Therapeutic Bispecific Antibody Formats: A Patent Applications Review (1994-2017)

Expert Opinion on Therapeutic Patents
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Therapeutic bispecific antibody formats: a patent

applications review (1994-2017)
Marie Godar, Hans de Haard, Christophe Blanchetot & Jacobus Rasser
To cite this article: Marie Godar, Hans de Haard, Christophe Blanchetot & Jacobus Rasser (2018):
Therapeutic bispecific antibody formats: a patent applications review (1994-2017), Expert Opinion
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EXPERT OPINION ON THERAPEUTIC PATENTS, 2018
https://doi.org/10.1080/13543776.2018.1428307
REVIEW
Therapeutic bispecific antibody formats: a patent applications review (1994-2017)

a,b,c
Marie Godar , Hans de Haarda, Christophe Blanchetot*a and Jacobus Rasser*a
a
argenx BVBA, Zwijnaarde, Belgium; bVIB-UGent Center for Inflammation Research, Ghent, Belgium; cDepartment of Internal Medicine, Ghent
University, Ghent, Belgium
ABSTRACT ARTICLE HISTORY

Introduction: Bispecific antibodies have become increasingly of interest by enabling new therapeutic Received 1 November 2017
applications such as retargeting cellular immunity towards tumor cells. About 23 bispecific antibody Accepted 11 January 2018
platforms have therefore been developed, generating about 62 molecules which are currently being KEYWORDS
evaluated for potential treatment of a variety of indications, such as cancer and inflammatory diseases, Bispecific antibody; BiTE;
among which three molecules were approved. This class of drugs will represent a multi-million-dollar cancer; CrossMAb;
market over the coming years. Many companies have consequently invested in the development of immunoglobulin gamma;
bispecific antibody platforms, creating an important patent activity in this field. inflammatory disease;
Areas covered: The present review gives an overview of the patent literature over the period Knobs-into-Holes;
1994–2017 of different immunoglobulin gamma-based bispecific antibody platforms and the molecules nanobody; Triomab
approved or in clinical trials. quadroma
Expert opinion: Bispecific antibodies are progressively accepted as potentially superior therapeutic
molecules in a broad range of diseases. This frantic activity creates a maze of hundreds of patents that
pose considerable legal risks for both newcomers and established companies. It can consecutively be
anticipated that the number of patent conflicts will increase. Nevertheless, it can be expected that
patents related to the use of a bispecific antibody will have tremendous commercial value.
1. Introduction to mimic a natural ligand (Figure 1B) [7], hijacking a transcytosis

mechanism for the delivery of antibodies (Ab) to the brain
Since the approval of the first therapeutic monoclonal antibody
(Figure 1C) [8], or even enhancing Ab binding affinity by multiple
(mAb) muromonab-CD3 by the United States Food and Drug
epitopes binding (Figure 1D) [4,5,9–11].
Administration (FDA) for the treatment of organ transplant-asso-
Some applications require both Ag-binding activities to be
ciated acute rejections in 1986, a total of about 62 mAbs have been
included in a single molecule, such as retargeting cellular
approved for clinical use [1,2]. Indeed, the FDA has approved an
immunity (Figure 1A) [12]. By contrast, other applications do
average of two to three mAbs each year over the past 25 years. At
not require both binding activities to be present in the same
this current approval rate, about 70 mAbs will be on the market by
molecule, such as inhibiting two signaling pathways by either
2020, and combined world-wide sales will be nearly $125 billion
blocking two receptors or two ligands (Figure 1E) [4,5,9–11].
[1]. However, despite the success, the limitations of mAb therapy
These latter applications can be achieved with combination
are also not negligible. For example, the majority of patients who
therapy or use of mixtures, which provide a great flexibility in
initially respond to mAb treatment, which works through blocking
the ratio and number of Abs [5,9]. Nevertheless, dual targeting
signaling pathways and the induction of cell death, eventually
with BsAbs has emerged as a powerful alternative to combi-
relapse due to extensive cross-talk among some signaling path-
nation therapy or mixtures. From a technological and regula-
ways [3].
tory perspective, this makes the development less complex
To overcome the shortcomings of mAb therapy and improve
because manufacturing, preclinical and clinical testing is
the treatment effectiveness, immunoglobulin gamma (IgG)-based
reduced to a single molecule [5,9]. Therapy with a single
bispecific antibodies (BsAb) have become increasingly of interest
dual-targeting drug rather than combinations should also be
by presenting advantages over the mAbs. Indeed, while mAbs are
less complicated for the administration of the therapeutic
monospecific, IgG-based BsAbs recognize simultaneously two dif-
Ab [5,9].
ferent epitopes either on the same or on different antigens (Ag) [4],
A major challenge in the engineering of IgG-based BsAbs is
enabling new therapeutic applications (Figure 1) [5]. Initially, ther-
to generate a pure BsAb without the presence of contaminat-
apeutic applications focused mainly on effector cell retargeting for
ing Abs (non-functional or monospecific molecules) [4,5,9–11].
cancer therapy, including T cells, which cannot be recruited to
Boosted by the Triomab quadroma technology (Frenesius
tumor cells by mAbs (Figure 1A) [6]. However, during the past
Biotech, Trion Pharma), which was described in a patent appli-
decade many other therapeutic strategies based on BsAbs have
cation filed in 1994 (Table 1) [13], it became possible to
been established [5], such as forcing the dimerization of co-factors
generate pure IgG-based BsAbs by fusion of two Ab-
CONTACT Christophe Blanchetot CBlanchetot@argenx.com

*
These authors contributed equally to this work.
© 2018 Informa UK Limited, trading as Taylor & Francis Group
2 M. GODAR ET AL.
candidates that enter phase I trials successfully ends up as a

Article highlights marketed product, and this high failure rate contributes sub-
● This review gives a comprehensive overview of the patent literature stantially to the high costs of biologic development [14]. To
over the period 1997-2017 of different immunoglobulin gamma- recover the expenses invested into research and development
based bispecific antibody platforms and the molecules approved or of new therapeutic BsAbs, patents are an indispensable tool, as
in clinical trials.
● Immunoglobulin gamma-based bispecific antibodies are increasingly they provide an exclusive right with respect to the protected
accepted as potentially superior therapeutic molecules in a broad subject matter. Indeed, only the patentee, or his licensee, is
range of diseases. allowed to exploit the invention commercially, e.g. by market-
● About 23 bispecific antibody platforms have generated about 62
molecules that are currently being evaluated for potential treatment ing the protected compound [15]. Additionally, patent protec-
of a variety of indications, such as cancer and inflammatory diseases, tion plays a crucial role in the biotechnology industry due to its
among which three molecules have been approved. reliance on rapidly changing technology [15]. Interestingly, it
● The quick advancements of bispecific antibody technologies require a
steady adaptation of patent strategies, to ensure that future products can be expected that patents related to the use of a BsAb will
will still be protected by intellectual property rights. have tremendous commercial value. This expectation is rein-
● This frantic activity creates a maze of hundreds of patents that pose forced by the fact that financial analysts expect sales of over
considerable legal risks for both newcomers and established
companies. $300 million for the second approved BsAb, blinatumomab
(Amgen, Micromet), by 2019, according to Thomson Reuters
This box summarizes key points contained in the article. Cortellis [16]. Not surprisingly, many companies have devel-
oped their own BsAb platforms, which has resulted in an impor-
tant patent activity in this field.
producing hybridomas, thereby yielding so-called hybrid With new formats, constantly emerging in this extremely fast-
hybridomas. Next, new platforms were developed to generate growing field, keeping track is a challenging task [5]. We opted for
pure BsAbs, and found their way into patent applications. clarity and have not included every single format that currently
About 23 BsAb platforms have been developed, allowing the exists. Only BsAb formats that have made it into the clinic are
generation of about 62 molecules which are currently being described in this review. Thus, our review gives a comprehensive
evaluated for potential treatment of a variety of indications, overview of the patent literature over the period 1997–2017 of
including cancer, chronic inflammatory diseases, autoimmu- different IgG-based BsAb platforms and the molecules approved
nity, neurodegeneration, bleeding disorders and infections, or in clinical trials.
with three molecules having been approved [4,5,9–11].
Nevertheless, translation of IgG-based BsAbs into treatments
is challenging, time-consuming and expensive as illustrated by 2. Patent review
the 9-year gap between the first patent application and the
European Medicines Agency’s approval of the first approved 2.1. New antibody formats patent protection
BsAb in 2009, catumaxomab, generated by using the Triomab The structure of an IgG molecule consists of two heavy chains
quadroma technology (Frenesius Biotech, Trion Pharma, (HC, comprising the CH3, CH2, hinge, CH1, and VH domains)
Table 2) [10]. Moreover, generally only about 1 in 10 therapeutic and two light chains (LC, comprising the CL and VL domains)
(a) (b) (c) (d)

Effector cell
Factor IXa Factor X
Blood Brain
Target cell Target cell
Blood-brain-barrier
(e) (f) (g) (h)

(b)
(a) Toxin or
antibody-drug conjugate
Drug-loaded nanoparticle
Target cell Target cell Target cell Target cell Target cell
Figure 1. Schematic representation of some therapeutic applications enabled by bispecific antibodies. (A) Retargeting cellular immunity (e.g., effector cells such as T
or natural killer cells) towards the target cell (e.g., a tumor cell). (B) Forcing the dimerization of co-factors to mimic a natural ligand (e.g., replacing missing factors
such as factor VIII to restore its activity in hemophilia A). (C) Delivering in the central nervous system by hijacking a transcytosis mechanism through binding with
one arm to a blood-brain-barrier receptor and targeting subsequently a specific target in the brain with the other arm. (D) Enhancing antibody binding affinity by
multiple epitopes binding (e.g., biparatopic antibody). (E) Modifying the host response by simultaneously inhibiting two distinct (a) membranes or (b) soluble
proteins. (F) Increasing killing specificities through simultaneous binding of both arms to cells expressing the two different targets. (G) Targeting of a bispecific toxin
(immunotoxin) or a bispecific antibody-drug conjugate to the target cell. (H) Targeting of a drug-loaded nanoparticle/liposome to the target cell. Strategies are
exemplified with a heteromeric immunoglobulin gamma-based bispecific antibody.
Table 1. Patent applications of immunoglobulin gamma-like asymmetric bispecific antibody formats classified by priority date.
Solutions to solve the light chain/heavy chain (LC/HC)
and/or heavy chain/heavy chain (HC/HC) mispairing Publication Priority Publication
Format problems Company number date date Inventor(s) Applicant(s) Ref.
Triomab quadroma LC/HC (species-specific LCs) and HC/HC (species-specific Frenesius Biotech, WO1995033844 03 June 14 December ● Horst Lindhofer [DE] ● GSF – [13]
protein A affinities) mispairing problems solved Trion Pharma A1 1994 1995 ● Stephan Thierfelder [DE] Forschungszentrum für
Umwelt und
Gesundheit GmbH [DE]
● Horst Lindhofer [DE]
● Stephan Thierfelder
[DE]
Knobs-into-Holes HC/HC mispairing problem solved by introducing Genentech WO1996027011 01 March 06 September ● Paul J. Carter [US] Genentech, Inc. [US] [32]
(KiH) mutations to create symmetric-to-asymmetric steric A1 1995 1996 ● Leonard G. Presta [US]
complementarity design: ● John B. Ridgway [US]
● CH3 domain a (knobs): T366Y
● CH3 domain b (holes): Y407T
HC/HC mispairing problem solved by introducing Genentech WO1998050431 02 May 14 January ● Robert Arathoon [US] Genentech, Inc. [US] [33]
mutations to create symmetric-to-asymmetric steric A3 1997 1999 ● Paul J. Carter [US]
complementarity design: ● Anne M. Merchant [US]
● Leonard G. Presta [US]
● CH3 domain a (knobs): S354C, T366W
● CH3 domain b (holes): Y349C, T366S, L368A, Y407V
Asymmetric Re- LC/HC (common LCs by framework/complementarity- Chugai WO2006106905 31 March 12 October ● Tomoyuki Igawa [JP] ● Chugai Pharmaceutical [37]
engineering determining regions shuffling) and HC/HC mispairing A1 2005 2006 ● Hiroyuki Tsunoda [JP] Co., Ltd [JP]
Technology- problems solved by introducing mutations to create ● Tomoyuki Igawa [JP]
Immunoglobulin electrostatic steering effects: ● Hiroyuki Tsunoda [JP]
(ART-Ig)
● CH3 domain a (positive charges): E356K, E357K,
D399K
● CH3 domain b (negative charges): K439E, K370E,
K409D
● Purification solved by isoelectric point engineering Chugai WO2007114325 31 March 11 October ● Tomoyuki Igawa [JP] ● Chugai Pharmaceutical [37]
A1 2006 2007 ● Hiroyuki Tsunoda [JP] Co., Ltd [JP]
● Tomoyuki Igawa [JP]
● Hiroyuki Tsunoda [JP]
CrossMAb LC/HC mispairing problem solved with: Roche WO2009080251 21 December 02 July ● Christian Klein [DE] ● F. Hoffmann-La Roche [35]
VH-VL A1 2007 2009 ● Wolfgang Schaefer [DE] Ag [CH]
● CrossMAb by exchanging the VH and VL ● Christian Klein [DE]
domains ● Wolfgang Schaefer [DE]
● CrossMAbCH1-CL by exchanging the CH1 and CL
domains
● CrossMAbFab by exchanging the VH-CH1 and VL-CL
domains
BiMAb LC/HC (common LCs) and HC/HC mispairing problems Oncomed WO2010129304 27 April 24 February ● Austin L. Gurney [US] ● Oncomed [38]
solved by introducing mutations to create A3 2009 2011 ● Aaron K. Sato [US] Pharmaceuticals, Inc.
electrostatic steering effects, e.g. in a human IgG2: [US]
● Austin L. Gurney [US]
● CH3 domain a: K249E, K288E ● Aaron K. Sato [US]
● CH3 domain b: E236K, D278K
FcΔAdp LC/HC (common LCs) and HC/HC mispairing problems Regeneron WO2010151792 26 June 29 December ● Samuel Davis [US] ● Regeneron [26]
solved by introducing mutations to create differential A1 2009 2010 ● Eric Smith [US] Pharmaceuticals, Inc.
protein A affinity: ● Douglas MacDonald [US] [US]
● Kara L. Olson [US] ● Samuel Davis [US]
EXPERT OPINION ON THERAPEUTIC PATENTS
● CH3 domain a: H435R ● Eric Smith [US]

● CH3 domain b: none ● Douglas MacDonald
[US]
3
● Kara L. Olson [US]
(Continued )
4
Table 1. (Continued).
Solutions to solve the light chain/heavy chain (LC/HC)
and/or heavy chain/heavy chain (HC/HC) mispairing Publication Priority Publication
Format problems Company number date date Inventor(s) Applicant(s) Ref.
M. GODAR ET AL.
XmAb LC/HC (Fab-scFv-Fc) and HC/HC mispairing problems Xencor WO2011028952 02 september 10 March ● Gregory A. Lazar [US] ● Xencor, Inc. [US] [45]
solved by introducing mutations (HA-TF): A1 2009 2011 ● Gregory L. Moore [US] ● Gregory A. Lazar [US]
● Gregory L. Moore [US]
● CH3 domain a: S364H, F405A
● CH3 domain b: Y349T, T394F
DuoBody LC/HC (controlled Fab-arm exchanged) and HC/HC Genmab WO2011131746 20 April 29 December ● Aran F. Labrijn [NL] ● Genmab A/S [DK] [48]
mispairing problems solved by introducing A3 2010 2011 ● Joyce Meesters [NL] ● Aran F. Labrijn [NL]
mutations: ● Ewald Van Den Bremer ● Joyce Meesters [NL]
[NL] ● Ewald Van Den Bremer
● CH3 domain a: F405L ● Joost J. Neijssen, [NL]
● CH3 domain b: K409R ● Patrick Van Berkel [NL] ● Joost J. Neijssen,
● Bart De Goeij [NL] ● Patrick Van Berkel [NL]
● Tom Vink [NL] ● Bart De Goeij [NL]
● Jan Van De Winkel [NL] ● Tom Vink [NL]
● Janine Schuurman [NL] ● Jan Van De Winkel [NL]
● Paul Parren [NL] ● Janine Schuurman [NL]
● Paul Parren [NL]
Azymetric LC/HC (orthoFab-Ig) and HC/HC mispairing problems Zymeworks WO2012058768 05 November 28 June ● Eric E. Cabrera [CA] ● Zymeworks, Inc. [US] [43]
solved by introducing mutations (ZW1): A8 2010 2012 ● Thomas S. Von ● Eric E. Cabrera [CA]
Kreudenstein [CA] ● Thomas S. Von
● CH3 domain a: T350V, L351Y, S400E, F405A, Y407V ● Surjit B. Dixit [CA] Kreudenstein [CA]
● CH3 domain b: T350V, T366L, N390R, K392M, T394W ● Paula I. Lario [CA] ● Surjit B. Dixit [CA]
● David K.Y. Poon [CA] ● Paula I. Lario [CA]
● Igor E. P. D’angelo [CA] ● David K.Y. Poon [CA]
● Igor E. P. D’angelo [CA]
Bispecific LC/HC (scFv-Fab) and HC/HC mispairing problems Glenmark WO2012131555 25 March 27 December ● Stanislas Blein [CH] ● Glenmark [51]
Engagement by solved by introducing residues: A3 2011 2012 ● Darko Skegro [CH] Pharmaceuticals S.A.
Antibodies based ● Paul Wassmann [CH] [CH]
● CH3 domain a: residues from T-cell receptor α
on the T-cell ● Stanislas Blein [CH]
receptor (BEAT) interface ● Darko Skegro [CH]
● CH3 domain b: residues from T-cell receptor β inter- ● Paul Wassmann [CH]
face
Biclonics LC/HC (common LCs generated by using the transgenic Merus WO2013157953 20 April 24 October ● Cornelis A. De Kruif [NL] ● Merus B.V. [NL] [41]
mouse MeMo and phage display libraries) and HC/HC A1 2012 2013 ● Linda J.A. Hendriks [NL] ● Cornelis A. De Kruif [NL]
mispairing problems solved by introducing ● Ton Logtenberg [NL] ● Linda J.A. Hendriks [NL]
mutations: ● Ton Logtenberg [NL]
● CH3 domain a: T366K (+L351K)
● CH3 domain b: L351D or E or D at Y349, L368, or
Y349 + R355
Table 2. Immunoglobulin gamma-like asymmetric bispecific antibodies approved or in clinical trials.
Publication number, priority date, publication
Format Molecule Targets Mode of action Indication Status of clinical trials Developed by date, inventor(s), and applicant(s) Ref.
Triomab quadroma Catumaxomab ● EpCAM T cell recruitment, Malignant ascites Marketed, approved in Neovii Biotech, Trion ● WO2002020039 A3 [31]
● CD3 Fc-mediated 2009 by the Pharma ● 04 september 2000
effector function European Medicines ● 25 July 2002
Agency ● Horst Lindhofer [DE]
● Horst Lindhofer [DE]
Asymmetric Re- Emicizumab, ● FIX 2-Factor Hemophilia A Marketed, approved in Chugai, Roche ● WO2006109592 A1 [39]
engineering ACE-910, ● FX dimerization 2017 by the United ● 08 April 2005
Technology- RG-6013, States Food and ● 19 October 2006
Immunoglobulin RO-5534262 Drug Administration ● Kunihiro Hattori, Tetsuo Kojima, Hiroyuki
(ART-Ig) Saito, Taro Miyazaki, Tetsuhiro Soeda
● Chugai pharmaceutical Co., Ltd [JP]
ERY-974 ● CD3 T-cell recruitment Solid tumors I Chugai ● WO2011078332 A1 [17]
● GPC3 ● 25 December 2009
● 30 June 2011
● Tomoyuki Igawa [JP], Zenjiro Sampei [JP],
Tetsuya Wakabayashi [JP], Eriko Ito [JP]
● Chugai pharmaceutical Co., Ltd [JP],
Tomoyuki Igawa [JP], Zenjiro Sampei [JP],
Tetsuya Wakabayashi [JP], Eriko Ito [JP]
CrossMAb and/or Vanucizumab, ● Angiopoietin 2 2-Ligand Colorectal cancer II Roche ● WO2011117329 A1 [36]
Knobs-into-Holes RG-7221, ● VEGF inactivation ● 26 March 2010
(KiH) RO-5520985 ● 29 September 2011
RG-7716, ● Angiopoietin 2 2-Ligand Diabetic macular II Roche ● Monika Baehner [DE], Sabine Imhof-Jung [18]
RO-6867461 ● VEGF inactivation edema, wet age- [DE], Anita Kavlie [NO], Hubert
related macular Kettenberger [DE], Christian Klein [CH],
degeneration Joerg T. Regula [DE], Wolfgang Schaefer
[DE], Juergen M. Schanzer [DE], Werner
Scheuer [DE], Kay-Gunnar Stubenrauch
[DE], Markus Thomas [DE]
● F. Hoffmann-La Roche Ag [CH], Monika
Baehner [DE], Sabine Imhof-Jung [DE],
Anita Kavlie [NO], Hubert Kettenberger
[DE], Christian Klein [CH], Joerg T. Regula
[DE], Wolfgang Schaefer [DE], Juergen M.
Schanzer [DE], Werner Scheuer [DE], Kay-
Gunnar Stubenrauch [DE], Markus Thomas
[DE]
RG-7802, ● CD3 T-cell recruitment Solid tumors I Roche ● WO2013026833 A1 [19]
RO-6958688 ● CEA ● 23 August 2011
● 28 February 2013
● Oliver Ast [CH], Peter Bruenker [CH], Tanja
Fauti [CH], Anne Freimoser-Grundschober
[CH], Christiane Jaeger [CH], Christian Klein
[CH], Ekkehard Moessner [CH], Pablo
Umana [CH]
● Roche Glycart Ag [CH], Oliver Ast [CH],
Peter Bruenker [CH], Tanja Fauti [CH], Anne
Freimoser-Grundschober [CH], Christiane

Jaeger [CH], Christian Klein [CH], Ekkehard
Moessner [CH], Pablo Umana [CH]
5
(Continued )
6
RG-7386, ● FAP Tumor site-specific Solid tumors I Roche ● WO2014161845 A1 [20]
RO-6874813 ● DR5 cell apoptosis ● 03 April 2013
M. GODAR ET AL.
(programmed ● 09 October 2014

cell death) ● Peter Bruenker [CH], Sherif Daouti [US],
Ningping feng [CA], Koller C. Ferrara [CH],
Guy Georges [DE], Sandra Grau-Richards
[CH], Ralf Hosse [CH], Christian Klein [CH],
Maximiliane Koenig [DE], Joerg Moelleken
[DE], Ekkehard Moessner [CH], Huifeng Niu
[US], Kathryn E. Packman [US], Valeria
Runza [DE], Stefan Seeber [DE], Pablo
Umana [CH], Inja Waldhauer [CH],
Huisheng Wang [US], Barbara Weiser [DE]
● Roche Glycart Ag [CH]
RG-6026 ● CD3 T-cell recruitment Relapsed or refractory I Roche ● WO2016020309 A1 [21]

● CD20 non-Hodgkin’s ● 04 August 2014
lymphoma ● 11 February 2016
● Oliver Ast [CH], Marina Bacac [CH], Sabine
Imhof-Jung [DE], Christiane Jaeger [CH],
Christian Klein [CH], Stefan Klostermann
[DE], Michael Molhoj [DE], Joerg T. Regula
[DE], Wolfgang Schaefer [DE], Pablo Umaña
[CH]
● F. Hoffmann-La Roche Ag [CH], Hoffmann-
La Roche Inc. [US]
RG-7828, ● CD3 T-cell recruitment Chronic lymphocytic I Genentech ● WO2016204966 A1 [5]
BTCT-4465A ● CD20 leukemia, non- ● 16 June 2015
Hodgkin’s ● 22 December 2016
lymphoma ● Chingwei V. Lee [US], Mark S. Dennis [US],
Germaine Fuh [US]
● Genentech, Inc. [US]
BiMAb Navicixizumab, ● DLL4 2-Ligand Colorectal cancer, I Oncomed, Celgene ● WO2013044215 A9 [38]
OMP-305B83 ● VEGF inactivation fallopian tube ● 23 September 2011
cancer, ovarian ● 03 April 2014
cancer, peritoneal ● Austin L. Gurney [US], Aaron K. Sato [US],
cancer, solid tumors Christopher J. Bond [US]
● Oncomed Pharmaceuticals, Inc. [US]
FcΔAdp REGN-1979 ● CD3 T-cell recruitment Acute lymphoblastic I Regeneron ● WO2014047231 A1 [40]
● CD20 leukemia, B cell ● 21 September 2012
lymphoma, chronic ● 27 March 2014
lymphocytic ● Eric Smith [US], Nicholas J. Papadopoulos
leukemia, non- [US]
Hodgkin’s ● Regeneron Pharmaceuticals, Inc. [US]
lymphoma
(Continued )
Biclonics MCLA-117 ● CD3 T-cell recruitment Acute myeloid I/II Merus ● WO2014051433 A1 [22]
● CLEC12A leukemia ● 27 September 2012
● 03 April 2014
● Alexander B. H. Bakker [NL], Pieter F. Van
Loo [NL], Ton Logtenberg [NL]
● Merus B.V. [NL]
MCLA-128 ● HER2/ErbB-2 2-Receptor Solid tumors I/II Merus ● WO2015130173 A1 [42]

● HER3/ErbB-3 tyrosine kinase ● 28 February 2014
inactivation ● 03 September 2015
● Cecilia A. W. Geuijen [NL], Cornelis A. De
Kruif [NL], Mark Throsby [NL], Ton
Logtenberg [NL], Alexander B. H. Bakker
[NL]
● Merus B.V. [NL]
Fab-scFv-Fc MEDI-3902 ● PsI 2-Antigen Nosocomial II MedImmune ● WO2014074528 A8 [23]
● PcrV inactivation pneumonia ● 06 November 2012
● 21 May 2015
● Antonio Digiandomenico [US], Paul
Warrener [US], Charles Stover [US], Bret
Sellman [US], Ralph Minter [GB], Sandrine
Guillard [GB], Steven Rust [GB], Miaden
Tomich [US], Vignesh Venkatraman [GB],
Reena Varkey [US], Li Peng [US], Melissa
Damschroder [US], Partha Chowdhury [US],
Nazzareno Dimasi [US], Ryan Fleming [US],
Binyam Bezabeh [US], Changshou Gao [US]
● MedImmune, Llc [US]
DuoBody JNJ-61186372 ● EGFR 2-Receptor Non-small cell lung I Janssen, Genmab ● WO2014081954 A1 [49]
● cMET tyrosine kinase cancer ● 21 November 2012
inactivation ● 30 May 2014
● Mark Chiu [US], Sheri Moores [US], Joost
Neijssen [NL], Paul Parren [NL], Janine
Schuurman [NL]
● Janssen Biotech, Inc. [US]
JNJ-63709178 ● CD3 T-cell recruitment Acute myeloid I Janssen, Genmab ● WO2016036937 A1 [50]
● CD123 leukemia ● 05 September 2014
● 10 March 2016
● Francois Gaudet [US], Ricardo Attar [US],
Benjamin C. harman [US], Yingzhe Li [US],
Jinquan Luo [US], Ronan Mcdaid [US],
Steven C. Pomerantz [US], Susan H. Tam
[US], Alexey Teplyakov [US], John Wheeler
[US], Sheng-Jiun Wu [US], Jennifer F.
Nemeth [US]
● Janssen Pharmaceutica N.V. [BE]
(Continued )
7
8 M. GODAR ET AL.
that fold into a complex quaternary Y-shaped structure
[53]
[44]
[46]
Ref.
(Figure 2A) [4,5,9–11]. IgGs can be further dissected into the
Ag-binding site (Fab), the hinge region and the stalk that forms
Matthew Bernett, Seung Chu [US], Rumana

Nina E. Weisser [CA], Gordon Y. K. Ng [CA],
Grant R. Wickman [CA], Surjit B. Dixit [CA],
Gregory Moore [US], John Desjarlais [US],

Eric Escobar-Cabrera [CA], Mario Sanches
the crystallisable fragment (Fc) region, comprising the CH2 and
Stanislas Blein [CH], Romain Ollier [CH],

date, inventor(s), and applicant(s)
Samuel Hou [CH], Darko Skegro [CH]

CH3 domains. The Fc region bears recognition motifs for bind-
Glenmark Pharmaceutical S.A. [CH]
Rashid [US], Umesh Muchhal [US]

ing innate immune receptors (Fc gamma receptors, C1q com-
plement and neonatal Fc receptor, FcRn) on an effector cell, and
is therefore responsible for mediating immune effector func-
Zymeworks, Inc. [US]

tions and in vivo IgG stability (Figure 2A) [24]. This part has been
09 September 2016
04 November 2013
27 November 2013
26 November 2014
WO2015077891 A1
WO2016086189 A3
WO2015063339A1
a prime molecular engineering target for either enhancing or
Xencor, Inc. [US]

04 June 2015
07 May 2015
inhibiting the immune response, including Ab-dependent cel-

lular cytotoxicity (ADCC), Ab-dependent cell-mediated phago-
[CA]
cytosis (ADCP), and complement-dependent cytotoxicity (CDC,
Figure 2A). Another important receptor of IgG-Fc is the FcRn,
●
●
●
●
●
●
●
●
●
●
●
●
●
which protect IgG and albumin from catabolism, which explains
the prolonged half-life of these two proteins compared with
other Igs and liver synthesized proteins. FcRn is also known to
Developed by
Novartis, Xencor
Novartis, Xencor
mediate bidirectional transcytosis of IgG across epithelial cells
Zymeworks
as well as membrane recycling. FcRn-IgG interaction also func-

Glenmark
tions in Ag presentation and cross-presentation in macrophage

and dendritic cells. Each of these functions has important impli-
cations in therapeutic Ab development (Figure 2A) [25]. This
Status of clinical trials
region also contains the binding site for protein A, a bacterial

protein produced by Staphylococcus aureus, extensively used
for the purification of Abs and recombinant proteins containing
I
the Fc fragment (Figure 2A) [26].

Therapeutic proteins that are derived from this general
IgG concept are commonly called ‘new Ab formats’ in a
patent point of view. Established formats such as chimeric
chronic lymphocytic
HER2/ErbB-2-positive
Abs (e.g. WO9007861A1 assigned to Protein Design Labs),

leukemia, non-
B cell lymphoma,
humanized Abs (e.g. WO1991009967A1 assigned to

Indication
malignancies
Ovarian cancer
Hematological
Celltech), Fab, and single chain variable fragments (scFv,

lymphoma
Hodgkin’s
cancers
e.g. WO8801649A1 assigned to Enzon) were, strictly speak-

ing, considered ‘new Ab formats’ when first introduced. The
basic concept of rearranging and recombining different com-
ponents of IgG was further pursued in the past decades and
T-cell recruitment
T-cell recruitment
T-cell recruitment
Receptor tyrosine
Mode of action
applied to BsAbs [15].

inactivation
kinase
2.2. Various bispecific antibody formats patented

IgGs are bivalent molecules comprising two identical Ag-bind-
Non-overlapping
ing sites. By contrast, IgG-based BsAbs are generally mono-

HER2/ErbB-2
HER2/ErbB-2
epitopes of
Targets
valent for each Ag and are artificial molecules, per se not

CD123
CD20
found in nature (except for human IgG4 [27]). They must,

CD3
CD3
CD3
therefore, be generated by biochemical, molecular, or genetic

●
●
●
●
●
●
means. The generation of IgG-based BsAbs is difficult because

the Ag-binding sites are built by the variable domains of the
LC and HC (VL and VH, respectively, Figure 2A) [4,5,9–11]. Two
Molecule
XmAb-14045
XmAb-13676
challenges of generating BsAbs are to direct and to force

GBR-1302
correct pairing of correct binding sites, i.e. HCs and LCs, and
ZW-25
of the HCs. Various strategies have been developed to solve

this problem and have been the subject of patent applications
over the past decades. With many companies in the BsAb field,
Antibodies based
Engagement by
receptor (BEAT)
Azymetric (ZW1)
and with many potential targets to hit (made possible thanks

on the T-cell
to the therapeutic applications allowed by BsAbs, Figure 1),

Bispecific
about 23 different IgG-based BsAb formats have been devel-

Format
XmAb
oped, which can be discriminated by the presence or absence

of an Fc region (Figure 2A) [4,5,9–11].
EXPERT OPINION ON THERAPEUTIC PATENTS 9
HC
(a) LC
(b)
VH Antigen-binding site
Fab Fv CH1
VL VH
(50 kDa)
Peptide linker
CL Fc gamma receptor binding ADCC and ADCP
Hinge VL
CH2 C1q binding CDC
Fc
(50 kDa) FcRn Half-life
CH3 Protein A Purification
Immunoglobulin gamma Single-chain Fv
(150 kDa) (25 kDa)
Figure 2. Schematic representation of a human immunoglobulin gamma and a derived format. (A) The structure of an immunoglobulin gamma molecule consists of
two heavy (HC, comprising the CH3, CH2, hinge, CH1 and VH domains) and two light chains (LC, comprising the CL and VL domains) that fold into a complex
quaternary Y-shaped structure. An immunoglobulin gamma molecule can be further dissected into the antigen-binding site (Fab), the hinge region and the stalk
that forms the crystallisable fragment (Fc) region, comprising the CH2 and CH3 domains. The Fc region bears recognition motifs for binding innate immune
receptors (Fc gamma receptors, C1q complement and neonatal Fc receptor proteins, FcRn) on an effector cell, and is therefore responsible for mediating immune
effector functions and in vivo IgG stability. This region also contains the binding site for protein A, extensively used for the purification of Abs and recombinant
proteins containing the Fc fragment. (B) The single chain variable fragment (scFv) format is the most commonly used derivative of the VH and VL domains
(connected in a flexible manner through a peptide linker), representing the minimal antigen-binding site of an antibody.
BsAbs that include an Fc region can be further divided into mouse IgG2a and rat IgG2b hybridomas, solving the LC/
those that exhibit a structure resembling that of an IgG mole- HC mispairing problem [13]. Because rat Abs cannot bind to
cule and those that contain additional binding sites, i.e. with an protein A whilst mouse Abs have a high affinity to protein A,
appended or modified IgG-like structure [4,5,9–11]. The differ- undesired homologous rat HC pairings are removed when the
ent BsAbs will have either a symmetric (homodimeric BsAbs) or quadroma products flow through the protein A purification
an asymmetric (heterodimeric BsAbs) architecture (Figures 3A column. Undesired homologous mouse HC pairings tightly
and 3B). Most bispecific IgG molecules are asymmetric, while bound to protein A by their two sites of two mouse HCs, the
IgG fusion proteins are often symmetric in their molecular desired rat/mouse BsAbs are moderately bound to protein A.
composition [4,5,9–11]. These two approaches enable to keep Thus, rat/mouse BsAbs can be easily eluted whereas parental
the native IgG structure (immunogenicity and half-life), while mouse Abs are stably retained in the protein A column.
still providing flexibility in terms of how to achieve bispecificity. Therefore, through the purification by protein A, the HC/HC
By contrast, BsAbs lacking the Fc region (e.g. Fc-mediated mispairing problem is solved. Nevertheless, these molecules
effector functions, Figure 2A) have also been engineered. This suffered from low production yields, heterogeneity, and
approach allowed for example the development of scFv-based human anti-mouse Ab response that accelerated clearance
molecules (Figures 2B and 3C). Each of these three different and therefore quickly decreased efficacy in patients [30].
BsAb formats brings different properties in binding valency for However, one such BsAb catumaxomab (EpCAM × CD3,
each Ag, geometry of Ag-binding sites, pharmacokinetic half- Neovii Biotech, Trion Pharma, Table 2), was the first BsAb
life and in some cases effector functions [4,5,9–11]. approved by the European Medicines Agency in 2009 for the
treatment of patients with epithelial cancer-associated malig-
nant ascites [31].
2.3. Immunoglobulin gamma-like asymmetric bispecific
antibodies 2.3.2. KiH
2.3.1. Triomab quadroma A breakthrough in solving the HC/HC mispairing problem was
The idea to combine the halves of two different Y-shaped Ab the emergence of HC engineering for heterodimerization. HCs
molecules seemed promising, but some problems were were first engineered for heterodimerization in the 1990s
encountered with producing and/or purifying the desired using the ‘Knobs-into-Holes’ (KiH) technology (Genentech),
BsAb. Indeed, all HCs can pair with each other and every LC protected by a patent application filed in 1995 (Table 1)
can associate randomly with two regions at the top of the HCs, [32,33]. The interaction between the CH3 domains is the key
causing HC/HC and LC/HC mispairing problems, respec- factor that promotes the assembly of two HCs and the forma-
tively [4,5,9–11]. To counter these mispairing problems, IgG- tion of two interchain disulfide bonds in the hinge (Figure 2A),
based BsAbs were initially generated by the fusion of two which forms the basis of engineering of the CH3 domain to
different hybridoma cells harboring different specificities, realize the KiH technology. Indeed, KiH-BsAbs rely on the
which resulted in a ‘quadroma’ cell line. These hybrid cells introduction of a bulky residue into the CH3 domain of one
potentially produce 16 different combinations (10 different HC and acts similarly to a key, whereas a ‘hole’ is formed in the
molecules) with only one being bispecific and the remaining CH3 domain of the other HC which, being able to accommo-
pairings resulting in non-functional or monospecific molecules date this bulky residue, mimics a lock, thereby creating a
[4,28]. The rat/mouse Triomab quadroma technology symmetric-to-asymmetric complementarity design. In this
(Frenesius Biotech, Trion Pharma) for which a patent applica- way, the HCs of parental mAbs that are expressed in the
tion was filed in 1994, solved both LC/HC and HC/HC mispair- same cell can correctly pair and assemble into the BsAbs
ing problems (Figure 3Aa and Table 1) [13,29]. Indeed, these (Figure 3Ab) [32,33]. KiH technology enables, therefore, correct
BsAbs consist of two half Abs that originate from parental HCs association, but it cannot prevent the undesired pairing of
10 M. GODAR ET AL.
Human immunoglobulins gamma (A) Asymmetric bispecific antibodies

a b a b a b a b a b a b
VHa VHb VHa VHb VHa VLb VHa VHb VHa VHb VHa VHb
VLa VLb VLa VLb VLa CLb VHb
CH1b
Mouse Rat
IgG2a IgG2b +- Fc* *
+-
(a) Triomab quadroma (b) KiH (c) CrossMAbCH1-CL/KiH (d) ART-Ig (e) BiMAb (f) FcΔAdp
a a b a a b a
VHa VHb b VHa VHb VHa VHb
VHa b VHa b
VLa VLb VLa VHb VLa VLb VLa VHb
VLb VLb
+-
Controlled
Fab-arm TCRα TCRβ
** ** ** exchanged ** **
a a b b
VHa VHb (g) Biclonics (h) Azymetric (i) XmAb (j) Duobody (k) BEAT
VLa CH1a VLb CH1b
(B) Symmetric bispecific antibodies
CLa CLb
CH2a b b
CH2b VHb a a a/b a/b
CH3a CH3b VHa
VLb VHa VHa/b
IgGa IgGb VLa VLa VLa/b
Fynomer
b
(l) DVD-Ig (m) FinomAb (n) Two-in-one/DAF
(C) Fragment-based bispecific antibodies

a
a a
VHa a VHa
a b VHa VLa a
VLa b VLa a VLb
VHa VHb VLb VHb VHb
VLa VHa Cβ Vβ T-cell
VHb VLb Cα Vα receptor
VLa-S-S- VLb b VLa
VHb VLb VLa VHa VHa
VLb VLa VHa
b VLb
VHb
b a
VHb
Camelid heavy-chain only b
(o) DNL-Fab3 (p) DART (q) TandAb (r) BiTE (s) Adaptir (t) ImmTAC (u) EDV
antibodies
a a b b a
VHHa VHHb VHHa
CH2a CH2b VHHb

CH3a CH3b b
IgGa IgGb (v) Nanobody-based
Figure 3. Schematic representation of different immunoglobulin gamma-based bispecific antibody formats. (A) Asymmetric bispecific antibodies are designed such
that the variable domains (VL and VH) at the tip of each arm bind to a different target, resulting in bivalent heterodimeric molecules. Some examples of asymmetric
technologies: (a) Triomab quadroma, (b) Knobs-into-Holes (KiH), (c) CrossMAb/KiH, (d) Asymmetric Re-engineering Technology-Immunoglobulin (ART-Ig), (e) BiMAb,
(f) FcΔAdp, (g) Biclonics, (h) Azymeric, (i) XmAb, (j) DuoBody and (k) Bispecific Engagement by Antibodies based on T-cell receptor (BEAT). (B) Symmetric bispecific
antibodies are generated by fusion of an additional binding site to the heavy/light chains, such as the (l) Dual Variable Domain-Immunoglobulin (DVD-Ig) and (m)
FinomAb formats, or by making differential but overlapping use of the light and heavy complementarity determining regions as main contacts for each antigen,
such as the (n) Two-in-one/Dual Action Fab (DAF) format. (C) Alternatively, antigen-binding fragments can be connected through peptide linkers, resulting in
fragment-based bispecific antibodies. Some examples of fragment-based bispecific technologies: (o) Dock-aNd-Lock-Fab3 (DNL-Fab3), (p) Dual-Affinity Re-Targeting
proteins (DART), (q) Tandem diAbody (TandAb), (r) Bispecific T-cell Engager (BiTE) and (s) Adaptir. Antigen-binding fragments can also be coupled to T-cell receptors,
such as the (t) Immune-mobilizing monoclonal T-cell receptors Against Cancer (ImmTAC) format, or with drug-loaded particles, such as the (u) EngeneIC Delivery
Vehicle (EDV) format. Otherwise, the smallest intact functional antigen-binding fragment of camelid heavy-chain only antibodies, also called single-domain VHHs or
nanobodies, can be used to create bispecific antibodies, such as the (v) nanobody-based bispecific antibody format.
the LCs associated with the two different HCs, because the together to overcome both LC/HC and HC/HC mispairing
molecular architectures of VH and VL, and CH1 and CL problems. For example, vanucizumab (Ang-2 × VEGF), for
domains in both arms of a BsAb are identical (Figure 2A). which a patent application was filed in 2010 and which is
currently in phase II for the treatment of colorectal cancer
2.3.3. CrossMAb (Table 2) [36], carries the original unmodified anti-VEGF bev-
The CrossMAb technology, developed by Roche and described acizumab Fab on the knob arm, whereas the anti-Ang-2 LC06-
in a patent application filed in 2007 (Table 1), is a technology bearing arm is a Fab carrying a CH1-CL domain crossover [35].
that solves the LC/HC mispairing problem by exchanging LC
and HC domains within the Fab of one half of the BsAb 2.3.4. ART-Ig and BiMAb
[4,34,35]. CrossMAbs have no linkers and prevent association Another solution to create heterodimers of BsAbs by solving
of the LCs to its heavy counterparts to prevent unwanted side the HC/HC mispairing problem was identified by rational
products. This ‘crossover’ still retains the original Ag-binding design of electrostatic steering mutations, i.e. the introduction
affinity, but makes the two arms so different that only the of mutations in the CH3 domains to create altered charge
specific interaction can take place. There are three ways for polarity across the interface region of the Fc. For example,
this change: the exchange of VHb and VLb, the exchange of the Fc of an IgGa is positively charged and the Fc of an IgGb is
CH1b and CLb, depicted in Figure 3Ac, and the exchange of negatively charged. Thus, one half of IgGa and the other half
VHb-CH1b and VLb-CLb, all of which occur at the gene level of IgGb preferentially form heterodimers through favorable
[4,34,35]. The CrossMAb and KiH technologies are used attractive interactions, while unfavorable repulsive charge
interactions suppress unwanted Fc homodimer formation. homodimers [43]. This Fc design, termed Azymetric
Several companies found such mutations and filed patent (Figure 3Ah), was developed by Zymeworks and described
applications, such as Chugai in 2005 with the Asymmetric in a patent application that was filed in 2010 (Table 1) [43].
Re-engineering Technology-Immunoglobulin (ART-Ig, Table 1 An orthoFab-Ig BsAb, termed ZW-25, targeting non-overlap-
and Figure 3Ad) [37] and Oncomed in 2009 with the BiMAb ping epitopes of HER2/ErbB-2, has been generated by using
technology (Table 1 and Figure 3Ae) [38]. Notably, the LC/HC this technology and is currently in phase I for the treatment
mispairing problem was solved for both technologies by using of ovarian cancer (Table 2) [44].
common LCs. These platforms led for example to the genera-
tion of ART-Ig emicizumab (FIX × FX), for which a patent
application was filed in 2005 and which was approved by 2.3.8. XmAb
the FDA in 2017 to restore the function of activated factor Otherwise, use of structure- and sequence-based
VIII, which is deficient in persons with hemophilia A (Table 2) approaches to explore energies of paired variant combina-
[39], and BiMAb navicixizumab (DLL4 × VEGF), for which a tions at the interface across the CH3 dimer yielded a HA-TF
patent application was filed in 2011 and which is currently in variant, termed XmAb (Figure 3Ai) and developed by Xencor
phase I for the treatment of different cancers, such as color- (patent application filed in 2009, Table 1) [45]. This technol-
ectal and ovarian cancers (Table 2) [38]. ogy led to the generation of a Fab-scFv-Fc molecule, XmAb-
14045, co-targeting CD3 and CD123, which is currently in
2.3.5. FcΔAdp phase I for the treatment of hematological malignancies
Another technology using common LCs, termed FcΔAdp, (Table 2) [46].
was developed by Regeneron and described in a patent
application filed in 2009 [26]. The technology is based on 2.3.9. DuoBody
a single common LC and two distinct HCs, which combine Inspired by the natural process of the Fab arm exchange
to form the heterodimeric BsAb. One of the HCs contains a of human IgG4 isotype in human serum [27], a technology
chimeric Fc sequence form (called Fc*, Table 1 and was developed by exploiting the CH3/CH3 dimer interface
Figure 3Af) that ablates binding to protein A via the con- of IgG4 [47]. This technology allows the generation of a
stant region. After the co-expression of the two HCs and the stable bispecific IgG4 [47] but also IgG1 [48], termed con-
common LC, three products are created, two of which are trolled Fab-arm exchange, and commercialized as the
homodimeric for the HCs and one that is the desired het- DuoBody platform (Genmab, Figure 3Aj) [48], for which a
erodimeric BsAb. The Fc* sequence allows selective purifica- patent application was filed in 2010 (Table 1). The method
tion of the FcFc* bispecific product on commercially involves the separate expression of two IgG1 mAbs, each
available affinity columns, due to intermediate binding affi- one containing a single matched point mutation at the
nity for protein A compared to the high avidity FcFc HC CH3-CH3 domain interface. The parental Abs are then
homodimer, or the weakly binding Fc*Fc* homodimer [26]. mixed and subjected to controlled reducing conditions in
Using this technology, REGN-1979, targeting the B cell mar- vitro that separate the Abs into half-molecules and allow
ker CD20, and the CD3 component of the T-cell receptor reassembly and reoxidation to form pure IgG1 BsAbs.
(TCR), which triggers redirected killing of B cells, has been Indeed, the matched mutations drive the efficient recom-
generated and is currently in phase I for treatment of acute bination of binding arms in a unidirectional way because
lymphoblastic leukemia (Table 2) [40]. the mutations are selected to weaken the non-covalent
CH3-CH3 interaction in the parental IgG1 mAbs favoring
2.3.6. Biclonics heterodimeric interaction [48]. Two Duobody BsAbs, JNJ-
To facilitate BsAb generation with a common LC, Merus devel- 61186372 (EGFR × cMET) [49] and JNJ-63709178 (CD3 ×
oped a transgenic mouse, termed MeMo, engineered to gen- CD123) [50], are currently in phase I for non-small cell lung
erate BsAbs with a single human common LC and diverse cancer and acute myeloid leukemia, respectively (Table 2).
human HCs. The MeMo technology, combined with their
own CH3-domain engineered heterodimeric Fc technology,
allows them to generate intact IgG-format BsAbs, called 2.3.10. BEAT
Biclonics (Figure 3Ag), for which a patent application was Glenmark’s technology also turned to nature to solve the
filed in 2012 (Table 1) [41]. Consecutively, a Biclonics-based HC/HC mispairing problem and designed a bio-mimicry
BsAb, MCLA-128, targeting HER2/ErbB-2 × HER3/ErbB-3, is cur- strategy to establish a stable heterodimer. Indeed, hijacking
rently being evaluated in phase I/II for patients with solid the TCR constant domain alpha/beta interface found on
tumors (Table 2) [42]. natural T-cells and grafting it onto the CH3 homodimer
interface results in two different HCs that readily assemble
2.3.7. Azymetric [51,52]. This technology, described in a patent application
Further, structure-guided design assisted with the computa- filed in 2011 (Table 1), is commercialized as the Bispecific
tional algorithm and optimized energy function in between Engagement by Antibodies based on the TCR (BEAT,
the CH3 partner chains were used to improve biophysical Figure 3Ak) platform. One such BEAT BsAb, GBR-1302 (CD3
properties of bispecific Ig molecules, i.e. resulting in a set of × HER2/ErbB-2), for which a patent application was filed in
mutations that were reported to have a high thermal stabi- 2013, is currently in phase I for HER2-positive cancers
lity and to form pure heterodimers with no detectable (Table 2) [53].
12 M. GODAR ET AL.
2.4. Immunoglobulin gamma-like symmetric bispecific
[54]
[66]
[65]
Ref.
antibodies
2.4.1. DVD-Ig
Abbott Laboratories [US]
Jenny M. Bostrom [US]

Besides asymmetric BsAbs, homodimerized BsAbs have been
Richard W. Dixon [US]

Jochen G. Salfeld [US]
Genentech, Inc. [US]
getting increasing attention and thereby forming the subject
Germaine Fuh [US]

Chengbin Wu [US]
Tariq Ghayur [US]
Applicant(s)
of patent applications [4,5,9–11]. Monospecific IgGs can be
Covagen Ag [CH]
engineered for bispecificity by appending either the amino
or carboxy termini of either LCs or HCs with additional Ag-
binding units. One such appended IgG format, which has
reached clinical development is the Dual Variable Domain-Ig
●
●
●
●
●
●
●
●
(DVD-Ig, Figure 3Bl), protected by a patent application filed by
Abbott in 2006 (Table 3) [54]. DVD-Ig have been generated by
Susann König-Friedrich [DE]

Isabella Attinger-Toller [CH]
appending the VL and VH domains of an IgG with similar
Dragan Grabulovski [CH]
Ulrike Von Der Bey [DE]

Julian Bertschinger [CH]
Jenny M. Bostrom [US]
Richard W. Dixon [US]
Roger Santimaria [CH]

Jochen G. Salfeld [US]
domains from a second Ab via short peptide linkers. DVD-Ig
Kristina Klupsch [CH]
Helen Hachemi [CH]

Richard Woods [CH]
Michela Silacci [CH]
Patricia Henne [CH]

Ulrich Wüllner [CH]
Irene Zbinden [CH]

Germaine Fuh [US]
Chengbin Wu [US]
Fabian Buller [CH]

Inventor(s)
are bispecific and bivalent for each Ag specificity, preserving
Tariq Ghayur [US]
Simon Brack [CH]

the natural Ab avidity to cell surface receptors or dimeric Ags
and providing an extended distance between the bound epi-
topes by placing the additional binding domains at the distal
ends of the HCs [55]. One potential advantage of appended
bispecific IgGs is that they can enable the simultaneous bind-
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
ing of Ag to all variable domains and hence provide a higher
specific binding capacity [56]. This may be useful in targeting
04 September 2008
18 December 2008
Publication date
23 October 2014
low abundance proteins such as cytokines and may enable
longer dosing intervals. For example, DVD-Ig lutikizumab, tar-
geting the cytokines IL-1α × IL-1β and for which a patent
Table 3. Patent applications of immunoglobulin gamma-like symmetric bispecific antibody formats classified by priority date.
application was filed in 2006, is currently in phase II for the

treatment of osteoarthritis (Table 4) [57].
18 August 2006
30 August 2006
2.4.2. Finomab
Priority date
19 April 2013
With various scaffold proteins being developed as alternatives
to Abs, there are opportunities to generate appended IgG-
based BsAbs by fusing these scaffold proteins to the HCs or
LCs of an IgG [5]. An example is the Fynomer, a small 7 kDa
globular proteins derived from the SH3 domain of human Fyn
Publication number
WO2008024188 A3
WO2008027236 A3
WO2014170063 A1
kinase, which lacks disulfide bonds and is very stable (≈70°C

thermal melting points [64]). Fusion of an IL-17A-specific
Fynomer to either the amino or carboxy terminus of the HCs
or LCs of the anti-TNF adalimumab resulted in a BsAb, called
FynomAb COVA-322 developed by Covagen (Figure 3Bm).
COVA-322, described in a patent application filed in 2009
(Table 4), is currently being evaluated in a phase I/II for treat-
Genentech
Company
Covagen
ment of plaque psoriasis [65].

Abbott
2.4.3. Two-in-one/DAF
The LC/HC and HC/HC mispairing problems can also be over-
Dual Variable Domain-Immunoglobulin (DVD-Ig)
come by using a single kind of HCs and LCs and engineering

the variable domains to recognize two unrelated Ags via two
different binding sites on the Ab. Such so called ‘two-in-one’
Abs, also known as Dual Action Fab (DAF), have been devel-
Two-in-one/Dual Action Fab (DAF)
oped by Genentech and described in a patent application filed

in 2006 (Table 3 and Figure 3Bn), make differential but over-
lapping use of the LC and HC complementarity determining
regions as main contacts for each Ag [66]. Attractive features
of two-in-one Abs are that they have a single kind of LC and
HC and are amenable to standard production processes devel-
FinomAb
oped for mAbs. A downside is that sophisticated upfront

Format
engineering is required to create these formats, which is not

invariably successful. Two-in-one BsAbs have overlapping Ag-
Table 4. Immunoglobulin gamma-like symmetric bispecific antibodies in clinical trials.
Status
of
clinical Publication number, priority date, publication date, inventor(s) and
Format Molecule Targets Mode of action Indication trials Developed by applicant(s) Ref.
Dual Variable Domain- Lutikizumab, ● IL-1α 2-Ligand Osteoathritis II Abbott ● WO2008082651 A3 [57]
Immunoglobulin (DVD-Ig) ABT-981 ● IL-1β inactivation Laboratories, ● 29 December 2006
AbbVie ● 09 April 2009
● Chung-Ming Hsieh [US], Bradford L. Mcrae [US], Yuliya Kutskova [US],
John E. Memmott, Michael Roguska [US], Ian Tomlinson [US], Mihriban
Tuna [US], Steven Grant [US], Carrie Enever [US]
● Abbott Laboratories [US], Chung-Ming Hsieh [US], Bradford L. Mcrae
[US], Yuliya Kutskova [US], John E. Memmott, Michael Roguska [US], Ian
Tomlinson [US], Mihriban Tuna [US], Steven Grant [US], Carrie Enever
[US]
ABT-165 ● DLL4 2-Ligand Solid tumors I AbbVie ● WO2014071074 A3 [58]
● VEGF inactivation ● 01 November 2012
● 26 June 2014
● Jonathan A. Hickson [US], Deanna L. Haasch [US], Supriya Gupta [US],
Ravi Chari [US], Camellia Zamiri [US], Jijie Gu [US], Dominic J. Ambrosi
[US], Susan E. Lappe [US], Yingchun Li [US], Louie Naumovski [US],
Xianhua Cao [US]
● AbbVie, Inc. [US]
Remtolumab, ● TNFα 2-Ligand Psoriatic arthritis, II Abbott ● WO2014144280 A3 [59]
ABT-122 ● IL-17A inactivation rheumatoid Laboratories, ● 2013-03-15
arthritis AbbVie ● 2015-01-15
● Tariq Ghayur [US], Jijie Gu [US], Maria Harris [US], Carrie Goodreau [US],
Sonal Saluja [US]
● AbbVie, Inc. [US]
Dual Variable Domain- SAR-156597 ● IL-4 2-Ligand Idiopathic II Sanofi ● WO2009052081 A4 [60]
Immunoglobulin (DVD-Ig) ● IL-13 inactivation pulmonary ● 15 October 2007
like (tetravalent bispecific fibrosis, ● 23 July 2009
tandem Ig) systemic ● Ercole Rao [DE], Vincent Mikol [FR], Danxi Li [US], Jochen Kruip [DE],
scleroderma Matthew Davison [US]
● Sanofi-Aventis [FR], Ercole Rao [DE], Vincent Mikol [FR], Danxi Li [US],
Jochen Kruip [DE], Matthew Davison [US]
Two-in-one/Dual Action Fab Duligotuzumab, ● EGFR 2-Receptor Head and neck, II Genentech, Roche ● WO2010108127 A1 [67]
(DAF) RG-7597, ● HER3/ErbB-3 tyrosine kinase colorectal ● 20 March 2009
RO-5541078 inactivation cancers ● 23 September 2010
● Germaine Fuh [US], Gabriele Schaefer [US], Lauric Haber [US], Mark X.
Sliwkowski [US]
● Genentech, Inc. [US], Germaine Fuh [US], Gabriele Schaefer [US], Lauric
Haber [US], Mark X. Sliwkowski [US]
FinomAb COVA-322 ● TNFα 2-Ligand Plaque psoriasis I/II Covagen ● WO2011023685 A1 [65]
● IL-17A inactivation ● 27 August 2009
● 03 March 2011
● Dragan Grabulovski [CH], Michela S. Melkko [CH], Frédéric Mourlane
[CH], Simon S. Brack [CH], Julian Bertschinger [CH]
● Covagen Ag [CH], Dragan Grabulovski [CH], Michela S. Melkko [CH],
Frédéric Mourlane [CH], Simon S. Brack [CH], Julian Bertschinger [CH]
(Continued )
13
14
Status
of
clinical Publication number, priority date, publication date, inventor(s) and
Format Molecule Targets Mode of action Indication trials Developed by applicant(s) Ref.
scFv-Fc-(Fab)-fusions Istiratumab, ● HER3/ErbB-3 2-Rceptor tyrosine Pancreatic cancer II Adimab, ● WO2011047180 A1 [61]
MM-141 ● IGF1R kinase Merrimack ● 14 October 2009
M. GODAR ET AL.
inactivation ● 21 April 2011

● Birgit Schoeberl [US], Ulrik Nielsen [US], Arthur J. Kudla [US], Arumugam
Muruganandam [IN], David Buckler [US], Alexey Alexandrovich [US]
Lugovskoy [US], Jonathan B. Fitzgerald [US], Lihui Xu [US], Neeraj Kohli
[US]
● Merrimack Pharmaceuticals, Inc. [US], Birgit Schoeberl [US], Ulrik Nielsen
[US], Arthur J. Kudla [US], Arumugam Muruganandam [IN], David Buckler
[US], Alexey Alexandrovich [US] Lugovskoy [US], Jonathan B. Fitzgerald
[US], Lihui Xu [US], Neeraj Kohli [US]
LY-3164530 ● EGFR 2-Receptor Cancer I Eli Lilly ● WO2015100104 A1 [62]
● cMET tyrosine kinase ● 23 December 2013
inactivation ● 02 July 2015
● Hector Aldaz [US], Barrett Allan [US], Ling Liu [US], Ying Tang [US],
Sheng-Hung R. Tschang [US], Pia P. Yachi [US], Jirong Lu [US]
● Eli Lilly and Company [US]
MEDI-7352 ● NGF 2-Ligand Osteoarthritis I MedImmune, ● WO2015114150 A1 [5]
● TNFα inactivation pain AstraZeneca ● 02 February 2014
● 06 August 2015
● Darren Schofield [GB], Matthew A. Sleeman [GB], Iain P. Chessell [GB],
Jonathan Hatcher [GB], David Lowe [GB]
● MedImmune, Ltd [GB]
Bispecific monoclonal LY-3114062 ● TNFα 2-Ligand Arthritis I Eli Lilly ● WO2014137961 A1 [5]
antibody (BsmAb) ● IL-17A inactivation ● 08 March 2013
● 12 September 2014
● Barrett Allan [US], Andrew L. Glasebrook [US], Donmienne D. M. Leung
[US], Jirong Lu [US], Ying Tang [US], Andrew C. Vendel [US], Derrick R.
Witcher [US], Pia P. Yachi [US]
● Eli Lilly And Company [US]
Immunoglobulin gamma RO-7040551, ● FGFR1c Receptor Type 2 diabetes I Genentech ● WO2015100366 A1 [5]
(IgG)-assembled from half RG-7992, ● KLB inactivation mellitus ● 23 December 2013
monoclonal antibodies BFKB-8488A ● 02 July 2015
(mAbs) ● Yongmei Chen [US], James Ernst [US], Hok S. Kim [US], Junichiro Sonoda
[US], Christoph Spiess [US], Scott Stawicki [US], Yan Wu [US]
● Genentech, Inc. [US], F. Hoffmann-La Roche Ag [CH]
RO-7040547, ● IL-13 2-Ligand Allergic asthma I Genentech, ● WO2015127405 A3 [5]
RG-7990, ● IL-17 inactivation Novimmune ● 21 February 2014
BITS-7201A ● 15 October 2015
● Lawren Wu [US], Joseph R. Arron [US], Michael Dillon [US], David F. Choy
[US], Sue Sohn [US], Christoph Spiess [US], Whitney Shatz [US]
● Genentech, Inc. [US], F. Hoffmann-La Roche Ag [CH]
(Continued )
binding sites that result in variable valency. They can poten-
[63]
Ref.
tially bind monovalently to both Ags or alternatively they can
Gao [US], Dorin Toader [US], Lakshmaiah Gingipalli [US], Fengjiang Wang
bind bivalently to a single Ag [66]. Duligotuzumab, which is
Damschroder [US], Changshou Gao [US], Godfrey Rainey [US], Cuihua

Publication number, priority date, publication date, inventor(s) and
currently being evaluated in phase II for the treatment of head
[US], Ryan Fleming [US], Binyam Bezabeh [US], Andy Q. Yuan [US],
John Li [US], Nazzareno Dimasi [US], Steven Coats [US], Melissa
and neck, and colorectal cancers, is a two-in-one phage-
derived humanized BsAb, which binds to EGFR and HER3/
ErbB-3, resulting in the inhibition of the downstream HER/
ErbB signaling pathways (Table 4) [67].
2.5. Fragment-based bispecific antibodies

applicant(s)
2.5.1. DNL-Fab3
In parallel, a growing repertoire of different fragment-based
BsAb formats have been described that lack some or all Ab
Srinath Kasturirangan [US]
constant domains [4,5,9–11]. Strong and specific natural pro-

MedImmune Llc [US]
tein-protein interactions have been exploited in the Dock-

WO2015157592 A1
15 October 2015
aNd-Lock method (DNL) to create BsAbs with high valency in

11 April 2014
the 1990s [68]. Indeed, the DNL technology, described in a

patent application filed in 1994 by Immunomedics (Table 5),
exploits the natural interaction between the dimeric cyclic
AMP-dependent protein kinase and the monomeric A-kinase
●
●
●
●
anchoring proteins. These proteins have two α-helical pep-

Developed by
tides that bind specifically with each other and these peptides
can be recombinantly fused or chemically attached to com-
MedImmune
pounds or proteins of interest. The helices provide a linker

module for ‘docking’ two modified components into a quasi-
stable structure, which is then ‘locked’ into a stable structure.
clinical
The BsAb is created by dimerizing a monospecific Fab dimer

Status
trials
of
with a second Fab molecule via the DNL [68]. The format has
been further expanded by placing the DNL domains at the
carboxy terminus of an Ab to create a hexavalent BsAb, called
Breast, gastric
Indication
DNL-Fab3 (Figure 3Co) [69]. Consecutively, an anti-CEACAM5/

cancers
anti-hapten DNL-Fab3, TF2-IMP288, described in a patent

application filed in 2013, is currently being investigated in
phase II for the treatment of colorectal, breast, non-small
tyrosine kinase
Mode of action
and small cell lung, and thyroid cancers (Table 6) [70].

Cell division
inactivation
Receptor
blocked
2.5.2. DART
Alternatively, for many BsAb fragments LCs and HCs are con-
●
nected with peptide linkers, allowing efficient expression of

ker, to the cyto-
agent tubulysin,
Conjugated, via
a cleavable lin-
the BsAb in a single host cell [10]. A resulting format is the

with potential
antineoplastic
HER2/ErbB-2
HER2/ErbB-2
microtubule
(domain IV)
(domain II)
scFv format, which is the most commonly used derivative of

Targets
toxic anti-
activity
the human VL and VH domains representing the minimal Ag-

binding site of an Ab (Figure 2B) [83,84]. The construction of
scFv fragments with short (1–10 amino acids) linkers permits
●
inter-chain but not intra-chain pairing of VL and VH domains

Molecule
[10]. Co-expression of two such scFv fragments can be used to

MEDI-4276
form a bispecific fragment known as a diabody that is mono-

valent for each Ag [85]. Additional refinements of the diabody
format include the introduction of a connecting linker
between chains to create a single-chain diabody [86,87]. A
disulfide bond engineered into the diabody can improve sta-
scFv-Fc-antibody-drug
bility as in the Dual-Affinity-ReTargeting (DART) format devel-

oped by Macrogenics, and for which the patent application
conjugate
was filed in 2007 (Figure 3Cp and Table 5) [88]. The anti-CD3/
anti-CD123 flotetuzumab has, for example, entered phase I for
Format
the treatment of acute myeloid leukemia and myelodysplastic

syndromes (Table 6) [89].
16
Table 5. Fragment-based bispecific antibody format patent applications classified by priority date.
Publication
Format Company number Priority date Publication date Inventor(s) Applicant(s) Ref.
Dock-aNd-Lock (DNL) Immunomedics WO1996004313 A1 05 August 1994 15 February 1996 David M. Goldenberg [US] Immunomedics, Inc. [US] [68]
M. GODAR ET AL.
Adaptir (previously SCORPION) Trubion, Emergent WO2002056910 A1 17 January 2001 25 July 2002 ● Jeffrey A. Ledbetter [US] ● Trubion [94]
BioSolutions, Aptevo ● Martha Hayden-Ledbetter Pharmaceuticals, Inc.
[US] [US]
● Jeffrey A. Ledbetter [US]
● Martha Hayden-
Ledbetter [US]
Tandem diAbody (TandAb) Affimed WO2003048209 A1 14 November 2001 12 June 2003 ● Sergey Kipriyanov [DE] ● Affimed Therapeutics [87]
● Fabrice Le Gall [DE] AG [DE]
● Melvyn Little [DE] ● Sergey Kipriyanov [DE]
● Holger Schäfer [DE] ● Fabrice Le Gall [DE]
● Gerhard Moldenhauer [DE] ● Melvyn Little [DE]
● Björn Cochlovius [NO] ● Holger Schäfer [DE]
● Gerhard Moldenhauer
[DE]
● Björn Cochlovius [NO]
Payload-packaged EDV (EngeneIC Delivery Vehicle) nanocells EngeneIC WO2005056749 A2 09 December 2003 13 October 2005 ● Himanshu Brahmbhatt [AU] ● EngeneIC Molecular [97]
actively targeted with tandem ● Jennifer Macdiarmid [AU] Delivery Pty, Ltd [AU]
scFvs ● Himanshu Brahmbhatt
[AU]
● Jennifer Macdiarmid
[AU]
Bispecific T-cell Engager (BiTE) Amgen WO2005061547 A3 22 December 2003 24 November 2005 ● Peter Kufer [DE] ● Micromet Ag [DE] [12]
● Meera Berry [DE] ● Peter Kufer [DE]
● Patrick Baeuerle [DE] ● Meera Berry [DE]
● Christian Itin [DE] ● Patrick Baeuerle [DE]
● Christian Itin [DE]
Nanobody-based Ablynx WO2009030285 A1 07 September 2007 12 March 2009 ● Edward Dolk [NL] ● Ablynx N.V. [BE] [5]
● Michael J. S. Saunders [BE]
● Johannes J. W. De Haard
[NL]
● Renee De Bruin [NL]
Dual-Affinity-ReTargeting (DART) Macrogenics WO2008157379 A8 21 June 2007 14 January 2010 ● Leslie S. Johnson [US] ● Macrogenics Inc. [US] [88]
● Ling Huang [US] ● Leslie S. Johnson [US]
● Ling Huang [US]
Immune-mobilizing monoclonal T-cell receptors Against Immunocore WO2010133828 A1 20 May 2009 25 November 2010 ● Bent K. Jakobsen [GB] ● Immunocore Ltd [GB] [95]
Cancer (ImmTAC) ● Annelise B. Vuidepot [GB] ● Bent K. Jakobsen [GB]
● Annelise B. Vuidepot
[GB]
(Continued )
2.5.3. TandAb
[71]
Ref.
Another derived format is the tetravalent tandem diabody
(TandAb, Figure 3Cq), developed by Affimed and described
company Biocad [RU]

in a patent application filed in 2001 (Table 5), in which two
Applicant(s)
pairs of VL and VH domains are connected in a single poly-
Closed joint stock

peptide chain. The resulting molecule is bispecific and bivalent
for each Ag [87]. Several TandAbs are currently being evalu-
ated in clinical trials, such as the anti-CD19/anti-CD3 AFM-11
[90] and anti-CD30/anti-CD16A AFM-13 [91], for the treatment
of acute lymphoblastic leukemia and non-Hodgkin’s lym-
Stanislav R. Evdokimov [RU]
Ekaterina V. Sofronova [RU]

Timothy A. Nemankin [RU]
Dmitriy V. Korzhavin [RU] phoma, and Hodgkin’s disease, respectively (Table 6).
Tatyana V. Chernovskaya
Olga V. Goncharova [RU]
Victoria M. Ekimova [RU]

Dmitriy V. Morozov [RU]
Yakov Y. Ustyugov [RU]

Valeriy V. Soloviev [RU]
Roman A. Ivanov [RU]

Yulia S. Chernyh [RU]
Andrey B. Ulitin [RU]
Inventor(s)
2.5.4. BiTE
One format well represented in clinical trials is the Bispecific
T-cell Engager (BiTE, Figure 3Cr), described in a patent applica-
tion filed in 2003 by Micromet (Table 5) [12]. This format is a
[RU]
type of tandem scFv, monovalent for each Ag, with one scFv
●
●
●
●
●
●
●
●
●
●
●
●
●
binding to a T cell-specific molecule, usually CD3, while the

second scFv binds to a tumor-associated Ag, efficiently enhan-
Publication date
cing the patient’s immune response to tumors. Indeed, the

22 September
activated T cells release cytokines to kill tumor cells when the

2016
synapse is formed between T cells and the tumor cells. Due to

its positive effect in clinical trials, a BiTE that directs CD3-
positive T cells against CD19-positive B cells, blinatumomab,
WO2016048188 A8 26 September 2014
received an accelerated approval from the FDA for the treat-

Priority date
ment of Philadelphia chromosome-negative relapsed or refrac-

tory acute lymphoblastic leukemia in 2014 (Table 6) [92]. Four
other BiTEs are currently in clinical trials, among which the
most advanced, an anti-CD3/anti-CEA MT-111, described in a
patent application filed in 2003 (Table 6), is being evaluated in
phase III for the treatment of gastrointestinal cancer [93].
Publication
number
2.5.5. Adaptir
Another scFv derived format leads to the development of
Adaptir multi-specific therapeutics (Figure 3Cs), developed by
Aptevo (a spin-off of Emergent BioSolutions) and described in
a patent application filed in 2001 (Table 5). Adaptir are single
Company
chain polypeptides that comprise two separate binding

domains (VL and VH), a hinge segment, and an effector
domain based on a human Fc region, which are produced as
Biocad
disulfide-linked dimers [94]. These proteins may act similarly to

mAbs by mediating complement dependent toxicity and Fc
dependent cytotoxicity. For example, a scFv-Fc-scFv APVO-414
(CD3 × PSMA), currently being evaluated in phase I, redirects
T-cell cytotoxicity toward prostate cancer cells by specifically
Unknown design (immunoglobulin gamma-based)
targeting T cells through CD3 to prostate cancer cells expres-

sing PSMA (Table 6) [94].
2.5.6. ImmTAC
Alternatively, BsAb fragments can be linked to other proteins
to add additional functionality or specificity. For example,
Immune-mobilizing monoclonal TCRs Against Cancer format
(ImmTACs, Figure 3Ct), developed by Immunocore and

described in a patent application filed in 2009 (Table 5), com-
prises an anti-CD3 scFv linked to a synthetic, soluble and
affinity-matured TCR that recognizes target human leukocyte
Format
Ag-presented peptides [95]. ImmTACs enable circulating

T-cells to selectively identify and kill diseased cells [10]. One
18
Table 6. Fragment-based bispecific antibodies approved or in clinical trials.

Status of clinical Publication number, priority date, publication date,
Format Molecule Targets Mode of action Indication trials Developed by inventor(s) and applicant(s) Ref.
M. GODAR ET AL.
Bispecific T-cell Engager Blinatumomab, AMG- ● CD3 T-cell recruitment Acute Marketed, Amgen, Micromet ● WO1999054440 A1 [92]
(BiTE) 103, ● CD19 lymphoblastic approved in ● 21 April 1998
MT-103 leukemia 2014 by the ● 28 October 1999
United States ● Bernd Dörken [DE], Gert Riethmüller [DE], Peter Kufer
Food and Drug [DE], Ralf Lutterbüse [DE], Ralf Bargou [DE], Anja Löffler
Administration [DE]
and in 2015 by ● Micromet Gesellschaft Für Biomedizinische Forschung
the European Mbh [DE], Bernd Dörken [DE], Gert Riethmüller [DE],
Union Agency Peter Kufer [DE], Ralf Lutterbüse [DE], Ralf Bargou [DE],
Anja Löffler [DE]
MEDI-565, ● CD3 T-cell recruitment Gastrointestinal III Amgen, ● WO2005040220 A1 [93]
AMG-211, ● CEA cancer MedImmune, ● 16 October 2003
MT-111 Micromet ● 06 May 2005
Pasotuxizumab, ● CD3 T-cell recruitment Prostate cancer I Amgen, Bayer, ● Robert Hofmeister [DE], Birgit Kohleisen [DE], Ulla [72]
AMG-212 ● PSMA Micromet Lenkkeri-Schütz [DE], Christian Itin [DE], Patrick Bäuerle
BAY-2010112, [DE], Francis J. Carr [GB], Anita A. Hamilton [GB], Stephen
MT-112 Williams [GB]
● Micromet Ag [DE], Robert Hofmeister [DE], Birgit
Kohleisen [DE], Ulla Lenkkeri-Schütz [DE], Christian Itin
[DE], Patrick Bäuerle [DE], Francis J. Carr [GB], Anita A.
Hamilton [GB], Stephen Williams [GB]
AMG-420, ● CD3 T-cell recruitment Multiple Myeloma I Amgen, ● WO2014140248 A1 [73]
BI-836909 ● BCMA Boehringer ● 15 March 2013
Ingelheim, ● 18 September 2014
Micromet ● Peter Kufer [DE], Tobias Raum [DE], Patrick Hoffmann
[DE], Roman Kischel [DE], Ralf Lutterbuese [DE], Doris
Rau [DE], Paul Adam [DE], Eric Borges [DE], Barbara
Hebeis [DE], Susanne Hip [DE]
● Amgen Research (Munich) Gmbh [DE], Boehringer
Ingelheim International Gmbh [DE]
AMG-330 ● CD3 T-cell recruitment Acute myeloid I Amgen ● WO2015036583 A3 [74]
● CD33 leukemia ● 13 September 2013
● 02 July 2015
● Roland B. Walter [US], Marion Subklewe [DE], Christina
Krupka [DE]
● Amgen Inc. [US], Amgen Research (Munich) Gmbh [DE]
(Continued )
Tandem diAbody AFM-11 ● CD3 T-cell recruitment Acute I Affimed ● WO2003025018 A3 [90]
(TandAb) ● CD19 lymphoblastic ● 14 September 2001
leukemia, non- ● 28 August 2003
Hodgkin’s ● Fabrice Le Gall [DE], Sergey Kipriyanov [DE], Uwe Reusch
lymphoma [DE], Gerhard Moldenhauer [DE], Melvyn Little [DE]
● Affimed Therapeutics Ag [DE], Fabrice Le Gall [DE],
Sergey Kipriyanov [DE], Uwe Reusch [DE], Gerhard
Moldenhauer [DE], Melvyn Little [DE]
AFM-13 ● CD16A Immune cell Hodgkin’s disease II Merck, Affimed ● WO2006125668 A3 [91]
● CD30 recruitment ● 26 May 2005
● 22 March 2007
● Karin Hoffmann [DE], Sergej Kiprijanov [DE], Stefan H. J.
Knackmuss [DE], Fabrice Le Gall [DE], Melvyn Little [DE],
Uwe Reusch [DE]
● Affimed Therapeutics AG [DE], Karin Hoffmann [DE],
Sergej Kiprijanov [DE], Stefan H. J. Knackmuss [DE],
Fabrice Le Gall [DE], Melvyn Little [DE], Uwe Reusch [DE]
Nanobody-based Ozoralizumab, ATN- ● TNFα PK-modulated Rheumatoid II Eddingpharm, ● WO2004041865 A3 [104]
103 ● Albumin ligand arthritis Pfizer, Taisho ● 08 November 2002
antagonist Pharmaceutical, ● 15 July 2004
Ablynx ● Karen Silence [BE], Marc Lauwereys [BE], Torsten Dreier
[DE]
● Ablynx N.V. [BE], Karen Silence [BE], Marc Lauwereys
[BE], Torsten Dreier [DE]
BI-1034020 ● Beta amyloid 2-Protein Alzheimer’s I Boehringer ● WO2006040153 A3 [5]
Aβ40 inactivation disease Ingelheim, ● 13 October 2004
● Beta amyloid Ablynx ● 19 April 2007
Aβ42 ● Marc Lauwereys [BE], Fred Van Leuven [BE], Ingrid Van
Der Auwera [BE], Stefaan Wera [BE], Pascal Merchiers
[BE]
● Ablynx N.V. [BE], Marc Lauwereys [BE], Fred Van Leuven
[BE], Ingrid Van Der Auwera [BE], Stefaan Wera [BE],
Pascal Merchiers [BE]
Vobarilizumab, ALX- ● IL-6R PK-modulated Rheumatoid II Ablynx ● WO2008020079 A1 [105]
0061 ● Albumin receptor arthritis, ● 18 August 2006
antagonist systemic lupus ● 21 February 2008
erythematosus ● Joost A. Kolkman [BE], Els Beirnaert [BE]
● Ablynx N.V. [BE], Joost A. Kolkman [BE], Els Beirnaert [BE]
BI-836880 ● VEGF 2-Ligand Solid tumors I Boehringer ● WO2012131078 A1 [5]
● Ang2 inactivation Ingelheim, ● 01 April 2011
Ablynx ● 04 October 2012
(Albumin) ● Andreas Gschwind [AT], Rene G. Ott [AT], Joachim
Boucneau [BE], Marie-Ange Buyse [BE], Erik Depla [BE]
● Boehringer Ingelheim International Gmbh [DE], Andreas
Gschwind [AT], Rene G. Ott [AT], Joachim Boucneau [BE],
Marie-Ange Buyse [BE], Erik Depla [BE]
(Continued )
19
20
ALX-0761, ● IL-17A 2-Ligand Psoriasis I Merck Serono, ● WO2012156219 A9 [75]
M-1095, ● IL-17F inactivation Ablynx ● 05 May 2011
MSB-0010841 ● 25 July 2013
(Albumin) ● Heidi Rommelaere [BE], Joost A. Kolkman [BE], Michael J.
S. Saunders [BE], Ann Union [BE], Yolande Chvatchko
M. GODAR ET AL.
[CH], Amanda E.I. Proudfoot [FR], Alain Vicari [FR], Denis

Bruniquel [FR], Laurent Chevalet [FR], Olivier Leger [FR]
● Ablynx N.V. [BE], Merck Patent Gmbh [DE], Heidi
Rommelaere [BE], Joost A. Kolkman [BE], Michael J. S.
Saunders [BE], Ann Union [BE], Yolande Chvatchko [CH],
Amanda E.I. Proudfoot [FR], Alain Vicari [FR], Denis
Bruniquel [FR], Laurent Chevalet [FR], Olivier Leger [FR]
Immune-mobilizing IMC-gp100, ● Monoclonal T T-cell recruitment Malignant II Immunocore, ● WO2011001152 A1 [96]
monoclonal T-cell ImmTAC-gp100 cell receptor melanoma, MedImmune ● 03 July 2009
receptors Against anti-CD3 scFv uveal melanoma ● 06 January 2011
Cancer (ImmTAC) fusion protein ● Bent K. Jakobsen [GB], Naomi Harwood [GB], Nathaniel
R. Liddy [GB]
● Immunocore Ltd [GB], Bent K. Jakobsen [GB], Naomi
Harwood [GB], Nathaniel R. Liddy [GB]
Dual-Affinity-ReTargeting MGD-009 ● CD3 T-cell recruitment Solid tumors I MacroGenics ● WO2012162067 A3 [76]
(DART)/ ● B7-H3 ● 21 May 2011
Dual-Affinity- ● 31 January 2013
ReTargeting-Fc (DART- ● Ling Huang [US], Leslie S. Johnson [US]
Fc) ● MacroGenics, Inc. [US], Ling Huang [US], Leslie S.
Duvortuxizumab, ● CD3 T-cell recruitment Solid tumors II Janssen Biotech, Johnson [US] [77]
MGD-011, ● CD19 MacroGenics
JNJ-64052781
MGD-010 ● CD32B B-cell modulation Autoimmune I MacroGenics, ● WO2015021089 A1 [78]
● CD79B diseases Takeda ● 09 August 2013
● 12 February 2015
● Leslie S. Johnson, Ling Huang [US], Kalpana Shah [US],
Ezio Bonvini [US], Paul A. Moore [US], Wei Chen [US]
● MacroGenics, Inc. [US]
Flotetuzumab, ● CD3 T-cell recruitment Acute myeloid I MacroGenics, ● WO2015026892 A1 [89]
MGD-006, ● CD123 leukemia, Servier ● 23 August 2013
S-80880 myelodysplastic ● 26 February 2015
syndromes ● Ezio Bonvini [US], Leslie S. Johnson [US], Ling Huang
[US], Paul A. Moore [US], Gurunadh R. Chichili [US], Ralph
F. Alderson [US]
● Macrogenics, Inc. [US]
MGD-007 ● CD3 T-cell recruitment Colorectal cancer I MacroGenics, ● WO2015026894 A3 [5]
● gpA33 Servier ● 23 August 2013
● 16 April 2015
● Paul A. Moore [US], Jonathan Li [US], Francine Z. Chen,
Leslie S. Johnson [US], Kalpana Shah [US], Ezio Bonvini
[US]
(Continued )
MGD-013 ● PD-1 2-Ligand Solid tumors/Heme I MacroGenics ● WO2015200119 A8 [79]
● LAG-3 inactivation ● 26 June 2014
● 30 March 2017
● Ezio Bonvini [US], Leslie S. Johnson [US], Kalpana Shah
[US], Ross La Motte-Mohs [US], Paul A. Moore [US], Scott
Koenig [US]
PF-6671008 ● CD3 T-cell recruitment Solid tumors I Pfizer, ● WO2016001810 A1 [80]
● P-cadherin MacroGenics ● 01 July 2014
● 07 January 2016
● Chad M. May [US], Adam R. Root [US], William A. Brady
[US], Lioudmila G. Tchistiakova [US], Lidia Mosyak [US],
Laird Bloom [US], Paul A. Moore [US], Leslie S. Johnson
[US]
● Pfizer, Inc. [US]
Payload-packaged EDV Doxorubicin ● EGFR Delivery of Glioblastoma, solid I/II EngeneIC ● WO2015049589 A1 [81]
(EngeneIC Delivery encapsulated ● Doxorubicin nanoparticles tumors ● 04 October 2013
Vehicle) nanocells nanocells ● 09 April 2015
actively targeted with (EGFREDVDoxorubicin) ● Himanshu Brahmbhatt [AU], Jennifer Macdiarmid [AU]
tandem MicroRNA ● EGFR Delivery of Mesothelioma I/II EngeneIC ● EngeneIC Molecular Delivery Pty, Ltd [AU] [98]
scFvs encapsulated ● miR-16 nanoparticles
nanocells
(EGFREDVmiR-16)
Mitoxantrone ● EGFR Delivery of Central nervous I EngeneIC [97]
encapsulated ● Mitoxantrone nanoparticles system cancer,
nanocells solid tumors
(EGFREDVMitoxantrone)
Small interfering RNA ● EGFR Delivery of Adrenocortical I EngeneIC [99]
encapsulated ● RRM1 nanoparticles carcinoma, solid
nanocells tumors
(EGFREDVRRM1)
Small interfering RNA ● EGFR Delivery of Adrenocortical I EngeneIC [97]
encapsulated ● PLK nanoparticles carcinoma, solid
nanocells tumors
(EGFREDVPLK)
Dock-aNd-Lock (DNL)- TF2-IMP288 ● CEACAM5 Payload delivery Colorectal, breast, I/II Immunomedics, ● WO2015069430 A3 [5]
Fab3 ● Hapten non-small and Nantes ● 05 November 2013
small cell lung, University ● 19 November 2015
thyroid cancers Hospital, IBC ● Hans J. Hansen [US], David M. Goldenberg [US]
Pharmaceuticals ● Immunomedics, Inc. [US]
(Continued )
21
22 M. GODAR ET AL.
such ImmTAC, termed ImmTAC-gp100, a monoclonal TCR anti-
Ref.
[71]
[94]
[82]
CD3 scFv fusion protein described in a patent application filed
in 2009 (Table 6), is currently being investigated in phase II for
Chernovskaya [RU], Timothy A. Nemankin [RU], Roman A.

Goncharova [RU], Dmitriy V. Korzhavin [RU], Tatyana V.
Daniel A. Vallera [US], Jeffrey S. Miller [US], Regents of

Valeriy V. Soloviev [RU], Yulia S. Chernyh [RU], Olga V.
the treatment of malignant melanoma and uveal mela-
Publication number, priority date, publication date,
Andrey B. Ulitin [RU], Stanislav R. Evdokimov [RU],
Ekimova [RU], Ekaterina V. Sofronova [RU], Yakov

noma [96].
Ivanov [RU], Dmitriy V. Morozov [RU], Victoria M.
Emergent Product Development Seattle Llc [US]

John Blankenship [US], Elaine T. Sewell [US]
inventor(s) and applicant(s)
Closed joint stock company Biocad [RU]

2.5.7. EDV
Another platform based on an immunotherapy technology for
The University of Minnesota [US]

the treatment of cancers, termed EnGeneIC’s bacterially-
derived EDV (EnGeneIC Delivery Vehicle) nanocell, has been
described in a patent application filed in 2003 (Figure 3Cu and
Yurevich Ustyugov [RU],
Table 5) [97]. The technology is built on payload-packaged

26 September 2014
22 September 2016
29 September 2016
WO2016048188 A8
WO2016130819 A3
WO2017062604 A1
11 February 2015
06 October 2015
EDV that can be coated with a BsAb, which attaches through
13 April 2017
Ab-Ag interaction to the EDV at one end, and has specificity to
cancer cells on the other end. EDV nanocells are therefore
coated with EGFR, and BsAb designs incorporated scFv frag-
ments derived from an anti-EGFR Ab, ABX-EGF [97]. EDV not
●
●
●
●
●
●
●
●
●
●
●
● only delivers toxic payloads to tumors, it also stimulates the
adaptive immune system to augment the anti-tumor response
Developed by
Therapeutics,
University of
Biosolutions
Morphosys,
[97]. Different categories of drugs-loaded nanocells are cur-

Oxis Biotech,
Minnesota
Emergent
rently being investigated in clinical trials, all targeting EGFR

Aptevo
BioCad
and incorporating either a microRNA [98], mitoxantrone, a

chemotherapy drug [97], or small interfering RNAs (RRM1
[99] and PLK [97]) (Table 6).
Status of clinical
trials
II
I
I
2.5.8. Nanobody-based
Finally, single domain Ab fragments (dAbs) can be used to
reduce molecular size further. DAbs like nanobodies have
been obtained from llama and camel HC-chain only Abs
B cell lymphoma,
Prostate cancer
(Figure 3Cv). Nanobodies are readily combined by short lin-

Indication
Rheumatoid
kers, providing a way to vary their Ag-binding valency and to

leukemia
arthritis
introduce bispecificity [100]. Interestingly, the small size of

nanobodies can allow efficient tissue penetration and provides
access to epitopes on some targets that may be sterically
T-cell recruitment
inaccessible to Abs in IgG format [101]. However, one weak-

Mode of action
inactivation
ness is their short in vivo half-life due to the absence of the Fc-
Toxin fusion
protein
located binding site of the FcRn [102]. Fusion to human serum

2-Ligand
albumin or albumin binding proteins can therefore be used to

extend the serum half-life of dAb fragments [103]. Such nano-
body-based BsAbs targeting albumin and either TNFα (ozor-
alizumab [104], Table 6) or IL-6R (vobarilizumab [105], Table 6),
Targets
for example, are currently in phase II for the treatment of

IL-17A
PSMA
CD19
CD22
TNFα
CD3
rheumatoid arthritis.
●
●
●
●
●
●
MOR-209/ES-414
3. Expert opinion
Molecule
DT-2219ARL
During the past two decades, BsAbs have been transforming

APVO-414,
BCD-121
therapeutic concepts and changing the standard of care for

different indications, attracting attention from industry and
the investment community. Indeed, about 23 BsAb platforms
(Tables 1, 3, and 5) have been developed, generating about 62
molecules (Tables 2, 4, and 6), which are currently being

Two scFvs linked to
(immunoglobulin
diphtheria toxin
gamma-based)
Unknown design
evaluated for potential treatment of a variety of indications,

including cancer and inflammatory diseases, among which
three molecules were approved for clinical use, catumaxomab
Adaptir
Format
(Table 2), emicizumab (Table 2) and blinatumomab (Table 6)

[4,5,9–11].
Having a diversity of formats is a critical driver and necessity for Interestingly, BsAbs start to be combined with other
the advancement of BsAb therapeutics [5]. These BsAbs represent advances in Ab engineering such as Ab-drug conjugates
different formats, which can be clustered under IgG-like symmetric (MEDI-4276, a scFv-Fc-Ab drug conjugate, Table 4). In addi-
or asymmetric BsAbs, and fragment-based BsAbs. In fact, all these tion, many other formats are being developed, which could
three major format clusters are represented in several molecules enter the clinic soon. For example, the asymmetric κλ-body
that have advanced into the clinic (Tables 2, 4 and 6). It has format developed by NovImmune, which is based on a com-
therefore become clear that, even if there were no freedom-to- mon HC and two different LCs, a lambda and a kappa [107].
operate or intellectual property restrictions or competitive/strate- Another example of asymmetric format is the SEEDbody
gic challenges, ‘one size fits all’ cannot be applied to the plethora developed by EMD Serono, which consists of alternating
of desired functionalities and applications of BsAbs [5]. Instead, the human IgA and IgG protein sequences in the CH3 domain
bispecific formats collectively serve as a valuable source of diver- to generate asymmetric but complementary domains [108].
sity that can be applied to the development of therapeutics for Additionally, mAb2 is a symmetric format developed by
various indications [5]. F-Star, which is a full-length Ab with two additional binding
The quick advancements of BsAb technologies require a sites engineered into the CH3 domain [109]. Otherwise, addi-
steady adaptation of patent strategies to ensure that future tional therapeutic applications are also anticipated given the
products will still be protected by intellectual property rights. widespread interest in BsAbs, the expanding repertoire of
Moreover, because of high upfront disbursements for research alternative formats and the growing body of clinical experi-
and lengthy clinical development and approval procedures, ence with BsAbs. Finally, BsAbs seem likely to emerge as an
with the respective outcomes by no means predictable, the important class of therapeutics in the near future. This expec-
biotechnology industry depends on efficient patent protection tation is reinforced by the fact that financial analysts expect
to assure a sufficient return of investment. More precisely, apart sales of over $300 million for blinatumomab (Table 6) by
from the very important decision when to file the first applica- 2019, according to Thomson Reuters Cortellis [16].
tion, there is another reason why right from the beginning, a
comprehensive patent strategy is required [106]. The patent
Funding
term (i.e. the lifetime of a patent) is 20 years after the filing
date and 21 years after the priority date. Up to 5.5 years of This work was supported by the Agentschap voor Innovatie door
additional protection are available in Europe by applying for a Wetenschap en Technologie (IWT, grant #IWT130849).
Supplementary Protection Certificate if a market authorization
is obtained for a product falling under the claims of a granted
patent. Similar provisions for patent term extensions exist in the
Declaration of interest
United States and other countries. The requirements and scope The authors are employed by Argenx BVBA. The authors have no other
of these extensions differs from country to country, and in relevant affiliations or financial involvement with any organization or
entity with a financial interest in or financial conflict with the subject
many cases, the additional scope of protection is smaller than
matter or materials discussed in the manuscript apart from those dis-
that of the original patent and may be restricted to the mar- closed. Peer reviewers on this manuscript have no relevant financial or
keted product. Moreover, it takes usually many years until a other relationships to disclose.
market authorization is obtained, with the consequence that
there are only a few remaining years of patent protection to
recoup the investment and make a profit [106]. Companies
ORCID
have consecutively first established, and protected, the basic Marie Godar http://orcid.org/0000-0002-5477-7726
enabling technologies related to the new format as such
(Tables 1, 3, and 5). In a subsequent step, specific drug candi-
dates have been developed, thereby forming the subject of References
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Therapeutic Bispecific Antibody Formats: A Patent Applications Review (1994-2017)

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ISSN: 1354-3776 (Print) 1744-7674 (Online) Journal homepage: http://www.tandfonline.com/loi/ietp20

Therapeutic bispecific antibody formats: a patent

Marie Godar, Hans de Haard, Christophe Blanchetot & Jacobus Rasser

To link to this article: https://doi.org/10.1080/13543776.2018.1428307

Published online: 25 Jan 2018.

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Therapeutic bispecific antibody formats: a patent applications review (1994-2017)

ABSTRACT ARTICLE HISTORY

1. Introduction to mimic a natural ligand (Figure 1B) [7], hijacking a transcytosis

CONTACT Christophe Blanchetot CBlanchetot@argenx.com

candidates that enter phase I trials successfully ends up as a

(a) (b) (c) (d)

Factor IXa Factor X

(e) (f) (g) (h)

● CH3 domain a: H435R ● Eric Smith [US]

● Kara L. Olson [US]

Freimoser-Grundschober [CH], Christiane

(programmed ● 09 October 2014

RG-6026 ● CD3 T-cell recruitment Relapsed or refractory I Roche ● WO2016020309 A1 [21]

MCLA-128 ● HER2/ErbB-2 2-Receptor Solid tumors I/II Merus ● WO2015130173 A1 [42]

that fold into a complex quaternary Y-shaped structure

Matthew Bernett, Seung Chu [US], Rumana

Gregory Moore [US], John Desjarlais [US],

Stanislas Blein [CH], Romain Ollier [CH],

Samuel Hou [CH], Darko Skegro [CH]

Glenmark Pharmaceutical S.A. [CH]

Rashid [US], Umesh Muchhal [US]

Zymeworks, Inc. [US]

a prime molecular engineering target for either enhancing or

Xencor, Inc. [US]

inhibiting the immune response, including Ab-dependent cel-

as well as membrane recycling. FcRn-IgG interaction also func-

tions in Ag presentation and cross-presentation in macrophage

region also contains the binding site for protein A, a bacterial

the Fc fragment (Figure 2A) [26].

Abs (e.g. WO9007861A1 assigned to Protein Design Labs),

humanized Abs (e.g. WO1991009967A1 assigned to

Celltech), Fab, and single chain variable fragments (scFv,

e.g. WO8801649A1 assigned to Enzon) were, strictly speak-

applied to BsAbs [15].

2.2. Various bispecific antibody formats patented

ing sites. By contrast, IgG-based BsAbs are generally mono-

valent for each Ag and are artificial molecules, per se not

found in nature (except for human IgG4 [27]). They must,

therefore, be generated by biochemical, molecular, or genetic

means. The generation of IgG-based BsAbs is difficult because

challenges of generating BsAbs are to direct and to force

of the HCs. Various strategies have been developed to solve

and with many potential targets to hit (made possible thanks

to the therapeutic applications allowed by BsAbs, Figure 1),

about 23 different IgG-based BsAb formats have been devel-

oped, which can be discriminated by the presence or absence

Human immunoglobulins gamma (A) Asymmetric bispecific antibodies

(l) DVD-Ig (m) FinomAb (n) Two-in-one/DAF

(C) Fragment-based bispecific antibodies

VHHa VHHb VHHa

CH2a CH2b VHHb

2.4. Immunoglobulin gamma-like symmetric bispecific

Abbott Laboratories [US]