Добавил:
Sekretar
kiopkiopkiop18@yandex.ru
t.me/Prokururor I Вовсе не секретарь, но почту проверяю
Опубликованный материал нарушает ваши авторские права? Сообщите нам.
Вуз:
Предмет:
Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5981_Библиотеки_им_академика_М_И_Перельмана
.pdf
(a) IgG-like molecules (Knob-into hole, DAF, Duobody, common light chains [CLCs],
CrossMab, etc.)
IgG-like PVB formats resemble human IgGs with minimal structural or sequence
deviations.
The similarities between these IgG-like molecules and mAbs are expected to
alleviate some of the possible disadvantages of the fragment-based formats such as
poor PK properties while enabling standard mAb manufacturing process. In
addition, this format contains Fc-binding properties that are similar to conventional
mAbs. As a result, Fc-mediated effector functions may be specifically increased or
decreased to gain the desired activ ity using similar genetic engineering techniques
as are used for conventional mAbs.
Knob-into-hole technology has been used to develop monovalent and bispecific
IgG antibodies for clinical use (Giese et al., 2017). In this approach,
heterodimerization (knob/hole) is promoted by introducing a “knob” into one CH3
domain (e.g., replacing a small amino acid with a large amino acid) and
introducing a “hole” into another CH3 domain (e.g., replacing one or more large
amino acids with small amino acids in the CH3 domain of the second heavy chain)
(Ma et al., 2021). Another approach consists of generating a “two-in-one” antibody,
also known as the dual-acting Fab (DAF), that has an unmodified IgG and a
modified Fa b such that each antigen-binding site within each Fab has the capacity
to specifically bind two different proteins (Bostrom et al., 2009 ; Schaefer et al.,
2011; Ma et al., 2021).
Duobody is a popular platform for IgG-like molecules (Fig. 2.1c). The Fab arm
of the IgG4 antibody can be dynamically recombined and exchanged in vivo to
form BsAbs, in a process referred to as named Fab-arm exchange (FAE)
(Scheuermann and Racila, 1995; Viardotetal.,2011; Rivera et al., 2008).
Controlled Fab-arm exchange (cFAE) is the core technology of the Duobody
platform that utilizes matched and destabilizing mutations in CH3 to increase
FAE yield. Other examples of this format include CLC (Fig. 2.1c)thathasCLCs
but different heavy chains. However, similar to TrioMabs, the CLC format
requires multiple purification steps. Kappa-lambda bodies (Fig. 2.1c)have
common heavy chain but different light chains to enable simpler purification
process. Additionally, the CrossMabs platform (Fig. 2.1c) involves swapping of
either the variable or the constant domains between light and heavy chains to
create two asymmetric Fab arms.
(b) IgG-modified molecules (appended and/or substituted, Fc-based) (scFv-Fab-Fc,
HLE-BiTE, bi DART-Fc, tetra. DART-Fc, VHH4-Fc, CrossMABs (2:1 or 2:2), FIT-Ig,
DVD-IgG, etc.)
IgG-modified formats (Fig. 2.1d) are the dominant PVB class due to the important advan-
tages in manufacturing and therapeutic application as a result of structural variation.
Substituting one Fab region of an IgG mAb w ith a synth etic scFv results in s cFv-Fab-Fc,
whereas t he substitution of both Fab-regions with scFv gives the h alf-life extended (HLE)
BiTE, while substitution with bivalent dual-affinity retargeting proteins yields bi DART-
2. Nonclinical development of monovalent and polyvalent biopharmaceuticals26
I. Development of biopharmaceuticals
Fc. These formats are very popular as they combine the advantages of fragment-based bis-
pecifics with the longer half-life of Fc-based constructs. For example, half-life extension
from 6 h for a canonical BiTE to 44e167 h for HLE-BiTE has been reported (Arevedson
et al., 2017).
Substitution of the Fab region of IgG with a single domain, that is, VHH results in Fab-VH-
Fc, which is more stable under denaturing conditions and less likely to aggregate when mul-
tiple fragments are fused together.
The increase in the number of binding sites, that is, valency for the TAA results in
increased avidity, enhanced specificity, and the ability of these constructs to compete with
monospecific, naturally bivalent antibodies. Therefore, multivalent (trivalent) formats
currently predominate in clinical trials (Fig. 2.1d; gray area). The simplest way to produce
a multivalent antibody is by chemical cross-linking of two different IgG antibodies
(IgGeIgG).
With increasing use of genetic engineering, most multivalent antibodies are generated by
adding antigen-binding elements, either as (multiple) substitutes for one or more Fab arms, or
as appendages to any basic bivalent format. Fab appendages are a relatively simple and pop-
ular way to achieve multivalency and include several varieties including Fab3-Fc (with one
additional Fab to IgG); trivalent (2:1) and tetravalent (2:2) CrossMabs (contain an additional
Fab or a CrossFab fused to either the C- or N-terminus of the heavy or light chains); FIT-Ig
(Fabs-in-tandem; light chains of the additional Fabs are fused in tandem with the heavy
chains of the core IgG construct); and Fab2-scFv-Fc (an additional anti-CD3 scFv alongside
two identical Fab regions of IgG).
Additionally, formats with two additional scFv attached to various positions on the heavy
or light chains are also in the clinic and include scFV attached to C-terminus (IgG(H)-scFv2)
or N-terminus (scFv2-(H)IgG) of the heavy chain or appended on the C-terminus of the light
chains (IgG(L)-scFv2). The addition of two variable domains pairs to IgG results in the dual
variable domain immunoglobulin (DVD-Ig) format.
Multiple substitutions of variable domains for one or more Fab arms are increasingly pop-
ular. Some examples include scFv2-Fc-scFv2 (2 scFv pairs joined with a silenced IgG1 Fc, each
linked to the N- or C-terminus) and tetra-DART-Fc (2 DARTs attached to an Fc region). A
functionally similar result can be achieved by attaching two pairs of nanobodies to Fc in a
symmetric manner (VHH4-Fc) and substitution of one Fab arm with one pair of identical
nanobodies (Fab-VH2-Fc). Another version is introduction of two new binding sites directly
in the Fc region (IgG(H)-Fcab2) while simultaneously maintaining Fc binding to the Fc and
FcRn receptors.
The functionality of PVBs can further be extended through fusion with other non-Ig pro-
teins. Some of these formats include IgG fusion proteins (e.g., TGF beta trap, a fusion protein
that contains an extracellular domain of TGF Beta receptor fused to anti-PD-L1 IgG1,
Fig. 2.1f); fragment or antibody drug conjugates for payload delivery (Fig. 2.1g), EnGeneIC
Delivery Vehicles (EDV, bacterially derived nanocells coated with bispecific antibodies for
the targeted del ivery of cytotoxics or other cargo, Fig. 2.1g) and a bispecific tri-Fab antibody
format for that binds to tumor antigen and the radiolabel to enable pretargeted radioimmu-
notherapy (Fig. 2.1g)(Elshiaty et al., 2021).
Technology and formats 27
I. Development of biopharmaceuticals
General challenges and considerations
In general, similar challenges apply for the development of nonclinical safety strategies for
MVBs and PVBs, except that engagement of two or more specific target pathways must be
considered for PVBs. As one can envision, these chal lenges increase as the complexity of
the desired attributes of PVBs increase. By design, PVBs target and modulate separate anti-
gens/epitopes as part of their mechanism of action. Therefore, all of the binding regions of
the molecule must be considered. Moreover, the ability to specifically introduce unique mech-
anisms of action (e.g., redirected immune effector cell lysis or enhanced transport to privi-
leged sites) as part of the desired properties of multitargeting therapeutics further
increases the complexity for nonclinical safety evaluation. The end result is that the chal-
lenges that we continue to face in deve loping traditional mAbs are multiplied for these
new PVB formats (Fig. 2.1).
As described in the previous sections, development of several novel multitargeting formats
and technologies has increased over the past decade as a result of advances in manufacturing
and purification processes. Each technology or platform will create its own unique advan-
tages and challenges that must be taken into consideration when developing the nonclinical
safety asse ssment strategy to support various clinical indications. For example, certain plat-
forms (e.g., antibody fragments) may allow for better tumor penetration yet may have rapid
PK clearance properties that could limit exposure because of their small size. If prolonged
exposure is required for a specific disease indication, one could potentially overcome this
PK liability through the addition of a “stabilizer” segment to the molecule, the encapsulation
of the molecule in stabilizing material (i.e., liposomes), or continuous administration of the
molecule through a portable infusion pump rather than a bolus IV infusion. This, along
with other challenges, will be discussed in further detail in the following sections.
Understanding expression, regulation, and distribution of all targets
Understanding the expression, regulation, and distribution of the relevant target antigens
is a key component to safety assessment of MVBs and PVBs. In general, this starts with a re-
view of the known biology of all of the targets, in both healthy and diseased conditions. The
known biology of all of the targets, in both healthy and diseased conditio ns, is first reviewed
which generally correlates with the pharmacology of the therapeutic candidate. Additional
studies may include immunohistochemical surveys and gene expression profiling across
different tissues.
Alternative methods may be more appropriate for certain molecular platforms or target
molecules. For instance, platforms that employ T celleredirected lysis as a mechanism of ac-
tion will require a comprehensive understanding of the specificity of the PVB targeting arm
and the expression, distribution, and density of the tumor antigen. Such information is critical
to minimize the risk of nonspecific redirected T-cell lysis. Multispecific recombinant fusion
proteins or multitargeting agents that recognize conformational epitopes that are only pre-
sent on viable cells may also require novel assays to determine tissue and/or cellular expres-
sion, distribution, and density. Expression of certain molecular targets may profoundly differ
in healthy versus diseased tissues. For example, inflammatory mediators are likely expressed
at very low levels in healthy animals but are expressed at much higher levels in models of
2. Nonclinical development of monovalent and polyvalent biopharmaceuticals28
I. Development of biopharmaceuticals
inflammation such as rheumatoid arthritis, colitis, allergic airway inflammation, and chronic
dermatitis. Furthermore, some targets, such as viral and bacterial proteins, are completely ab-
sent in nonclinical species and only expressed in specific infection models. Thus, nonclinical
safety studies conducted in healthy animals may not accurately predict safety liabilities
related to on-target pharmacology. Therefore, it can be useful to consider the utility of infor-
mation from well-characterized specific disease models, transgenic models or more special-
ized models such as the T-cell-dependent antibody response (TDAR) used for
immunomodulators, as part of the safety assessment strategy. However, it is critical to under-
stand the limitations of each specific model, and weigh this against the potential translat-
ability and utility of any outcomes to the clinical setting.
Understanding target biology
It is important to understand the biology of targeted pathways and/or engineered effector
functions. In case of PVBs, the impact that altering multiple biological pathways has on po-
tential safety and tolerability needs to be balanced with the clear scientific advantages that
they offer over traditional mAbs. Targeting multiple clinically validated biological pathways
may allow one to assess the potential safety liabilities of the PVBs based on the available
nonclinical and clinical safety liability information associated with monotherapeutic interven-
tion of the specific pathways. In the situation where safety information is available for one
biological pat hway but lacking for the other(s), one could take an approach that directly com-
pares the safety liabilities of the PVB with that of a traditional mAb and evaluate for
enhanced, overlapping, or unique toxicities associated with the PVB. If little or no safety in-
formation is available for either target pathwa y, the nonclinical approach may reflect a strat-
egy used for traditional biopharmaceuticals, keeping in mind that any safety signal observed
must be evaluated for a potential relationship to all of the biological pathways, and would
likely become associated with the specific PVB molecule. Regardless of the scenario, the
more knowledge one has on biological pathways, the more likely it is that one can make
an informed scientific-based assessment of the potential liabilities associated with targeting
multiple pathways.
Effector function
One of the major limitations of the first-generation IgG-based bispecific formats (e.g.,
quadroma) or trifunctional antibodies, with specifically modified Fcg type I/III receptor,
was nonspecific activation of Fc receptoreexpressing accessory immune cells, resulting in se-
vere adverse events related to cytokine release syndrome. One trifunctional antibody, catu-
maxomab (Removab), which has specificity to CD3, EpCAM, and FcgRI/III, was
developed by TRION Pharma and Fresenius Biotech for patients with EpCAM-positive
ovarian cancers with symptomatic malignant ascites and received EU approval in 2009. Since
patients receive catumaxomab via intraperitoneal infusion after paracentesis, there may be
fewer Fc-expressing accessory cells present, which may partially explain the acceptable safety
profile. In contrast, intravenous infusion of catumaxumab was associated with a significant
cytokine release that produced grade 3 and 4 dose-limiting toxicities. Combining dexameth-
asone pretreatment with catumaxumab successfully reduced the cytokine-related adverse
General challenges and considerations 29
I. Development of biopharmaceuticals
effects. The toxicology evaluation conducted for the catumaxomab program will be discussed
in furthe r detail below.
Some of the early heterogeneous F(ab)
0
2 molecules, in which one of the Fab fragments spe-
cifically targeted CD64 (FcgRI) or CD16 (FcgRIII) were associated with dose-limiting toxicities
consistent with acute cytokine release (Weiner et al., 1995). A second bispecific F(ab)
0
2, this
time targeting CD64 (anti-HER2 anti-CD64; MDX-H210), activated monocyte/macrophage
effector cells in vivo and was also associated with severe adverse events consistent with acute
cytokine release including flu-like symptoms, fatigue, chest pain, dyspnea, fever, chills, hypo-
tension, myalgias, and allergic reactions (James et al., 2001) in several Phase I clinical trials.
Species selection
Similar to earlier generations of mAbs, the nonclinical safety program for a PVB will be
based on target epitopes, relevant pharmacology, clinical indication and proposed usage,
and specific molecular attributes and characteristics. As with conventional bio-
pharmaceuticals, the goal is to select appropriate nonclinical species where exposure to the
PVB results in the expected changes in cellular or molecular processes that are consistent
with an on-target pharmacologic response. Therefore, an in-depth examination of the expres-
sion, regulation and biology of the molecular targets in humans and animal species must be
conducted to compare target engagement and modulation between species. Nonclinical
development programs can then be designed on a “case-by-case” basis.
As with most mAbs, early generation PVBs have been designed to maximize specificity for
their human targets, sometimes to the exclusion of reactivity to the analogous target proteins
in the animal species typically considered for nonclinical safety evaluation. For PVB plat-
forms, the determination of an appropriate and pharmacologically relevant animal species
for toxic ity testing is complicated by having multiple target antigens. On ce it is determined
that all of the targets are expressed in the nonclinical species and have the expected relevant
downstream effects, the next consideration is whether there is cross-reactivity of the PVB to
the orthologous proteins. The multitargeting attributes can result in a number of different
cross-reactivity scenarios:
• Relevant cross-reactivity is confirmed for all of the target antigens in the nonclinical
species.
• Relevant cross-reactivity is confirmed for all of the target antigens, but the affinity is
different (often lower) in the nonclinical species for at least one target, as compared to
the affinity for the human targets.
• Relevant cross-reactivity can be confirmed for only a subset of the target antigens, with
no cross-reactivity to the other targets.
• No relevant cross-reactivity to any of the target antigens in the nonclinical species
If the results of the species-specificity analysis are consistent with either of the first two sce-
narios, a relevant nonclinical species has likely been identified in which to assess the PVB
nonclinically. However, in the second scenario, one may consider increasing the dose in
the nonclinical studies as a way to compensate for the difference in af finity between the
nonclinical species and humans. In contrast, if the PVB cross-reacts with only one of the target
antigens or does not cross-react with any of the target antigens in any nonclinical species, it is
2. Nonclinical development of monovalent and polyvalent biopharmaceuticals30
I. Development of biopharmaceuticals
likely that in vivo toxicity studies would be of limited utility. When no relevant species exists,
alternative in vivo approaches such as transgenic animal models expressing the human tar-
gets, or the use of surrogate PVBs can be considered. In the absence of any relevant in vivo
model, alternative approaches can be used to characterize potential safet y liabilities (e.g.,
in vitro cytokine release, tumor killing, inhibition of viral or bacterial entry, and receptor oc-
cupancy). However, in the absence of in vivo toxicity data, it is likely that the clinical devel-
opment plan will need to apply a “start low, go slow” paradigm.
Tissue cross-reactivity study
A tissue cross-reactivity (TCR) study is one approach to identify unexpected tissue binding
of the MVBs/PVB. However, the assay requires that an acceptable immunohistochemical
method can be developed, often utilizing expected positive and negative tissues or phenotyp-
ically characterized cell pellets expressing the target antigen(s). TCR assays are also typically
limited to molecules with a CDR (antibody-like). Historically, the TCR study has been con-
ducted on tissue panels from humans, with optional inclusion of selected or a full panel of
tissues from the species used for toxicity testing.
For PVBs, the full spectrum of tissue binding is relevant to the safety assessment of the drug.
Specific challenges to the more highly engineered molecules include optimizing binding for
multiple CDRs, which is not always possible in a single assay. In general, the same cautions
and limitations to the interpretation and use of these studies apply to PVBs as to more tradi-
tional mAbs (Leach et al., 2010; MacLachlan et al., 2021). Interpretation of results depends on
factors such as cross reactivity, presence of target antigen, nonspecific binding or artifacts
and as such, additional analyses may be needed to further characterize the nature and/or spec-
ificity of any unexpected staining.
TCR studies can provide relevant information with regard to recognition of specific bind-
ing patterns that can be supportive in the interpretation of nonclinical and clinical toxicity. It
is recognized that results of these studies should be used as one component of a broader
safety assessment of a specific molecule. A lack of tissue reactivity recognized in this assay
does not necessarily ensure a well-tolerated biopharmaceutical following in vivo administra-
tion. Likewise, identification of expected or unexpected spe cific staining may not translate to
toxicity in vivo. Finally, and consistent with ICH S6(R1) (ICH, 2011). TCR studies with mAbs
or, by extension, PVBs, are not specifically useful to select a relevant species for nonclinical
testing. Alternative approaches to the characterization of binding of MBVs/PVBs have also
been explored over the last decade. These include protein expression arrays to assess near-
target or cross-reactive binding that is not tissue specific (as reviewed in MacLachlan et al.,
2021). Such approaches are particularly useful when the intended target antigen(s) are ex-
pected to be widely distributed, and therefore, the typical TCR does not have high value
in identifying novel or unexpected tissue binding patterns. In some cases, both a TCR and
an expression array might be considered. Finally, for anticancer pharmaceuticals TCRs are
generally not conducted unless there is a specific concern, or lack of pharmacologically active
species, in which case a human TCR may be considered, in accordance with ICH S9 QA.
With awareness of these caveats, TCR studies can be a useful adjunct to the safety program
and may be an expected component of the FIH safety data package by regulators. However,
General challenges and considerations 31
I. Development of biopharmaceuticals
the value of such studies depends on the establishment of an interpretable assay, without
which, TCR studies may not be appropriate or useful for safety assessment.
Finally, TCR studies may provide only limited value if the targets are not expected to be
present in tissue used for immunohistochemistry (soluble factors, nonendogenous proteins or
must be expressed on viable cells), or if the clinical candidate PVB is a poor immunohisto-
chemical reagent for any of the intended targets. In the first case, if antigen is not ““expected
to be expressed in the tissue used for IHC, a TCR study on human tissues is still warranted to
reveal any potential unexpected binding to tissue. In the second case, if a robust immunohis-
tochemical method cannot be developed, there is no value in proceeding with the assay.
Therefore, each specific PVB must be evaluated individually to support the justification to
include, or not include, a TCR study as part of the safety assessment of the molecule.
Pharmacokinetics
The PK properties of MVB and PVBs are important factors when considering dosing
schedules in both the nonclinical and clinical settings. As discussed previously, several
different formats of PVBs exist with individual PK challenges associated with each one.
The presence and amount of target antigen play a key role in the bioavailability and terminal
elimination half-life. The format and molecular weight of the PVB will likely impact PK char-
acteristics such as exposure and disposition. PVB constructs that resemble IgG or proteins
that incorporate Fc, CH3, or other proteins as stabilizing moieties may have PK properties
similar to conventional mAbs. In contrast, PVBs based on fragment scaffolds, such as BiTE,
have a shorter half-life. Nonclinical studies need to account for these properties by adjusting
the dosing schedule and/or regimen (i.e., increased frequency, prolonged infusions, etc.) so
that exposure can be used as the basis for selecting an initial safe starting dose for clinical use.
Alternatively, the HLE-BiTE format is being explored to extend the half-life of a BiTE (see
earlier Section “Technology and Format” for details).
Molecular properties
For PVBs, selecting a candidate which has a desired therapeutic index is determined by a
multitude of factors such as the identity of target antigens, expression levels and distribution
in normal versus target tissues, and the specific properties of the antibody itself.
For CD3 PVBs, the molecular properties of the antibody such as affinity of each target arm,
avidity to the target, binding epitope, spatial orientation, and effectiveness to form the immu-
nological synapse can all be factors which influence the resulting potency and safety profile of
the molecule (reviewed in Ellerman, 2019). Ultimately, there is no universal formula that can
be applied across targets and the optimal properties should be investigated for each individ-
ual program. For a solid tumor target like HER2, which is widely expressed in normal tissues,
it is important to find a favorable Therapeutic Index (TI) as to have minimal impact on
healthy tissues. In this case, lower affinity binding to both target antigens (HER2/CD3)
showed a more favorable safety profile while maintaining high potency in vivo and
in vitro (Staflin et al., 2020). In addition, higher CD3 affinity is further associated with a bio-
distribution profile that favors secondary lymphoid tissues over tumor (Mandikian et al.,
2. Nonclinical development of monovalent and polyvalent biopharmaceuticals32
I. Development of biopharmaceuticals
2018), which is less optimal from a safety and efficacy point of view. A second approach to
increase the TI of a HER2 targeting molecule was applied by reducing the affinity and
increasing the avidity to the target (Slaga et al., 2018). The binding of this molecule is depen-
dent on the receptor density and increased the selectivity to tumor cells. No significant
toxicity or pharmacological activity was seen in in vivo toxicity studies, while the HER2/
CD3 molecule retained high potency against HER2 overexpressing tumors. For future appli-
cations, it may be possible to fine tune these molecules in more detail as well as potentially
separate the killing capacity and cytokine production to optimize the therapeutic window for
these highly potent molecules (Haber et al., 2021).
Immunogenicity
It is generally well accepted for human or humanized mAbs that immunogenici ty in
nonclinical species does not accurately predict immunogenicity in humans. This reflects the
fact that the human antibody is recognized as a fo reign protein in animals. In fact, the ICH
S6(R1) guidance document acknowledges that immunogenicity data in nonclinical safety
studies cannot be directly extrapolated to predict clinical immunogenicity. However, immu-
nogenic potential is a critical factor to consider, as it can substantially affect expo sure, phar-
macology, and toxicity profiling of biopharmaceuticals in nonclinical species.
Several factors may impact the immunogeni city of protein pharmaceuticals, such as the
amino acid sequences, post-translational modifications, higher order structure, product- or
process-related factors, as well as the mechanism of action. In addition, there are several
nonclinical and clinical determinants such as species, age, gender, immunological status,
and pre-existing antibodies (EMEA, 2017; Ducreta et al., 2022).
With PVBs, the added complexity of the molecule potentially multiplies these same gen-
eral challenges. Although clinical immunogenicity data is limited, CD3 dependent B-cell
depleting agents have shown relatively low ADA formation in the clinic presumably by elim-
inating antibody producing B-cells (Zhu et al., 2021) and/or mutated FC. T-cell engagers
which do not target B-cell malignancies show a wide range of clinical ADA rates, which
does not always correlate with clinical impact (Zhou et al., 2022). By design, many of the
new platform technologies used to generate PVBs have the potential to crea te novel epitopes
(cryptic epitopes or neoantigen formation) through the use of chemical crosslinkers, short
linker sequences, or half-life extenders such as human serum albumin. Generation of
molecule-specific reagents and development of appropriate assays such as in silico, in vitro
(e.g., DC maturation and T-cell specific assays) and in vivo tools can help assess the potential
immunogenicity risk during the candidate selection phases (Ducreta et al., 2022). Although
useful, it is difficult to translate and interpret this into a likelihood of ADA formation in
the clinic. Tools and assays for ADA detection are also utilized to measure antidrug anti-
bodies (ADAs) in the nonclinical studies to aid in study interpretation. To better characterize
the ADAs, assays may be needed that are specific for all components of the PVB and may
include assays that are platform-specific. Although the molecular attributes may increase
the immunogenicity of PVBs, and thus may limit nonclinical testing if exposure cannot be
maintained, the ability of nonclinical models to predict clinical immunogenicity remains
problematic. The high ADA formation in nonclinical studies with CD3 bispecifics may impact
General challenges and considerations 33
I. Development of biopharmaceuticals
exposures and limit the conduct of longer-term studies (Yadav et al., 2022; Campagne et al.,
2020; Chichili et al., 2015; Hosseini et al., 2020, BLA; 761324, BLA; 761291). Future consider-
ations with regards to conducting sub-/chronic studies may include a weight of evidence
approach with information around the existing clinical experience with the target and
nonclinical safety assessment as demonstrated in the case studies discussed below (Teclista-
mab, Epicoritamab).
One unique feature of PVBs is that recognition of target antigens is monovalent, regardless
of platform technology. The different epitopes recognized by a PVB may lie within the same
target antigen or be within two different cell-bound or soluble proteins. PVBs can also be
designed to recognize two antigens expressed on different cells such as platforms designed
to induce T celleredirected lysis (i.e., BiTE). ADAs that recognize and bind to one of the
CDRs will likely inhibit antigen binding and desired activity for that specific pathway. How-
ever, in the case of PVBs, the other CDR-binding sites are not likely to be affected, so their target
epitope is still recognizable and can maintain their desired biological activity, provided there is
no steric hindrance from the ADA-bound arm of the PVB. One important consequence of
ADAs is that in the case of fragment-based formats, the bivalent ADA could bind two separate
therapeutic molecules, thereby creating a multivalent molecule against one of the target anti-
gens. Other than impacting the desired activity, this situation may not have any safety conse-
quences. However, in platforms designed to induce T celleredirected lysis, the possibility
exists that ADAs that recognize and bind to the CDR that targets the tumor associated antigen
(i.e., CD20, CD19, EGFR, etc.) could dimerize two therapeutic molecules that still have the abil-
ity to bind to the CD3 complex of the T-cell receptor. This theoretical configuration could
resemble the structure and mimic the activity of a conventional anti-CD3 mAb, resulting in un-
desired activation of T cells in an antigen-independent manner. Importantly, it should be
emphasized that no such ADA response has been note d to date in the numerous clinical trials
with T celleredirecting biopharmaceuticals.
Selection of starting dose
As described in ICH S6(R1), nonclinical safety studies for biopharmaceuticals are designed
to identify potential safety liabilities associated with treatment under conditions that are
consistent with the proposed clinical development strategy. For biopharmaceuticals, both
in vitro and in vivo pharmacology and toxicology data from relevant species is considered
when determining the FIH dose. This includes but is not exclusive to, target biology, target
binding properties, receptor occupancy, species specific effects/differences, off-target toxic-
ities, and pharmacokinetics/pharmacodynamics. Nonclinical studies help determine a rele-
vant endpoint (e.g., No-observed-adverse-effect level (NOAEL), highest nonseverely toxic
dose (HNSTD), minimal anticipated biological effect level (MABEL) and pharmacologically
active dose (PAD), which guide HED determination with added safety factors (ICH S6; Saber
et al., 2016; Tibbitts et al., 2010). Depending on the targets, the data available upon comple-
tion of the nonclinical toxicology studies may be limited due to the absence of relevant
nonclinical species. For example, the PVBs may not cross-react with the nonclinical species
or the relevant targets may not be expressed in the nonclinical species (i.e., viral or bacterial
targets). In these cases, other approaches based on in vitro data (e.g., receptor occupancy or
2. Nonclinical development of monovalent and polyvalent biopharmaceuticals34
I. Development of biopharmaceuticals
redirected cell lysis) and incorporat ed modeling-based approaches can be considered when
determining appropriate first-in-human dose.
ICH S9 nonclinical evaluation of anticancer pha rmaceuticals suggests a minimal antici-
pated biological effect level (MABEL) to determine the FIH dose regarding targets with im-
mune agonistic properties or MOAs, which could enhance immune activation. This came as a
response following the TGN1412 clinic al trial incidence of near fatal systemic inflammatory
responses in healthy volunteers. Subsequently the FDA published a position paper (Saber
et al., 2017, US FDA, 2021) concluding that in vitro 10%e30% pharmacologic activity was
acceptable for CD3 engagers as not to exceed the MTD in the clinic. For CD3 bispecific mol-
ecules a MABEL approach is generally utilized using in vitro pharmacology data from T-cell
activation/proliferation, cytokine release or target lysis/apoptosis assessments. Depending
on the available nonclinical data, the in vitro results are interpreted in the context of the
in vivo pharmacology/pharmacokinetics information, in addition to in vitro and in vivo
safety data, to ensure that the selected FIH dose is considered safe.
For cancer patients treated with CD3 engagers, there is an additional need to refine and
optimize the FIH dose selection to minimize the number of patients treated at sub-
therapeutic dose levels.
Summary of challenges
The key challenge for the development of PVBs will continue to be the identification of clin-
ically viable targets that improve efficacy without increasing known or inducing novel adverse
effects. Selection of appropriate nonclinical species, characterization of target expression/dis-
tribution, effects of modulation of multiple pathwa ys, and PK and immunogenicity properties
all increase with the complexity of the PVB. Although a vast majority of the PVB platforms
currently in development have focused on oncology indications, application of these platforms
in additional non-life-threatening or chronic disease indications will put even more pressure on
the nonclinical safety assessment since they will likely require a larger therapeutic index for
establishing a safe starting dose in humans. There are several bispecifics being developed in
the nononcology space as reviewed in Mullard (2017). The first bispecific antibody approved
for nononcology indications, HEMLIBRA, is used for the treatment of hemophilia A and tar-
gets factor IXa and X. Careful nonclinical safety evaluation will continue to play an importan t
role in minimizing the risk to patients given these therapeutic platforms.
T celledependent bispecific antibody biotherapeutics
As discussed previously, desirable qualities for drug targets from a safety perspective
include low normal tissue expression and toxicology species cross-reactivity. As multiple
platforms of PVBs exist, this section will discuss several molecules that work via
T celleredirected tumor lysis as their mechanism of action and are mainly developed for
oncology therapeutics. Although this class also includes co-stimulatory bispecifics (e.g.,
CD28 and CD137) that are under development, CD3 bispecifics will be discussed here in
detail as there are several approved CD3 bispecifics in the clinic (e.g., Table 2.1), in addition
T celledependent bispecific antibody biotherapeutics 35
I. Development of biopharmaceuticals
Соседние файлы в папке Библиотека им академика М.И. Перельмана