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References 17
I. Development of biopharmaceuticals
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2
Nonclinical development of
monovalent and polyvalent
biopharmaceuticals
Karin Staflin
1
, Chandrashekhar K. Korgaonkar
2
,
Padma Kumar Narayanan
3
, Rodney A. Prell
1
,
Donna W. Lee
1
, Wendy G. Halpern
1
and Anu Connor
4
1
Safety Assessment, Genentech, South San Francisco, CA, United States;
2
Regeneron
Pharmaceuticals, Inc, Tarrytown, NY, United States;
3
Preclinical and Clinical Development
Sciences, Wave Life Sciences, Lexington, MA, United States;
4
Ipsen, Cambridge, MA, United States
OUTLINE
Introduction 20
Monovalent biopharmaceuticals 21
Polyvalent biopharmaceuticals 21
Scientific rationale for PVBs 21
Technology and formats 23
Chemical cross-linking and hybridoma
fusion approaches 23
Recombinant formats using engineered
protein scaffolds 24
General challenges and considerations 28
Understanding expression, regulation,
and distribution of all targets 28
Understanding target biology 29
Effector function 29
Species selection 30
Tissue cross-reactivity study 31
Pharmacokinetics 32
Molecular properties 32
Immunogenicity 33
Selection of starting dose 34
Summary of challenges 35
T celledependent bispecific antibody
biotherapeutics 35
Catumaxomab (EpCAM/CD3
multitargeting full-length
antibody) 36
MT110 (EpCAM/CD3 TDB, BiTE
format) 38
CHAPTER
19
Nonclinical Development of Biologics, Vaccines and
Specialty Biologics, Second Edition
https://doi.org/10.1016/B978-0-443-13344-2.00008-0
© 2025 Elsevier Inc. All rights are reserved, including those for text
and data mining, AI training, and similar technologies.
Case Example 4: Formation of ADAs
limited the conduct of sub-chronic (3-
month) or chronic toxicology studies 42
Dual-targeting antibodies 43
Oncology 43
Coagulation disorder 45
Hemophilia 45
Ocular disease 46
Age-related macular degeneration 46
Bispecific antibodies of endogenous
receptor ligands for CNS delivery of
biotherapeutics across bloodebrain
barrier 47
Combination therapeutics 49
Case study : SARS-CoV-2 antibody
cocktail 49
Summary 50
References 51
Introduction
The initial development of therapeutic monoclonal antibodies (mAbs) over 40 years ago
was built on advances in recombinant protein engineering and the recognized need for ther-
apeutics with increased efficacy and lower safety risks as compared to standard of care treat-
ments. Since then, several iterations of mAb development have moved the industry from
fully murine and chimeric versions to predominantly humanized or fully human antibody
biopharmaceuticals. Recent advances in antibody engineering efforts have allowed for design
of mAbs with lower rates of immunogenicity, increased half-life, and varying degrees of
effector functions including antibody-dependent cell-mediated cytotoxicity (ADCC),
antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity
(CDC), and neonatal Fc receptor (FcRn) binding. Extensive knowledge of disease biology,
target antigen, and antibody technology are thus essential in the design of mAbs with a
high probability of success in the clinic.
As a result of a better understanding of the underlying disease biology, it has become more
apparent that many diseases are multifactorial and treatment for these complex diseases re-
mains limited. In addition, the development of resistance due to escape mechanisms, limited
target expression, and sub-optimal effect or mechanisms, among other reasons, continues to
drive the need for more effective therapeutics with fewer dose-limiting toxicities. Therefore,
the development of polyvalent biopharmaceuticals (PVBs) that target multiple epitopes of a
single antigen or single epitopes of different antigens has intensified over the past decade
with the goal of overcoming some of the limitations of conventional mAbs.
Advances in antibody engineering include the development of antibody fragments (Fabs),
antibodyedrug conjugates (ADCs), bispecific and multispecific antibodies, immunocyto-
kines, and scaffold proteins (Jin et al., 2022). The goal of these efforts is to improve efficacy
and potency of a single therapeutic. However, like previous generations of mAbs, challenges
remain in these newer antibody formats. This chapter will introduce monovalent bio-
pharmaceuticals (MVBs) and PVBs and focus on the considerations and challenges associated
2. Nonclinical development of monovalent and polyvalent biopharmaceuticals20
I. Development of biopharmaceuticals
with the preclinical development of PVBs, with particular emphasis on how the unique char-
acteristics of each format can impact the nonclinical development strategy as well as the char-
acterization and interpretation of associated toxicities.
Monovalent biopharmaceuticals
MVBs such as mAbs (in the context of this discussion) bind to a single target with high
specificity. As a brief review, mAbs are tetrameric proteins consisting of two identical heavy
chains and two identical light chains with inter- and intrachain disulfide bonds (Fig. 2.1a).
Each mAb contains two antigen-binding sites formed by the N-terminal regions of the heavy
chains and light chains that determine the antigen recognition and binding properties of the
mAb. Sequence variability within the heavy- and light-chain antigen-binding sites
(complementarity-determining region; CDR) determines antig en specificity. The remaining
region of the mAb, known as the Fc region, functions to allow interactions with other im-
mune components and determines the class and isotype of the mAb Fig. 2.1a. Conventional
mAbs have been developed as drugs due to the highly desirable characteristics of high affin-
ity and target specificity coupled with low potential for off-target toxicity. These properties
have resulted in mAb therapeutics that are generally well tolerated and provide a favorable
therapeutic window in several disease indications such as oncology, autoimmunity, and in-
fectious disease. As a result, mAbs have been instrumental in leading the biologics revolution
with more than 100 Food and Drug Administration (FDA) approvals in the last 35 years
(Mullard, 2021). The nonclinical development considerations for MVBs are similar to PVBs
(see below) and have been discussed at length in the literature (Meyer-Tamaki, 2017;
Chapman et al., 2009; Hansel et al., 2010).
Polyvalent biopharmaceuticals
Scientific rationale for PVBs
It has become increasingly apparent that disease pathobiology is extremely complex and
involves the dysregulation of several redundant and distinct biological pathways. Because
combination therapy may provide a potential for greater efficacy while decreasing the poten-
tial for resistance mechanisms, targeting more than one pathway may provide improved and
sustained therapeutic benefit as highlig hted by the clinical success of mAbs targeting
different tyrosine kinases, antiangiogenesis com binations or checkpoint inhibitors (Konter-
mann, 2012). A growing number of clinical trials are testing the efficacy and tolerabi lity of
two or more investigational drugs, including combinations of two or more mAbs or mAbs
plus small molecules that target different pathways involved in the disease (Zhu et al.,
2021). Since these treatments may involve combinations of marketed and investigational
mAbs, a “Guidance for Industry: Co-development of Two or More Unmarketed Investiga-
tional Drugs for Use in Combination” was written, which outlines development consider-
ations for combination therapy (U.S. FDA, 2013). Development of innovative
biopharmaceutical modalities that target multiple pathways while maintaining specificity
Polyvalent biopharmaceuticals 21
I. Development of biopharmaceuticals
FIGURE 2.1 Polyvalent biopharmaceuticals (PVB) formats in clinical trials: (a) Basic IgG structure; (b) main
antibody components and approaches to multispecificity; (c) IgG-like “two-halves” bispecific formats; (d) IgG-
modified (appended and/or substituted, Fc-based) bispecific and multispecific antibodies; (e) fragment-based
(Fc-free) bispecific and multispecific antibodies; (f) IgG fusion protein; (g) bi-/multispecific constructs for payload
delivery. For the abbreviations, please see the respective section in the main text.
2. Nonclinical development of monovalent and polyvalent biopharmaceuticals22
I. Development of biopharmaceuticals
and tolerability offers an attractive alternative to avoid the complexity of developing two or
more new molecular entities or antibody mixtures.
PVBs may offer several advantages over conventional mAbs by offering combinatorial or
obligate functionality. With combinatorial functionality, the same activity can be obtained by
combining separate antibodies, whereas the activity is dependent on the physical linkage of
the two specificities (and cannot be obtained by combining separate antibodies with the
same specificities) for obligate functionality (Labrijin et al., 2019). For example, PVBs can be
designed so that they target multiple epitopes on the same antigen, multiple antigens involved
in a single disease mechanism, multiple antigens involved in different mechanisms of a given
disease, or multiple antigens on a single or different cell types (Fischer and Leger, 2007; Gu
and Ghayur, 2010; Fagete and Fischer, 2011). This flexibility provides an opportunity for
increased avidity, additive/synergistic activity, prevention of the development of resistance,
delivery of therapeutics to otherwise inaccessible sites, and/or the introduction of unique
modes of action otherwise unattainable via standard mAbs targeting a single antigen
(Fig. 2.1a). In addition to the obvious benefit of the need to develop only one drug, PVBs can
increase the specificity for the target cell, mitigate development of resistance to the drug, and
potentially increase the therapeutic index. Along with these advantages come several unique
challenges in the nonclinical development of PVBs as discussed in the section below.
Technology and formats
As recombinant technology continues to evolve, the number and complexity of PVBs have
increased dramatically. Currently there are many different formats being developed for
various indications, and readers are directed to a number of excellent reviews that have
been published (Gu and Ghayur, 2010; Beck et al., 2010; Lum and Thakur, 2011; Chames
and Baty, 2009; Muller and Kontermann, 2010; Elshiaty et al., 2021; Brinkmann and Konter-
mann, 2017; Jin et al., 2022). In this chapter, we have simplified these numerous formats into
general categories. The main components used for generation of a variety of PVB formats are
shown in Fig. 2.1b, and a variety of different PVB formats are visually depicted in Fig. 2.1
(Fig. 2.1cef). The following section notes the techniques used to generate PVBs and provides
a brief description of the key formats as well as some challenges that may be unique for the
specific format.
Chemical cross-linking and hybridoma fusion approaches
As the limitations of mAbs for certain disease indications became apparent, the develop-
ment of PVBs intensified. The first generation of bispecific antibodies was generated through
the covalent linking of two monospecific antibodies or their antigen binding (Fab) domains
using chemical crosslinking approaches. However, manufacturing and purification of the
desired bispecific species from the mixture of undesired monospecific spe cies represented a
major hurdle for this technology.
An alternative approach to the production of early generation bispecific antibodies was
through the fusion of two different hybridomas resulting in a mixed hybridoma (Fig. 2.1c).
These “quadromas” are produced through random assembly of the heavy and light chains,
Technology and formats 23
I. Development of biopharmaceuticals
which results in a number of different immunoglobulin species, including nonfunctional
monospecific and bispecific species. Thus, some of the early techniques for producing bispe-
cific antibodies were met with severe limitations in the manufacturing and purification pro-
cesses. Trioma antibodies (Triomab) are similar to quadromas in that they recognize two
distinct antigens via the CDR portions of the antibody (Fig. 2.1c). Triomas are considered tri-
functional because they preferentially engage activating Fcg receptors (FcgRI and FcgRIII)
but not inhibitory FcgRIIb on CD64þ and CD16þ accessory cells through their Fc region.
This results in a tricellular complex consisting of a tumor cell, T lymphocyte, and accessory
cell. Despite manufacturing and purification challenges, a few quadroma-based bispecific an-
tibodies have entered clinical trials, and one trioma antibody, catumaxomab, has been
approved in Europe for the treatment of malignant asc ites, a condition found in patients
with a variety of epithelial cancers (Seimetz et al., 2010).
Recombinant formats using engineered protein scaffolds
The advances in recombinant DNA technology and antibody engineering have paved the
way for the generation of numerous PVBs, with the common goal of targeting more than one
antigenic epitope.
The PVB formats can be generally classified into two main categories, that is, Fc-based
versus Fc-free (fragment-based-Fig. 2.1e). The Fc-based formats can be further broadly
divided into IgG-like “two-halves” bispecific format (Fig. 2.1c) and IgG-modified (appended
and/or substituted, Fc-baseddFig. 2.1d)(Brinkmann and Kontermann, 2017). In addition to
these main categories, IgG fusion proteins (Fig. 2.1f) and PVB constructs for payload delivery
(Fig. 2.1g) are also being explored (Elshiaty et al., 2021). Below are brief descriptions of key
PVB formats that are currently in development with some potential advantages and disad-
vantages for each (Elshiaty et al., 2021).
(1) Fc-free: Fragment-based molecules
Several potential advantages have been postulated for the small fragment-based
molecules. Their modular structure provides a tremendous amount of flexibility in size,
shape, and antigen specificity. While the fragment scaffolds offer several
manufacturing advantages including the ability for expression in nonmammalian cell
fermentation systems, manufacturing hurdles reported to date include low yields, poor
product quality, and poor stability ( Pirkalkhoran et al., 2023). Biologically, the smaller
size of these molecules may favor better tissue penetration when compared to whole
IgG molecules. However, this potenti al advantage may be offset by poor PK properties
due to the lack of an Fc region that can interact with the FcRn and lack of glomerular
preservation (Kontermann, 2009), potentially decreasing half-life. Approaches for
overcoming the poor PK properties are discussed below.
• Formats based on single-chain variable fragments (scFv, tandem scFv, diabodies,
tandem diabodies, BiTEs, nanobodies, ImmTAC)
The variable heavy (VH) chain and variable light (VL) chain domains of a mAb
are the building blocks for single-chain variable fragment-types of PVB. A single-
chain Fv (scFv) is an antibody fragment in which the VH and VL domains are
interconnected by a flexible linker that forces them into a stable configuration
2. Nonclinical development of monovalent and polyvalent biopharmaceuticals24
I. Development of biopharmaceuticals
(Fig. 2.1b). An example of this technology that will be discussed in detail later in
this chapter is the bispecific T-cell engager (BiTE). Shortening the flexible linker that
separates the VH and VL forces these domains to pair with the VH and VL regions
from a second polypeptide, resulting in the formation of bivalent molecule called a
diabody (Fig. 2.1b)(Choi et al., 2011). Linking two scFvs or two diabodies via an
additional flexible linker generates tandem scFv or tandem diabodies, respectively
(Fig. 2.1e). Further addition of scFvs enables the creation of trivalent scFvs that can
be either bispecific or trispecific in nature. The diabody-based dual affinity
retargeting molecule (DART) format (Fig. 2.1e) has cross-linked VH and VL chains
of different specificities as well as stabilization by disulfide bridges that provide
compact and stable structure.
Another variety in this format is known as nanobody or single-domain antibody
(sdAb) that consists only of a single variable heavy chain homodimer (VHH,
Fig. 2.1b). This format is based on the fact that the antigen specificity of most
antibodies shows a predominant dependence on the CDRs contributed primarily by
the VH rather than the VL domain. Nanobodies have a molecular weight 1/10th of
the IgG (w15 kDa) and yet retain high-antigen binding affinity. These are highly
soluble and more stable than conventional antibodies but have fast renal clearance
(half-life w 2 h) due to small size. They can survive a wide pH range (pH 3e9) and
chemical and thermal denaturing conditions. Recently, the first nanobody drug,
caplacizumab, an anti-von Willebrand factor humanized immunoglobulin was
approved for the treatment of thrombotic thrombocytopenic purpura (TTP) (Jin
et al., 2022).
Another popular format is ImmTAC (immune-mobilizing monoclonal TCRs
against cancer), a new class of T celleredirecting bispecific fusion proteins that
utilizes an affinity-enhanced soluble TCR fused to an anti-CD3 scFv to target a
tumor cell protein, including intracellular antigens (presented as a peptideeHLA
complex on the target-cell surface). An approved drug with this format is
tebentafusp-tebn (Kimmtrak) (Lowe et al., 2019).
• Trispecific and multispecific formats (TRiKE, TriTAC and DARPin)
Trispecific and multispecific formats aim for even higher efficacy based on further
increase in antibody specificity. An scFv-based TriKEs (trispecific killer engagers;
Fig. 2.1e) binds one TAA and CD16, while their third moiety is IL-15, which
activates the recruited NK cells through cross-linking of their IL-15 receptors. A
diabody-based “trispecific T-cell activation constructs” (TriTACs,
Fig. 2.1e) consist of
a nanobody targeting the respective TAA, joined with an anti-CD3 scFv, and a
nanobody against human serum albumin (HSA) to prolong the half-life. Another
format consists of designed ankyrin repeat proteins (DARPins, Fig. 2.1e) that are
antibody-mimetic (62 kDa) polypeptide chains containing four ankyrin domains
with specificity against HAS, TAA or T-cell antigen.
(2) Fc-based format:
Fc-based constructs are generally larger, have longer half-lives (typically a few
weeks) due to recycling by the FcRn and glomerular preser vation (Kontermann, 2009).
Additionally, they show improved solubility and stability and can be purified using
established chromatography techniques (Arora et al., 2017).
Technology and formats 25
I. Development of biopharmaceuticals
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