-SP34-CL Fab light

chain was generated by in-frame ligation of the amplified PCR

products to the pFuse-hIgG1-Fc backbone vector. The resulting

mammalian expression vectors were confirmed by DNA se-

quencing.

Expression and purification of fusion proteins. Fusion

proteins were expressed through transient transfection of Free-

Style HEK 293 cells with expression vectors (Table S1), according

to the manufacturer's protocol. Briefly, 28 mL FreeStyle HEK 293

cells containing 3×10

cells were seeded in a 125 mL shaking flask.

Defined amounts of plasmids encoding the light chain and heavy

chain (Table S1) were diluted in 1 mL Opti-MEM medium and

added to 1 mL Opti-MEM containing 60 µL 293fectin (Invitro-

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gen, Inc). After the plasmids were incubated with 293fectin for

30 min at room temperature, the lipoplex mixture was added to

the cell suspension. Cells were then shaken at 125 rpm in a 5%

environment at 37 ºC. Culture medium containing secreted

proteins was harvested every 48 hours for twice after transfec-

tion. Fusion proteins were purified by Protein G and size-

exclusion chromatography. Purified proteins were analyzed by

SDS-PAGE, mass spectrometry and size exclusion chromatog-

raphy.

In-vitro assay of EPO activity. Human TF-1 cells were cul-

tured at 37 °C with 5% CO

in RPMI-1640 medium containing

10% fetal bovine serum (FBS), penicillin/streptomycin (50

U/mL), and 2 ng/mL human granulocyte macrophage colony

stimulating factor (GM-CSF). To test the proliferative activity of

EPO fusion proteins, cells were washed three times with RPMI-

1640 medium plus 10% FBS, resuspended in RPMI-1640 medium

with 10% FBS at a density of 1.5×10

cells/ml, plated in 96-well

plates (1.5×10

cells per well) with various concentrations of EPO-

Herceptin-CL, EPO-Herceptin-CH1, Herceptin-hEPO-CDR3H

(positive control) and then incubated for 72 h at 37 °C with 5%

. Cells were then incubated with Alamar Blue (Invitrogen)

for 4 h at 37 °C. Fluorescence intensity measured with λ

= 570

nm and λ

= 595 nm is proportional to cell viability and plotted

versus protein concentrations. The EC

values were determined

by fitting data into a logistic sigmoidal function: y = A2 + (A

−

)/(1 + (x/x

)p), where A

is the initial value, A

is the final value,

is the inflection point of the curve, and p is the power.

In vitro PBMC-mediated cytotoxicity of Her2ScFv-SP34-

CL on breast cancer cells. For in vitro cytotoxicity assays,

PBMCs were purified from fresh healthy human donor blood

(from The Scripps Research Institute normal blood donor ser-

vice) by conventional Ficoll-Hypaque gradient centrifugation

(GE Healthcare). Purified PBMCs were washed and resuspended

in RPMI with 10% (vol/vol) FBS and were incubated with target

cells and different concentrations of Her2ScFv-SP34-CL for 24 h

at 37 °C. Cytotoxicity of each well was measured by LDH levels in

supernatant using the Cytotox-96 nonradioactive cytotoxicity

assay kit (Promega). Lysis solution provided in the same kit (10

μL) was added to wells containing only target cells to achieve the

maximum killing; and spontaneous killing was measured in wells

with effector and target cells treated with vehicle (10 μL PBS).

The absorbance at 490 nm was recorded using a SpectraMax 250

plate reader (Molecular Devices Corp.). Percent cytotoxicity was

calculated by:

% cytotoxicity

= (absorbance experimental − absorbance spontaneous aver-

age)/ (absorbance maximum killing average − absorbance spon-

taneous average).

Pharmacokinetics of Her2ScFv-SP34-CL in Mice. 8 mg/kg

Her2ScFv-SP34-CL in PBS (pH 7.4) was administrated by intra-

venous (i.v.) injection into CD1 mice (6 per group). Blood was

collected from 5 min, 15 min, 30 min, 1h, 2h, 4h, 6h, 8h, 24 hr, 32

hr, 48 hr after injection. 75µl of whole blood sample is collected

via retro-orbital sinus into a heparinized capillary tube and

stored on wet ice until processed. Each sample is spun at

12,000RPM for 3 min; the resulting plasma is placed into a

uniquely identified location of a 96 well plate. The plate is stored

at -80°C until analyzed. The plasma concentration of Her2ScFv-

SP34-CL is determined by ELISA against immobilized hErbB2-Fc

(R&D Systems) with HRP anti-human Kappa (Abcam) as sec-

ondary antibody. The half-lives for elimination phase were de-

termined by fitting the last four data points into the first-order

equation, A = A

−kt

, where A

is the initial concentration, t is

the time, and k is the first order rate constant.

In vivo efficacy study of Her2ScFv-SP34-CL. All procedures

were approved by The Scripps Research Institute Animal Care

and Use Committee and were performed according to national

and international guidelines for the humane treatment of ani-

mals. All efficacy studies were conducted with 6 to 8-week-old

female NOD-SCID-γ(NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice

(Jackson Laboratory). Human breast cancer cell lines Her2

(MDA-MB-453), and Her2

(MDA-MB-435)] were used to evalu-

ate the in vivo efficacy of Her2ScFv-SP34-CL.

For Her2

tumor model, 5×10

MDA-MB-453 cells in 50%

Matrigel (BD Bioscience) were subcutaneously implanted into

the right flank of mice. One day after that, 2×10

fresh PBMC

were injected into the intraperitoneal space. Meanwhile, human

PBMCs were activated with plate-bound anti-CD3 antibody

(clone OKT3, eBioscience) and 2 µg/mL of soluble anti-CD28

antibody (clone CD28.2, eBioscience), and maintained in RPMI-

1640 media supplemented with 10% FBS and 50 IU/mL of re-

combinant human IL-2 (R&D Systems). Eight days and eleven

days after tumor implantation, mice received 2×10

activated T

cells via intraperitoneal injection. Ten days after tumor inocula-

tion, when tumors reached a volume of 200-300 mm

, mice were

intravenously administered Her2ScFv-SP34-CL (1 mg/kg) or sa-

line daily for ten days.

For Her2

tumor model, 2.5×10

MDA MB435 cells in 50%

Matrigel were subcutaneously implanted to the right flank of

mice. One day after that, 2×10

fresh PBMC were injected into

the intraperitoneal space. Eight days and eleven days after tumor

implantation, mice received 2×10

activated PBMC cells via in-

traperitoneal injection. Ten days after tumor inoculation, when

tumors reached a volume of 200-300 mm

, mice were intrave-

nously administered Her2ScFv-SP34-CL (1 mg/kg) or saline daily

for ten days.

Tumors were measured twice weekly by calipers. Tumor vol-

ume was calculated based on width × length × height.

ASSOCIATED CONTENT

Supporting Information

The Supporting Information is available free of charge on the

ACS Publications website.

Detailed materials and methods, supplementary figures and

tables (PDF file).

AUTHOR INFORMATION

Corresponding Author

schultz@scripps.edu

fwang@calibr.edu

Author Contributions

The manuscript was written through contributions of all

authors. All authors have given approval to the final version

of the manuscript.

ACKNOWLEDGMENT

We thank Prof. Liangfang Zhang (UCSD) and his student Yue

Zhang in helping with the DLS measurement.

ABBREVIATIONS

CDR, complementarity determining region; EPO, Erythro-

poietin; scFv, single-chain variable fragment; ICK, inhibitor

cysteine knot; IgG, immunoglobulin G; Fab, fragment anti-

gen-binding; Fc, fragment constant; bsAb, bispecific anti-

body.

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... Herein, fusion protein technique is introduced to modify proteins. The fusion protein technique is aimed to synthesize fusion proteins through the target genes fusion and expression of protein [22]. The basic method of constructing fusion protein is to encode multiple specific functions coding to form a polypeptide sequence which lead to realize the common expression of two genes. ...

Telomerase is considered as a widely accepted cancer biomarker for early cancer diagnostics. Herein, we develop a simple, ultrahigh sensitivity method for detection of telomerase activity, which relied on that RecA-GFP fusion proteins wrapped around telomeric DNA to form fluorescence bouquets. RecA-GFP fusion protein was synthesized through fusion protein technology. In the presence of telomerase, telomerase elongation products are wrapped around by RecA-GFP fusion protein to form big fluorescent bouquets, which resulted in strong fluorescence. This method has the linear range from 50 to 1000 HeLa cells and the detection limit is 8 HeLa cells, based on a signal-to-noise ratio (S/N) of 3. Compared with conventional methods, this method has the advantages of low toxicity, outstanding sensitivity, and excellent selectivity. Hence, it provides a promising approach for the detection of telomerase activity and diagnosis of cancer.

... 88 Du et al generated bifunctional antibodies by grafting full-length proteins into constant region loops of a Fab, which showed that the fusion proteins retained antigen-binding activity of the parent antibody with an additional activity associated with the protein insert. 89 At the same time, other production methods, such as yeast, transgenic plant, and cell-free expression systems, provide new alternatives to facilitate generation of Fab antibodies. ...

Hui Chen
Jun-Sheng Chen
Pameila Paerhati
Yunsheng Yuan

With the advancement of genetic engineering, monoclonal antibodies (mAbs) have made far-reaching progress in the treatment of various human diseases. However, due to the high cost of production, the increasing demands for antibody-based therapies have not been fully met. Currently, mAb-derived alternatives, such as antigen-binding fragments (Fab), single-chain variable fragments, bispecifics, nanobodies, and conjugated mAbs have emerged as promising new therapeutic modalities. They can be readily prepared in bacterial systems with well-established fermentation technology and ease of manipulation, leading to the reduction of overall cost. This review aims to shed light on the strategies to improve the expression, purification, and yield of Fab fragments in Escherichia coli expression systems, as well as current advances in the applications of Fab fragments.

Pamela D. Garzone
Yow-Ming C. Wang

Biologics currently account for more than 25% of FDA approved entities, and this percentage is expected to increase. Biologics have been very successful in the treatment of major diseases; cancer therapeutics continue to be the top disease category since 2014. Information on proteins, monoclonal, and bi-specific antibodies currently marketed or under investigation and methodology used to assay macromolecules and interspecies scaling is discussed. Pharmacokinetic (PK) characteristics and pharmacodynamics (PD) of macromolecules are presented. Concepts of pharmacokinetics and pharmacodynamics in biosimilar drug development are presented.

Zui Zhang
Yuxiu Chu
Cheng Li
Changyou Zhan

Anti-PEG antibodies have been witnessed in patients and experimental animals, accelerating the blood clearance (termed ABC phenomenon) of PEGylated nanomedicines by activating complement after absorption on the nano-surface. The ABC phenomenon presents an obstacle to the clinical translation of PEGylated nanomedicines. Herein, an anti-PEG single-chain variable fragment (PEG-scFv) that possesses a low molecule weight (30 kDa) and high PEG binding affinity was exploited to ameliorate the ABC phenomenon of PEGylated liposomes (sLip). Pre-deposition of PEG-scFv on the surface of sLip was incompetent to activate complement due to the lack of Fc chains, exhibiting negligible influence on in vivo performance of sLip in naïve rats (without anti-PEG antibodies). However, PEG-scFv effectively competed the binding of anti-PEG IgM in rats that were pre-stimulated with low dose of sLip, thus ameliorated the ABC phenomenon of sLip. PEG- scFv was also effective to inhibit the binding of anti-PEG antibodies with sLip in human plasma and the consequent complement activation, presenting a promising tool to improve the performance of PEGylated nanomedicines and to mitigate individual difference occurred by the varying levels of anti-PEG antibodies in the clinic. The application of anti-PEG scFv paves a new avenue for the development of nanocarriers to achieve precise medication.

While most organisms utilize 20 canonical amino acid building blocks for protein synthesis, adding additional candidates to the amino acid repertoire can greatly facilitate the investigation and manipulation of protein structures and functions. In this study, we report the generation of completely autonomous organisms with a 21st noncanonical amino acid, 5-hydroxytryptophan (5HTP). Like 20 canonical amino acids, 5-hydroxytryptophan can be biosynthesized in vivo from simple carbon sources and is subsequently incorporated into proteins in response to the amber stop codon. Using this unnatural organism, we have prepared a single-chain immunoglobulin variable fragment conjugated with a fluorophore and demonstrated the utility of these autonomous cells to monitor oxidative stress. The creation of this and other cells containing the 21st amino acid will provide an opportunity to generate proteins and organisms with novel activities, as well as to determine the evolutionary consequences of using additional amino acid buildings.

Zhefu Dai
Qinqin Cheng
Yong Zhang

Cathepsin B (CTSB) is an abundant cysteine protease that functions in both endolysosomal compartments and extracellular regions. A considerable number of preclinical and clinical studies indicate that CTSB is implicated in many human diseases. Expression levels and activity of CTSB significantly correlate with disease progression and severity. Current inhibitors of CTSB are lack of adequate specificity and pharmacological activities. Through structure-guided rational design, we hereby designed and generated a humanized antibody inhibitor targeting human CTSB. This was achieved by genetically fusing the propeptide of procathepsin B, a naturally occurring inhibitor of CTSB, into the heavy chain complementarity determining region 3(CDR3H) of Herceptin that is used in clinic for treatment of breast cancer. The resulting antibody-propeptide fusion displayed high specificity for inhibiting CTSB proteolytic activity at nanomolar levels. Pharmacokinetic studies in mice revealed a plasma half-life of approximately 42 hours for this anti-CTSB antibody inhibitor, comparable to that of parental Herceptin scaffold. This study demonstrates a new approach for efficient generation of humanized antibody inhibitors with high potency and specificity for human CTSB, which may be extended to develop antibody inhibitors against other disease relevant cathepsin proteases.

We developed a repertoire approach to generate human antibody bispecifics. Using phage display selection of antibody heavy chains in the presence of a competitor light chain and providing a cognate light chain with an affinity handle, we identified mutations that prevent heavy/light chain mispairing. The strategy allows for the selection of human antibody chains that autonomously assemble into bispecifics.

Andres Lopez-Albaitero
Hong Xu
Hongfen Guo
Nai-Kong Cheung

T-cell based therapies have emerged as one of the most clinically effective ways to target solid and non-solid tumors. HER2 is responsible for the oncogenesis and treatment resistance of several human solid tumors. As a member of the HER family of tyrosine kinase receptors, its over-activity confers unfavorable clinical outcome. Targeted therapies directed at this receptor have achieved responses, although development of resistance is common. We explored a novel HER2/CD3 bispecific antibody (HER2-BsAb) platform that while preserving the anti-proliferative effects of trastuzumab, it recruits and activates non-specific circulating T-cells, promoting T cell tumor infiltration and ablating HER2(+) tumors, even when these are resistant to standard HER2 targeted therapies. Its in vitro tumor cytotoxicity, when expressed as EC50, correlated with the surface HER2 expression in a large panel of human tumor cell lines, irrespective of lineage or tumor type. HER2-BsAb mediated cytotoxicity was relatively insensitive to PD-1/PD-L1 immune checkpoint inhibition. In four separate humanized mouse models of human breast cancer and ovarian cancer cell line xenografts, as well as human breast cancer and gastric cancer patient-derived xenografts (PDXs), HER2-BsAb was highly effective in promoting T cell infiltration and suppressing tumor growth when used in the presence of human peripheral blood mononuclear cells (PBMC) or activated T cells (ATC). The in vivo and in vitro antitumor properties of this BsAb support its further clinical development as a cancer immunotherapeutic.

Ji Li
Nicola J. Stagg
Jennifer Johnston
Teemu T Junttila

The anti-FcRH5/CD3 T cell-dependent bispecific antibody (TDB) targets the B cell lineage marker FcRH5 expressed in multiple myeloma (MM) tumor cells. We demonstrate that TDBs trigger T cell receptor activation by inducing target clustering and exclusion of CD45 phosphatase from the synapse. The dimensions of the target molecule play a key role in the efficiency of the synapse formation. The anti-FcRH5/CD3 TDB kills human plasma cells and patient-derived myeloma cells at picomolar concentrations and results in complete depletion of B cells and bone marrow plasma cells in cynomolgus monkeys. These data demonstrate the potential for the anti-FcRH5/CD3 TDB, alone or in combination with inhibition of PD-1/PD-L1 signaling, in the treatment of MM and other B cell malignancies.

Therapeutic monoclonal antibodies have become molecules of choice to treat autoimmune disorders, inflammatory diseases, and cancer. Moreover, bispecific/multispecific antibodies that target more than one antigen or epitope on a target cell or recruit effector cells (T cell, natural killer cell, or macrophage cell) toward target cells have shown great potential to maximize the benefits of antibody therapy. In the past decade, many novel concepts to generate bispecific and multispecific antibodies have evolved successfully into a range of formats from full bispecific immunoglobulin gammas to antibody fragments. Impressively, antibody fragments such as bispecific T-cell engager, bispecific killer cell engager, trispecific killer cell engager, tandem diabody, and dual-affinity-retargeting are showing exciting results in terms of recruiting and activating self-immune effector cells to target and lyse tumor cells. Promisingly, crystallizable fragment (Fc) antigen-binding fragment and monomeric antibody or half antibody may be particularly advantageous to target solid tumors owing to their small size and thus good tissue penetration potential while, on the other hand, keeping Fc-related effector functions such as antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity, antibody-dependent cell-mediated phagocytosis, and extended serum half-life via interaction with neonatal Fc receptor. This review, therefore, focuses on the progress of Fc engineering in generating bispecific molecules and on the use of small antibody fragment as scaffolds for therapeutic development.

During the past two decades we have seen a phenomenal evolution of bispecific antibodies for therapeutic applications. The 'zoo' of bispecific antibodies is populated by many different species, comprising around 100 different formats, including small molecules composed solely of the antigen-binding sites of two antibodies, molecules with an IgG structure, and large complex molecules composed of different antigen-binding moieties often combined with dimerization modules. The application of sophisticated molecular design and genetic engineering has solved many of the technical problems associated with the formation of bispecific antibodies such as stability, solubility and other parameters that confer drug properties. These parameters may be summarized under the term 'developability'. In addition, different 'target product profiles', i.e., desired features of the bispecific antibody to be generated, mandates the need for access to a diverse panel of formats. These may vary in size, arrangement, valencies, flexibility and geometry of their binding modules, as well as in their distribution and pharmacokinetic properties. There is not 'one best format' for generating bispecific antibodies, and no single format is suitable for all, or even most of, the desired applications. Instead, the bispecific formats collectively serve as a valuable source of diversity that can be applied to the development of therapeutics for various indications. Here, a comprehensive overview of the different bispecific antibody formats is provided.

Christoph Spiess
Qianting Zhai
Paul J. Carter

Bispecific antibodies are on the cusp of coming of age as therapeutics more than half a century after they were first described. Two bispecific antibodies, catumaxomab (Removab(®), anti-EpCAM×anti-CD3) and blinatumomab (Blincyto(®), anti-CD19×anti-CD3) are approved for therapy, and >30 additional bispecific antibodies are currently in clinical development. Many of these investigational bispecific antibody drugs are designed to retarget T cells to kill tumor cells, whereas most others are intended to interact with two different disease mediators such as cell surface receptors, soluble ligands and other proteins. The modular architecture of antibodies has been exploited to create more than 60 different bispecific antibody formats. These formats vary in many ways including their molecular weight, number of antigen-binding sites, spatial relationship between different binding sites, valency for each antigen, ability to support secondary immune functions and pharmacokinetic half-life. These diverse formats provide great opportunity to tailor the design of bispecific antibodies to match the proposed mechanisms of action and the intended clinical application. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Xiaozhou Luo
Tao Liu
Ying Wang
Feng Wang

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infections in children. We have generated an epitope-specific RSV vaccine by grafting a neutralizing epitope (F-epitope) in its native conformation into an immunoglobulin scaffold. The resulting antibody fusion exhibited strong binding affinity to Motavizumab, an RSV neutralizing antibody, and effectively induced potent neutralizing antibodies in mice. This work illustrates the potential of the immunoglobulin molecule as a scaffold to present conformationally constrained B-cell epitopes.

The field of therapeutic antibodies has been revolutionized over the past decade, led by the development of novel antibody-modification technologies. Besides the huge success achieved by therapeutic monoclonal antibodies, a diversity of antibody derivatives have emerged with hope to outperform their parental antibodies. Here we review the recent development of methodologies to modify immunoglobulin domains and their therapeutic applications. The innovative genetic and chemical approaches enable novel and controllable modifications on immunoglobulin domains, producing homogeneous therapeutics with new functionalities or enhanced therapeutic profiles. Such therapeutics, including antibody-drug conjugates, bispecific antibodies, and antibody/Fc fusion proteins, have demonstrated great prospects in the treatment of cancer, auto-immune diseases, infectious diseases, and many other disorders. Copyright © 2015 Elsevier Ltd. All rights reserved.

Xiufeng Wu
Arlene Sereno
Flora Huang
Stephen J Demarest

A myriad of innovative bispecific antibody (BsAb) platforms have been reported. Most require significant protein engineering to be viable from a development and manufacturing perspective. Single-chain variable fragments (scFvs) and diabodies that consist only of antibody variable domains have been used as building blocks for making BsAbs for decades. The drawback with Fv-only moieties is that they lack the native-like interactions with CH1/CL domains that make antibody Fab regions stable and soluble. Here, we utilize a redesigned Fab interface to explore two novel Fab-based BsAbs platforms. The redesigned Fab interface designs limit heavy and light chain mixing when two Fabs are co-expressed simultaneously, thus allowing the use of two different Fabs within a BsAb construct without the requirement of one or more scFvs. We describe the stability and activity of a HER2ċHER2 IgG-Fab BsAb, and compare its biophysical and activity properties with those of an IgG-scFv that utilizes the variable domains of the same parental antibodies. We also generated an EGFRċCD3 tandem Fab protein with a similar format to a tandem scFv (otherwise known as a Bispecific T cell Engager or BiTE). We show that the Fab-based BsAbs have superior biophysical properties compared to the scFv-based BsAbs. Additionally, the Fab-based BsAbs do not simply recapitulate the activity of their scFv counterparts, but are shown to possess unique biological activity.

Tao Liu
Guangsen Fu
Xiaozhou Luo
Feng Wang

The bovine antibody (BLV1H12) which has an ultralong CDR3H provides a novel scaffold for engineering new func-tions into the antibody variable region. By modifying the β-strand "stalk" of BLV1H12 with sequences derived from natu-ral or synthetic protease inhibitors, we have generated anti-bodies that inhibit bovine trypsin and human neutrophil elastase (HNE) with low nanomolar affinities. We were also able to generate a humanized variant using a human immu-noglobulin scaffold that shares a high degree of homology with BLV1H12. Further optimization yielded a highly selec-tive humanized anti-HNE antibody with sub-nanomolar affinity. This work demonstrates a novel strategy for gener-ating antibodies with potent and selective inhibitory activi-ties against extracellular proteases involved in human dis-ease.

Tao Liu
Yong Zhang
Yan Liu
Feng Wang

On the basis of the 3D structure of a bovine antibody with a well-folded, ultralong complementarity-determining region (CDR), we have developed a versatile approach for generating human or humanized antibody agonists with excellent pharmacological properties. Using human growth hormone (hGH) and human leptin (hLeptin) as model proteins, we have demonstrated that functional human antibody CDR fusions can be efficiently engineered by grafting the native hormones into different CDRs of the humanized antibody Herceptin. The resulting Herceptin CDR fusion proteins were expressed in good yields in mammalian cells and retain comparable in vitro biological activity to the native hormones. Pharmacological studies in rodents indicated a 20- to 100-fold increase in plasma circulating half-life for these antibody agonists and significantly extended in vivo activities in the GH-deficient rat model and leptin-deficient obese mouse model for the hGH and hLeptin antibody fusions, respectively. These results illustrate the utility of antibody CDR fusions as a general and versatile strategy for generating long-acting protein therapeutics.

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Source: https://www.researchgate.net/publication/321376941_Engineering_Bifunctional_Antibodies_with_Constant_Region_Fusion_Architectures

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