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Tumor-localized costimulatory T cell engagement by the 4-1BB/HER2
bispecific antibody-Anticalin fusion PRS-343
Authors: Marlon J. Hinner1*
, Rachida Siham Bel Aiba1, Thomas Jaquin
1, Sven Berger
1,
Manuela Dürr1, Corinna Schlosser
1, Andrea Allersdorfer
1, Alexander Wiedenmann
1, Gabriele
Matschiner1, Julia Schüler
2, Ulrich Moebius
1, Christine Rothe
1, Louis Matis
1, Shane.A. Olwill
1*
Affiliations:
1 Research and Development, Pieris Pharmaceuticals GmbH, Freising, Germany.
2 In Vivo Operations, Oncotest GmbH, Freiburg, Germany.
*Corresponding authors: Shane A. Olwill, Pieris Pharmaceuticals GmbH, Lise-Meitner-Strasse
30, 85354 Freising, Germany. Phone: +49-8161-1411400; email olwill@pieris.com.
Marlon J. Hinner; email marlon.jakob@hinner.de.
Abstract:
Purpose: 4-1BB (CD137) is a key costimulatory immunoreceptor and promising therapeutic
target in cancer. To overcome limitations of current 4-1BB-targeting antibodies, we have
developed PRS-343, a 4-1BB/HER2 bispecific molecule. PRS-343 is designed to facilitate T-cell
costimulation by tumor-localized, HER2-dependent 4-1BB clustering and activation.
Experimental Design: PRS-343 was generated by the genetic fusion of 4-1BB-specific
Anticalin® proteins to a variant of trastuzumab with an engineered IgG4 isotype. Its activity was
characterized using a panel of in vitro assays and humanized mouse models. The safety was
assessed using ex vivo human cell assays and a toxicity study in cynomolgus monkeys.
Results: PRS-343 targets 4-1BB and HER2 with high affinity and binds both targets
simultaneously. 4-1BB-expressing T cells are efficiently costimulated when incubated with PRS-
343 in the presence of cancer cells expressing HER2, as evidenced by increased production of
proinflammatory cytokines (IL-2, GM-CSF, TNF- and IFN-In a humanized mouse model
engrafted with HER2-positive SK-OV-3 tumor cells and human PBMCs, PRS-343 leads to
tumor growth inhibition and a dose-dependent increase of tumor-infiltrating lymphocytes. In
IND-enabling studies, PRS-343 was found to be well tolerated, with no overt toxicity and no
relevant drug-related toxicological findings.
Conclusion: PRS-343 facilitates tumor-localized targeting of T cells by bispecific engagement
of HER2 and 4-1BB. This approach has the potential to provide a more localized activation of
the immune system with higher efficacy and reduced peripheral toxicity compared to current
monospecific approaches. The reported data led to initiation of a Phase 1 clinical trial with this
first-in-class molecule.
Running Title: Costimulatory T-cell engagement by the 4-1BB/HER2 bispecific PRS-343
molecule
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Translational Relevance
Immunotherapy with checkpoint inhibitors, such as anti-PD-1 monoclonal antibodies, has had a
major impact on cancer therapy. While such therapies afford durable responses or even cures,
additional therapeutic strategies are still required for the majority of patients who do not respond
or relapse. The activation of costimulatory pathways such as 4-1BB has long been acknowledged
to hold great promise, but clinical efforts thus far have failed to demonstrate broad efficacy and
have been associated with severe toxicity (1, 2). Herein we describe the HER2/4-1BB bispecific
molecule PRS-343, which aims to overcome these limitations by activating 4-1BB in a tumor-
localized manner, sparing the periphery of unwanted toxicity. The data presented herein confirm
the desired mode of action of PRS-343, providing a novel approach to target 4-1BB that may
prove both safer and more efficacious compared to monospecific targeting. PRS-343 has the
potential to offer an alternative therapeutic strategy to patients in multiple HER2-positive
indications including bladder, breast, and gastric cancer.
Introduction
4-1BB, also known as CD137, is a costimulatory immune receptor, a member of the tumor
necrosis factor receptor (TNFR) super-family, and is predominantly expressed on activated
CD4+ and CD8+ T cells, activated B cells, and natural killer (NK) cells (3). 4-1BB plays an
important role in the regulation of immune responses and together with the fact that it is
expressed on tumor infiltrating lymphocytes makes it a promising target for cancer
immunotherapy. The 4-1BB ligand (4-1BBL) is constitutively expressed on several types of
antigen-presenting cells (APC) (4). Upon pathway activation, 4-1BB facilitates enhanced
proliferation, cytokine production, and cytolytic activity of T and NK cells (5). Recent studies
have pointed to the pathway’s impact on mitochondrial capacity and biogenesis of T cells to
explain why 4-1BB agonism could help overcome the immunosuppressive landscape of the
tumor microenvironment (6). While most research has focused on 4-1BB in the context of T
effector cells it should be noted that 4-1BB is also expressed on T regulatory cells where its role
remains contentious and requires further elucidation (7) . 4-1BB has also been implicated in
promoting a central memory T cell response which may support therapeutic persistence of tumor
specific T cells and resistance to exhaustion in patients treated with a 4-1BB agonist (8, 9).
The potential therapeutic benefit of 4-1BB costimulation has been demonstrated in multiple
preclinical models. The forced expression of 4-1BBL on a tumor, for example, leads to tumor
rejection (10). Likewise, the forced expression of an anti-4-1BB single chain antibody fragment
(scFv) on a tumor leads to a CD4+ T-cell and NK-cell dependent elimination of the tumor (11-
13). A systemically administered anti-4-1BB antibody has also been demonstrated to lead to
retardation of tumor growth in mouse models (14).
Human ex vivo data support the potential of 4-1BB as a costimulatory receptor in cancer therapy.
It has been reported that for T cells isolated from human tumors, 4-1BB is a marker for those that
are tumor-specific (15). In line with this observation, anti-4-1BB antibodies can be utilized to
improve adoptive T-cell therapy (ACT) by augmenting the expansion and activity of CD8+
melanoma tumor-infiltrating lymphocytes (16). Further clinical evidence for the importance of 4-
1BB signaling for a sustained and successful anti-cancer T cell response is provided in ACT with
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chimeric antigen receptors (CARs), where inclusion of 4-1BB-signaling elements in the
cytoplasmic domain of CARs has been found to improve durability of clinical responses (17, 18).
Receptors from the TNF receptor family in general require higher-order clustering for efficient
downstream signaling activation in the TNF receptor expressing cell (19), which usually is not
afforded by the soluble ligand alone, but requires engagement of another cell expressing the
respective ligand on its surface. For example, despite being a homotrimer, soluble 4-1BBL is not
capable of efficiently activating 4-1BB downstream signaling (20). Antibodies targeting 4-1BB
are either inherently agonistic or often require a secondary means of clustering beyond bivalent
binding to allow for agonistic activity. It is hypothesized that in vivo TNF receptor agonism by
such antibodies is dependent on simultaneous binding to Fc receptor-positive cells (21, 22).
The intricate mode of activation of 4-1BB may underlie the modest clinical success obtained
with monospecific anti-4-1BB antibodies to date. BMS-663513 (urelumab) (23) has been
hampered by dose-limiting hepatotoxicity, likely due to its tendency to systemically activate 4-
1BB (1). Initial clinical trials with PF-05082566 (utomilumab) (24, 25) have not shown the same
safety issues as urelumab profile, yet preclinical characterization suggests it is a less potent 4-
1BB agonist (2).
To overcome the challenges of targeting the 4-1BB pathway systemically, we have generated
PRS-343, a bispecific molecule designed to activate the pathway in a tumor-localized and -
dependent manner. PRS-343 binds 4-1BB and the tumor associated antigen HER2, and was
generated by the recombinant fusion of a 4-1BB-specific Anticalin protein to a HER2-specific
antibody. Anticalin proteins are engineered variants of lipocalins, a family of natural
extracellular binding proteins. Lipocalins are characterized by a highly conserved tertiary
structure despite low amino acid identity. This property coupled to the fact that they possess free
C- and N-termini, which are not required for target recognition, allow facile fusion to other
proteins, defining a versatile basis for a multispecific biologics platform (26). The molecule,
PRS-343, is designed to promote 4-1BB clustering by bridging T cells with HER2-positive
tumor cells, providing a potent costimulatory signal to tumor antigen-specific T cells, further
enhancing T cell receptor (TCR)-mediated activity and leading to tumor destruction. PRS-343-
mediated 4-1BB activation is, therefore, biased towards locations in the body where T cells and
tumor cells are co-localized, such as in primary tumors with tumor-infiltrating lymphocytes
(TIL) or in lymph-nodes containing tumor metastases. Here, we describe the generation and
preclinical characterization of PRS-343 with regard to target engagement, biological activity and
safety.
Materials and Methods
Engineering and production of Anticalin proteins and bispecific fusion proteins
Recombinant 4-1BB (R&D Systems) was used as the target for phage display selection and
ELISA screening of cognate Anticalin protein candidates. A random library based on human
neutrophil-gelatinase associated lipocalin (NGAL) with high combinatorial complexity was
prepared by concerted mutagenesis of multiple amino acid positions (27). 4-1BB target
specificity was confirmed by ELISA screening and selected Anticalin proteins were optimized
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via partial mutagenesis of coding regions followed by biophysical and functional
characterization, which resulted in selection of a lead candidate J10.
Bispecific antibody-Anticalin fusion proteins were generated by the recombinant fusion of the 4-
1BB-specific Anticalin protein (J10) to either the heavy or light chain N- or C-terminus of a
modified variant of trastuzumab. Specifically, the isotype of trastuzumab was changed to an
IgG4 and additional mutations were introduced to reduce interactions with activating and
inhibitory Fc-receptors. These were the mutation S228P, which has been described to suppress
half-antibody exchange (28), and the mutations F234A and L235A which have been reported to
reduce binding to FcRI, were introduced (29, 30). Bispecific proteins were obtained by
recombinant expression in mammalian cells (HEK293 or CHO) using standard techniques.
Reagents and cell lines
Recombinant 4-1BB and HER2 protein (human and cynomolgus monkey) were obtained
from R&D Systems and Sino Biological. The cell lines HT-29, SKBR-3, SKOV-3, BxPC-3,
MCF-7, MDA-MB-231, MKN-7, NCI-N87, ZR-75-1, A375, A431, A549, JIMT-1, MDA-MB-
453, MKN45, SUM-225, and LoVo were obtained from ATCC. Primary cells (human cardiac
myocytes, human epidermal keratinocytes, human pulmonary fibroblasts, human cardiac
fibroblasts, human dermal microvascular endothelial cells, human umbilical vein endothelial
cells, human aortic smooth muscle cells and human bronchial smooth muscle cells) were
obtained from Promocell. The cells were expanded following cell bank instructions. The HER2
levels were confirmed by QifiKit (Dako) according to the provided protocol and were
standardized to HER2 levels expressed by SKBR3. The list of the cells, corresponding cell bank
catalogue number and their respective HER2 expression levels standardized to SKBR3 HER2
levels are summarized in Supplementary Table S1. CHO cells expressing human 4-1BB (h 4-
1BB) were established by stable transfection of h 4-1BB using the Flp-In system (Invitrogen).
An NF-ĸB-Luc2/4-1BB Jurkat cell line was obtained from Promega. Human peripheral blood
mononuclear cells (PBMCs) from healthy volunteer donors were isolated from buffy coats by
centrifugation through a polysucrose density gradient, and T cells were isolated from the
resulting PBMCs using a pan T cell isolation Kit (Miltenyi Biotec) according to the
manufacturer´s protocols. The 4-1BB agonist antibody 20H4.9 (corresponding to urelumab) was
obtained by recombinant expression in CHO cells using standard techniques.
Target-binding studies
Binding of PRS-343 to 4-1BB or HER2 proteins (human or cynomolgus monkey) was analyzed
by enzyme-linked immunosorbent assay (ELISA) or surface plasmon resonance (SPR).
In the target-binding ELISA, the extracellular domain of the target proteins, 4-1BB (human or
cynomolgus), HER2 (human) or control protein, was coated onto microtiter plates. Either PRS-
343, the 4-1BB specific reference Anticalin protein or the reference anti-HER2 antibody was
added and binding was detected with peroxidase conjugated anti-human IgG or anti-NGAL
antibody.
For the dual-binding ELISA, human HER2 (h HER2) was coated and PRS-343 binding was
detected using biotinylated h 4-1BB detected by peroxidase-labeled ExtrAvidin.
In the SPR affinity assay, biotinylated h 4-1BB or h HER2 was captured on a sensor chip CAP
using the Biotin CAPture Kit (GE). Determination of PRS-343 binding kinetics and affinity was
performed by applying four dilutions of PRS-343 to the chip surface with a flow rate of
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30 µL/min; the sample contact time was 180 s and dissociation time was 1800 s. Data were fit
with a 1:1 binding model.
Binding capability to primary cells, tumor cells, and target-positive engineered cell lines was
assessed by flow cytometry. Cells were incubated with PRS-343, reference antibody or h IgG4 as
negative control, and binding of the test molecules was detected using Alexa Fluor 488 labeled
goat anti-human IgG antibody.
An immunohistochemical assay was performed to assess cross reactivity of PRS-343 on human
tissue. Briefly, cryo-sections histologically prepared at a nominal 5 μm from a panel of 40 frozen
human tissues together with positive and negative control material were stained with biotinylated
PRS-343 or control. A detailed microscopic examination was performed to assess each tissue for any
sign of positive staining.
T-cell activation
The impact of HER2 receptor density on PRS-343-mediated T-cell activation was assessed in co-
culture experiments using a panel of cell lines expressing different levels of HER2. Cancer cell
lines representing a range of clinically relevant levels of HER2 receptor were tested for their
ability to mediate clustering of PRS-343 and subsequent activation of T-cells. To evaluate a
potential therapeutic window cell lines derived from healthy tissues known to express
background levels of HER2 were also included. The level of T-cell activation was measured by
quantification of human IL-2, using an electrochemiluminescence (ECL) immunoassay
(Mesoscale Discovery). Briefly, cancer cells or cells derived from healthy tissue pretreated with
10 µg/ml of mitomycin C (Sigma Aldrich) were seeded in culture plates precoated with anti-CD3
and incubated overnight at 37 °C in a humidified 5 % CO2 atmosphere. T-cell suspension
(5 x 104
cells) together with test article were added. The IL-2 concentration in the supernatant
was assessed by an ECL assay (using IL-2 DuoSet kit; R&D Systems) following 3 days
incubation.
Specific activation of the 4-1BB pathway by PRS-343 was assessed using a luciferase reporter
cell assay (Promega), where a 4-1BB-overexpressing reporter cell line (NF-ĸB-Luc2/4-1BB
Jurkat cells) was co-cultured with HER2-positive tumor cell lines and where 4-1BB pathway
activation was measured by luminescence.
Cytokine release assay
The potential of PRS-343 to induce cytokine release syndrome was evaluated in vitro using a
cytokine release assay (31), where soluble or coated (wet or dry coated) PRS-343 was incubated
with human PBMC for 72 hours followed by the quantification of a panel of pro-inflammatory
cytokines (MSD). The anti-CD3 monoclonal antibody OKT3 (Muromonab-CD3) was used as
positive control and a human monoclonal IgG4 isotype antibody was used as negative control.
Cynomolgus Toxicity Study
A 4-week repeated dose toxicity study (vehicle, 10 mg/kg or 120 mg/kg PRS-343 on days 1, 8,
15, 22, and 29) followed by a 4-week recovery period was performed in cynomolgus monkeys.
Serum levels of PRS-343 were assessed by an ECL assay. Assessment of safety and toxicity was
based on standard parameters.
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Mouse and cynomolgus monkey pharmacokinetics
Single dose pharmacokinetics studies were performed in mice and cynomolgus monkeys. Male
CD-1 mice were administered with an intravenous injection of PRS-343 (10 mg/kg).
Cynomolgus monkeys (Macaca fascicularis) received a 60-min infusion of PRS-343 (3 mg/kg)
For each study plasma was collected predose and at multiple timepoints post administration.
PRS-343 plasma concentrations were determined via an ECL assay (MSD) using a dual-binding
ELISA. The pharmacokinetic parameters were derived by non-compartmental analysis using
Phoenix WinNonlin.
In vivo tumor efficacy model
PRS-343 in vivo activity was evaluated in a SK-OV-3 ovarian cancer model in human PBMC-
reconstituted NOG female mice (NOD.cdPrkdcscid
IL2rgtm1.Sug
/JicTac, supplied by Taconic,
Denmark), aged 5-7 weeks. Tumor growth was monitored twice weekly and tumor volume was
calculated as follows: (length x width2)/2. At study end, plasma was collected for all animals and
lymphocyte phenotyping was carried out by flow cytometry assessing human cell markers such
as CD45 (Invitrogen), CD4 and CD8 (BD Biosciences). To assess tumor infiltrating
lymphocytes, tumors were also collected, fixed and paraffin-embedded followed by human
CD45, CD3, CD4 and CD8 staining. Stained tumor sections were digitally scanned, and the
resulting digitalized data were evaluated to determine the percentage of target positive (tumor-
infiltrating) cells.
Graft Versus Host Disease (GVHD) model
The potential for PRS-343 to induce systemic GVHD was assessed. 5-7-week-old female NOG
mice (NOD.cdPrkdcscidIL2rgtm1.Sug/JicTac, supplied by Taconic, Denmark) were injected
with fresh human PBMC and treated with PRS-343 or controls. As a read-out for signs of
GVHD, the animals were routinely checked for changes in body weight or general health. Mice
who were euthanized (based on predefined criteria) or spontaneously expired mice were recorded
for survival analysis by each treatment. Data were plotted using a Kaplan Meier curve.
Flow cytometry
Flow cytometry analyses were carried out on an iQue Screener (Intellicyt Corporation) equipped
with ForeCyt software or with an Attune Focusing Cytometer (blue (488 nm)/violet (405 nm)
laser configuration).
Ethical considerations
All animal experiments and protocols were approved by the institutional animal welfare body
and the relevant local authorities and were conducted according to all applicable international,
national and local laws and guidelines. All animal experiments were approved by the regional
Ethics Committees (Germany or the UK).
Statistical analysis
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All statistics were calculated using Graphpad Prism Version 5 for Windows. Statistical
significance was determined using one-way or two-way ANOVA to compare differences among
multiple groups. P values less than 0.05 were considered statistically significant.
Results
Generation of PRS-343 by Protein Engineering and Design
A 4-1BB binding Anticalin protein was obtained by phage display selection and optimized by
protein engineering as described in Materials and Methods. The 4-1BB specific Anticalin protein
(J10) has an affinity of 2 nM as determined by SPR, binds to human 4-1BB transfected CHO
cells and does not compete with the binding of 4-1BB ligand to its receptor (data not shown). An
ELISA screen of related TNFR superfamily proteins (4-1BB Ox40, GITR, TNFRI, TNFRII,
CD30, RANK) confirmed J10 Anticalin protein specifically binds to 4-1BB (Figure S1)
As the HER2-binding, tumor-targeting building block of a 4-1BB/HER2 bispecific, we utilized
a modified variant of trastuzumab. The formatting flexibility offered by the Anticalin technology
facilitated the generation of various Antibody-anticalin fusion formats as depicted in Figure S 1.
We hypothesized that the distance between T cell and tumor cell binding sites may impact the
ability of the 4-1BB/HER2 bispecific to appropriately cluster the 4-1BB receptor and thus
activate the pathway. Therefore, the 4-1BB-engaging Anticalin protein was genetically fused
either to the N- or C-terminus of the anti-HER2 antibody heavy or light chain resulting in the
generation of four different geometries of the fusion protein covering a range of binding
distances. The antibody Fc region was engineered (see Materials and Methods for details) to
exclude the risk of NK-driven ADCC against 4-1BB-positive cells or undesired, non-tumor-
target activation of 4-1BB-positive lymphocytes via FcR derived crosslinking. The interaction
with the neonatal Fc receptor (FcRn) was retained to support a prolonged terminal plasma half-
life.
While all formats behaved similarly from the perspective of biophysical and target binding
properties, we observed a marked difference in their ability to elicit T cell activation (Figure S2).
Based on these data a single format was selected and is described herein. The lead bispecific
fusion molecule, PRS-343, was constructed via the genetic fusion of the 4-1BB specific
Anticalin protein to the C-terminus of the heavy chain of the trastuzumab IgG4 variant,
connected by a flexible, non-immunogenic (G4S)3 linker sequence, as depicted in Figure 1.
Human target binding
The interaction of PRS-343 with its human targets was investigated by ELISA, surface plasmon
resonance (SPR) and FACS. As assessed by SPR, PRS-343 binds to recombinant human HER2
with high affinity (KD = 0.3 nM), similar to the parental antibody trastuzumab. The binding
affinity of PRS-343 to human monomeric 4-1BB was determined to be 5 nM (Figure 1).
In a FACS assay, PRS-343 was found to bind HER2-expressing MCF-7 cells with an EC50 of
7.4 nM. Binding to CHO cells transfected with human 4-1BB was determined with an EC50 of
6.2 nM (Figure 1).
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Using a dual binding ELISA, PRS-343 was found to be capable of binding both targets
simultaneously, which is a prerequisite for its envisioned mode of action.
PRS-343 displays reduced cross-reactivity to cyno 4-1BB in comparison to human 4-1BB as
shown by ELISA (Figure 1K). An SPR experiment also demonstrated that binding of PRS-343 to
cyno 4-1BB strongly depends on target densities (data not shown).
Further SPR experiments demonstrated that PRS-343 does not bind to Fc receptors but binding
to the neonatal Fc receptor (FcRn) is retained. In accordance with its lack of interaction with Fc
receptors, PRS-343 does not elicit any ADCC activity when co-incubated with human PBMCs
and HER2-positive breast carcinoma cells (data not shown).
A tissue cross-reactivity study using a panel of 40 human tissues (n=3 donors) each demonstrated
that PRS-343 binds as expected for a molecule targeting both HER2 and 4-1BB in a bispecific
manner. Epithelial cells of various tissues showed strong positivity for binding PRS-343 which is
in accordance with published data on HER2 expression in human tissues (32). The observed
lymphocyte staining is in agreement with the expected binding of PRS-343 to 4-1BB on this cell
type since 4-1BB has been reported to be (inducibly) expressed on lymphocytes (33).
In vitro activity of PRS-343
The ability of PRS-343 to costimulate T cells in a HER2-dependent manner was investigated in
vitro using a reporter assay.
The reporter cell assay was performed to investigate whether PRS-343 is capable of inducing
HER2-dependent 4-1BB clustering on T cells. In this assay, NF-ĸB-Luc2/4-1BB Jurkat cells
were co-cultured with a HER2-positive tumor cell line. Successful clustering of 4-1BB on the
Jurkat cell surface is expected to lead to TRAF-2 (and TRAF-1) mediated activation of Nf-B
(reviewed in reference (34)), which in the presence of a luciferase substrate can be detected by
increased luminescence.
Indeed, co-incubation of up to 10 nM PRS-343 with the Jurkat reporter cell line and the HER2-
positive NCI-N87 cell line led to a 20-fold increase of NF-κB luciferase reporter activity with an
EC50 of 50 pM (Figure 2A). Furthermore, in a wash-out experiment where PRS-343 was added
to various cancer cell lines overnight prior to a media exchange, HER2 specific 4-1BB activation
was maintained (Figure S4). When using the low HER2-expressing MKN-45 or Hep-G2 cell
lines, the NF-κB activity was similar to background and control values. A bell-shaped response
was observed when concentrations of up to 1µM PRS-343 were applied in this type of assay
(data not shown).
These results demonstrate that PRS-343 facilitates 4-1BB pathway activation only in the
presence of HER2-overexpressing cells.
HER2-dependent costimulatory T cell activation
To investigate whether this mechanism can be applied to effectively costimulate T cells, a co-
culture assay was developed using human T cells and HER2-expressing cell lines. Here, the T
cells receive a primary T cell receptor stimulus via anti-CD3 antibody activation, and PRS-343
induced costimulation can be quantified by measuring supernatant levels of pro-inflammatory
cytokines. Using a multiplex assay, we first investigated which cytokines were produced by T
cells co-stimulated by PRS-343 in the presence of HER2-expressing cells (Figure S5). The
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strongest fold increase over background was observed for IL-2. Statistically significant increases
in GM-CSF, IFN and TNFwereobserved. IL-4 and IL-6 supernatant levels were increased
albeit absolute cytokine levels were low. There was no PRS-343-dependent response observed
for IL-1, IL-8, IL-10 and IL-12. Given the robust nature of the IL-2 response it was selected for
as a marker of activation for future assays. In the presence of HER2 positive cell lines, a dose-
dependent induction of IL-2 was observed with PRS-343 (Figure 2B and C). When the
experiment was performed with cell lines expressing basal levels of HER2, no PRS-343-
dependent IL-2 induction was observed. To further confirm that PRS-343 activity is dependent
on HER2 binding, additional assays were performed in the presence of an excess of trastuzumab
(200 nM). Under these conditions, the excess trastuzumab competed for binding to HER2 and
was found to abrogate PRS-343 induced T cell costimulation, further supporting its envisaged
mode of action (Figure 2C), whereas the control 4-1BB agonistic antibody 20H4.9 consistently
induced IL2 secretion irrespective of the co-incubated cell lines (Figure 2C).
Impact of HER2 expression level
To examine the HER2 expression level threshold required for successful PRS-343-induced T cell
costimulation, the assay was performed with a panel of cell lines of variable HER2-positivity
(Table S1). The HER2 cell surface expression level was determined by quantitative FACS and is
reported as the specific anti-HER2-antibody binding capacity (sABC). For the purpose of
comparison, HER2 expression was normalized relative to a reference cell line, SK-BR-3. The
cancer cell lines selected represent a wide range of HER2 expression levels ranging from
approximately 1% to 200% of the reference value. In contrast, cell lines from healthy tissues
express HER2 at a much lower and more confined level, with relative HER2 expression around 1
% of HER2 expression on SK-BR-3 (Table S1). These cancer- and healthy tissue-derived cell
lines were then applied in the previously described costimulatory T cell activation assay to
identify a potential HER2 threshold required for T cell costimulation.
A selection of experimental results based on IL2 as the readout and utilizing tumor cell lines and
primary cell types is shown in Figure 3. These data confirm that IL2 secretion strongly correlates
with HER2 expression. Assessing the maximum level of IL2 secretion compared to control, we
show that cancer cells with a high level of HER2 induced significantly increased IL2 secretion.
For cell lines described as having a more intermediate level of HER2 (e.g. MCF-7 or MKN-45),
we could also observe significantly increased IL2 secretion in a proportion of the donors tested.
Both the cancer cell lines expressing basal levels of HER2 (such as Bx-PC-3 or MDA-MB-231)
and the tissue-derived primary cells did not induce a significant amount of IL2 secretion,
supporting the hypothesis that PRS-343 requires HER2 expression at supraphysiologic levels on
target cells to costimulate T cells.
Cytokine release assay
To investigate the risk of systemic cytokine release in patients induced by PRS-343, an in vitro
cytokine release assay (31) was carried out with peripheral blood mononuclear cells (PBMCs)
from 12 healthy control donors. In contrast to the anti-CD3 positive control antibody OKT3,
PRS-343 did not induce a significant increase of cytokines over background when wet coated
onto plates or when incubated with PBMCs in solution. When PRS-343 was air-dried, a modest
but statistically significant increase of the cytokines IFN-y, GM-CSF, IL-4 and IL-8 was
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observed only at the highest concentration (i.e.100 µg/mL). Relative to the maximum increase
induced by OKT-3 (10 µg/mL, air dried), the levels induced by PRS-343 were 0.2% for GM-
CSF, 0.1% for IFN-ɣ, 6% for IL-4 and 13% for IL-8 (Figure S3).
Pharmacokinetics in mice and cynomolgus monkeys
Pharmacokinetics was evaluated following a single i.v. administration of PRS-343 by bolus
injection of male CD-1 mice at 10 mg/kg, or upon a 60-minute i.v. infusion to male cynomolgus
monkeys at 3 mg/kg. The plasma concentrations in both species were found to decline in a
biphasic manner (Figure 4). The terminal elimination half-life was >14 days in mouse. In
cynomolgus monkeys, a terminal elimination half-life of approximately 4 days was observed. In
conclusion, the determined PK parameters for PRS-343 demonstrate that it behaves similar to a
conventional monoclonal antibody in preclinical models.
Humanized Mouse Tumor Model
The in vivo activity of PRS-343 was evaluated in a humanized mouse model using
immunocompromised mice subcutaneously engrafted with the SK-OV-3 cell line as a HER2-
positive tumor xenograft. This cell line was chosen based on HER2 positivity, trastuzumab
sensitivity, and the ability to grow homogenously as a subcutaneous xenograft in the presence of
human PBMC without being directly eliminated. Mice with established tumors (> 100mm3) were
grouped and treated with test articles on a weekly dosing schedule. Median tumor volumes over
time for each of the treatment groups are shown in Figure 5A. PRS-343 displayed dose-
dependent anti-tumor efficacy with doses ranging from 4µg to 100 µg (approximately 0.2 mg/kg
to 5 mg/kg), while the 200 µg dose (approximately 10 mg/kg) did not further enhance tumor
regression. Equimolar dosing of the trastuzumab-IgG4 control antibody displayed comparable
anti-tumor efficacy to that of PRS-343. The model has been previously described as being
responsive to anti-HER2 therapy and helps confirm that HER2-mediated anti-tumor activity is
preserved in PRS-343. Interestingly, the 4-1BB agonist (20H4.9) antibody was unable to block
tumor growth in this model.
Flow cytometry analysis of the human immune cell population in the peripheral blood of the
mice revealed very little differences between the PRS-343-treated groups and control groups.
However, in mice treated with the 4-1BB agonist antibody (20H4.9), a peripheral expansion of
human CD8-positive cells was observed (Figure 5B), demonstrating a systemic impact of the
agent.
To assess the 4-1BB-mediated effect of PRS-343, excised tumors from treated mice were
analyzed for immune infiltration. PRS-343 administration led to a significant increase in human
CD45-positive lymphocytes in tumor tissue (Figure 5C) compared to the control groups. While
assessing the immune cell subtypes infiltrated within the tumors, we could observe that PRS-
343-induced infiltrate consisted predominantly of CD8 positive T cells (Figure 5C). Importantly,
there was no significant increase in immune cell infiltrates in the excised tumors from mice
treated with the trastuzumab-IgG4 when compared to controls.
In immune-compromised mice engrafted with human PBMCs, graft-versus-host disease (GVHD)
is expected to occur due to the human PBMCs destroying mouse tissue and organs resulting in
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progressive weight loss and mortality (35). In this humanized xenograft study, we observed that
the 4-1BB agonist antibody accelerated GVHD-induced mortality, while the administration of
PRS-343 or the isotype control antibody did not influence weight loss or the median survival of
the mice. To confirm this observation, we carried out a dedicated experiment using larger group
sizes (n=15) of non-tumor bearing mice. This study confirmed that weight loss and survival
following PRS-343 treatment were comparable to that observed in the IgG4 isotype control
treated animals, while treatment with the systemically active 4-1BB agonist led to both
significantly greater weight loss and shorter survival compared to control (Figure 5D).
Taken together, these data show that PRS-343 provides dual activity by increasing the number of
tumor-infiltrating lymphocytes coupled with direct tumor growth inhibition by bispecific
targeting of 4-1BB and HER2. Notably, the tumor growth inhibition provided by targeting HER2
did not require any antibody directed cellular cytotoxicity (ADCC), as both PRS-343 and the
trastuzumab-IgG4 control lack the ability to interact with Fc-gamma receptors on NK cells that
ADCC would require.
Safety of PRS-343 in cynomolgus monkeys
The safety of PRS-343 was investigated in a 4-week GLP-compliant toxicity study in
cynomolgus monkeys. It is important to take into account the reduced cross-reactivity of PRS-
343 to cynomolgus 4-1BB which impacts the ability of this study to predict 4-1BB-related
toxicity. PRS-343 was administered weekly as an intravenous infusion of 120 min duration at
doses of 10 and 120 mg/kg over 4 weeks, followed by a recovery phase in the control and high
dose group to evaluate any potential delayed onset or reversibility of toxicity.
Overall, PRS-343 was well tolerated at both doses tested, with no significant findings. No
unscheduled deaths occurred. There were no changes in standard parameters such as clinical
observations, body weight, ophthalmology, rectal temperatures, clinical chemistry, hematology,
coagulation tests, urinalysis parameters or serum cytokines as well as ECG. Furthermore, no
toxicologically relevant organ weight or organ weight ratio changes, and no macroscopic or
microscopic findings were observed, indicating that treatment with PRS-343 over four weeks did
not lead to any systemic toxicity. In addition, no evidence of delayed onset toxicity was noted at
the end of the four-week recovery phase. Toxicokinetic analysis demonstrated dose-proportional
systemic exposure at both dose levels upon first and last dose. Two animals out of sixteen
developed ADA’s that persisted throughout the study while three others showed a transient ADA
response. No gender-related toxicity differences were noted in the study.
Discussion
4-1BB (CD137) is a key costimulatory immunoreceptor and a member of the TNF-receptor
(TNFR) superfamily. The preclinical and clinical demonstration of the potential therapeutic
benefit of 4-1BB costimulation has spurred the development of therapeutic antibodies targeting
4-1BB, utomilumab (24, 25) and urelumab (23). Utomilumab is a fully humanized IgG2
monoclonal antibody that binds 4-1BB in a manner that blocks the binding of endogenous 4-
1BBL to 4-1BB and that appears well tolerated as a monotherapy (36) and in combination with
rituximab; however, modest activity has been observed (37). Urelumab is an IgG4 monoclonal
antibody that, in contrast to utomilumab, binds 4-1BB in a manner that does not interfere with
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the 4-1BB / 4-1BBL interaction. While an initial trial reported modest clinical activity (38), a
follow-up study was stopped due to hepatotoxicity (1). These data indicate that systemic
activation of 4-1BB with a potent agonist may lead to prohibitive toxicity, supporting the
rationale for tumor-localized targeting of the pathway.
While multiple lines of evidence suggest that 4-1BB is a highly promising therapeutic target in
cancer, current systemic antibody-based approaches are not designed to achieve a tumor-target
driven activation and are likely to display toxicity due to peripheral T cell and NK cell
activation. We hypothesized that biotherapeutics addressing this pathway should efficiently
activate the immune costimulatory target, but its activation should be restricted to the tumor
microenvironment (TME) to avoid systemic effects and unwanted toxicity.
PRS-343 is an Anticalin-antibody fusion protein with dual specificity for both 4-1BB and the
tumor antigen HER2. PRS-343 was designed to promote 4-1BB clustering by bridging 4-1BB-
positive T-cells with HER2-positive tumor cells, thereby providing a potent costimulatory signal
to tumor antigen-specific T-cells. Based on its differentiated mechanism of action PRS-343 has
the potential to expand therapeutic options to HER2-positive tumors including those who may
not be responsive to conventional antibody or small molecule based HER2 inhibitors.
Anticalin® proteins are 18 kDa protein therapeutics derived from human lipocalins. We
utilized phage display to generate an Anticalin protein binding to 4-1BB with high affinity and
specificity. PRS-343 was generated by genetic fusion of the 4-1BB-specific Anticalin protein to
a variant of the HER2-targeting monoclonal antibody trastuzumab with an engineered IgG4
backbone. We demonstrated the benefits of our bispecific platform’s flexible formatting,
allowing for functional testing of multiple bispecific geometries and the demonstration that the
4-1BB Anticalin protein fused to the heavy-chain C-terminus of the antibody was functionally
the most active in cell-based assays.
Binding studies using SPR, ELISA and FACS showed that PRS-343 displays similar
potency against each target compared to parental building blocks. Simultaneous binding of both
targets was also demonstrated. PRS-343 displays cross-reactivity to cynomolgus HER2 similar to
trastuzumab but with reduced cross-reactivity to cynomolgus 4-1BB. PRS-343 induces 4-1BB
clustering and downstream signaling in a Jurkat Nf-kB reporter cell line in the presence of
HER2-positive cells with a potency of approximately 50 pM (EC50) as well as IL-2 production
in a costimulatory T cell activation assay in the presence of HER2-positive NCI-N87 cells, with
a potency of 35pM. We observed a bell-shaped response both in the Jurkat Nf-kB reporter assay
and the primary T-cell activation assay which is in accord with expectations as a response
requires the formation of a ternary complex of the tumor cell target HER2, the drug PRS-343 and
the T-cell receptor 4-1BB, that can be disrupted when components are individually saturated
with drug. The effect can be rationalized by a mathematical model recently described for ternary
complex formation in biological systems and depends on the concentrations of all binding
partners and their affinities to each other (39),
The pharmacology of PRS-343 was investigated by further ex vivo T-cell costimulation
assays based on mixed culture of human T-cells and cell lines. The cell panel was selected to
cover a broad range of HER2 expressing cells derived from cancer tissue as well as from
different healthy tissue origins. Of note, three of the cell lines capable of costimulating T-cells in
a HER2-dependent manner have been described as being resistant [(40-42)] in preclinical models
to trastuzumab (SUM-225) or even both trastuzumab and lapatinib (JIMT-1, MDA-MB-453),
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13
demonstrating the potential of PRS-343 to provide a therapeutic option for patients with innate or
acquired resistance to HER2-targeted therapy. The risk of PRS-343-mediated systemic 4-1BB
activation and concomitant toxicity was investigated in a cytokine release assay, indicating that
4-1BB clustering leads to negligible cytokine release by T-cells in the absence of a primary T-
cell receptor stimulus. Together with the costimulation experiments, these results indicate that
PRS-343 is able to activate T cells only when these are engaging a target cell that expresses
HER2 at a level that is usually only encountered in malignant, tumorous tissue, and when the T-
cells are activated at the same time via the T cell receptor, e.g. by recognizing a tumor antigen.
In vivo proof of concept data utilizing a humanized SK-OV-3 mouse model support the desired
mode of action of PRS-343 in vivo. PRS-343 exhibits both direct cytotoxicity via monospecific
targeting of tumor expressed HER2 and activates 4-1BB via bispecific targeting of tumoral
HER2 and 4-1BB on human lymphocytes. These data also highlight the differentiating features
over a monospecific 4-1BB-targeting constitutive agonist. PRS-343 leads to an increase in the
frequency of human lymphocytes in the tumor, but does not affect the human lymphocyte
frequency in the peripheral blood, which correlates with an unchanged time course of GVHD-
induced morbidity and mortality compared to controls. In contrast, the monospecific 4-1BB-
targeting agonist antibody 20H4.9 leads to an expansion of human lymphocytes in the peripheral
blood and a concomitant acceleration of GVHD-induced morbidity and mortality, despite lacking
any activity in the TME, as evidenced by no significant increase in T cell infiltration in the TME
when compared to control. The data on PRS-343 reported here support its further evaluation
either as a single agent or combination therapy. Indeed, a preclinical rationale for combining 4-
1BB agonism with checkpoint blockade has been demonstrated (43, 44). PRS-343 is the first
bispecific 4-1BB agonist to enter the clinic, and a Phase 1 dose escalation study in patients with
HER2-positive advanced or metastatic solid tumors is ongoing (NCT03330561). A clinical trial
evaluating PRS-343 in combination with atezolizumab (anti-PD-L1) has also commenced
(NCT03650348).
Disclosure of Potential Conflicts of Interest
All authors except JS are or were employees of Pieris Pharmaceuticals, Inc. or its subsidiaries
and received compensation from and hold ownership interest in Pieris Pharmaceuticals, Inc. or
its subsidiaries. JS is an employee of Oncotest GmbH, a subsidiary of Charles River, Inc., which
received funding to perform the mouse studies described in this publication.
Authors' Contributions
Conception and design:
Development of methodology:
Acquisition of data (provided animals, acquired and managed patients,
provided facilities, etc.):
Analysis and interpretation of data (e.g., statistical analysis, biostatistics,
computational analysis):
Writing, review, and/or revision of the manuscript:
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14
Administrative, technical, or material support (i.e., reporting or organizing
data, constructing databases):
Study supervision:
Acknowledgments
We would like to thank the extended Pieris team for their support in generating and reviewing
data related to the PRS-343 project. We would like to specifically thank Tanya Aneichyk for her
assistance in data interpretation and presentation.
Grant Support
The research funding was provided by Pieris Pharmaceuticals. The costs of publication of this
article were defrayed in part by the payment of page charges. This article must therefore be
hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this
fact.
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Figure 1. PRS-343 Design and target and cell binding. PRS-343 structure (A) and design (B). HER2 binding
studies were performed by ELISA (C) or by FACS (D) using MCF-7 cells. Similarly, 4-1BB binding studies
were performed by ELISA (F) or by FACS (G) using 4-1BB overexpressing CHO cells. Representative
sensograms of SPR experiments are shown using immobilized hHER2 (E) or immobilized h4-1BB (H) and
allowed determination of PRS-343 on-, off-rate and KD (I). Dual binding: PRS-343 is capable of binding both
targets at the same time as determined in a simultaneous binding ELISA (J). Cross-reactivity: PRS-343
displays reduced cross-reactivity to cynomolgus monkey 4-1BB (K).
Figure 2: PRS-343 cell-based activity. Concentration dependence of PRS-343 mediated T-cell activation.
Dose-dependent T-cell costimulation by PRS-343 against NCI-N87 (HER2 high), MKN45 (HER2 low) and
HepG2 (HER2 null) cell lines, or without tumor cells, using a 4-1BB over-expressing Jurkat Nf-kB reporter
cell line (A) or purified human T-cells (B). IL2 secretion induced by human T-cells costimulated by PRS-343,
isotype control, trastuzumab, PRS-343 in excess of trastuzumab and anti-4-1bb antibody in the presence of
cells lines with expressing various HER2 levels (NCI-N87; SK-BR-3 and SUM-225-CW (HER2 high); JIMT-1
(HER2 medium) and MCF-7, MKN45 and MDA-MB-231 (HER2 low) (C). All data depicted here are
representative illustration of experiments carried out with minimum two different donors. Statistical
analysis: * p<0.05; ** p<0.01 and *** p<0.001, using 1way ANNOVA with Dunnet’s Multiple comparison
test.
Figure 3: Co-culture system data showing correlation between HER2 expression level and T-cell activation
(IL2 secretion) via PRS-343. (A) Collated data from co-culture experiments show that significant T-cell
activation (as measured by increased IL2) is observed in the presence of HER2 positive cancer cell lines and
not primary tissue cell lines. Effect size (x-axis) and significance (y-axis) of PRS-343 on IL2 levels as
compared to isotype control. Dotted line shows the significance cut-off corresponding to p-value = 0.05. (B)
Data shows that increasing levels of HER2 correlate with enhanced IL2 secretion (R2=0.46; p = 0.002).
Shaded area indicates confidence interval for a linear fit.
Figure 4: Pharmacokinetic profile of PRS-343 in murine (A) and primate (B) species following single dose i.v.
administration.
Figure 5: PRS-343 mediated antitumor activity in human PBMCs reconstituted xenograft SK-OV-3 tumor-
bearing mice. (A) Tumor volume was measured at indicated times through Day 20 and plotted as group mean
± SEM. (B) Lymphocyte phenotyping of the peripheral blood for human cell markers CD45 and CD8 was
performed by flow cytometry. All data were plotted as group mean ± SEM. (C) Tumor infiltrating
lymphocytes were assessed by immunohistochemistry staining of excised tumors using the human markers
(CD45, CD3, CD4 and CD8). (D) For each cell type, fold increase infiltration over isotype control were plotted
as column graph plot ± SEM. For each condition, between 3 and 5 tumors were analyzed. (E) Kaplan Meir
curves plotting the survival of human PBMC reconstituted non-tumor bearing mice following weekly dosing
of PRS-343 or controls.
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A B
Figure 4
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Iso
typ
e c
trl
PR
S-3
43 (
5 m
g/k
g)
Tra
s- I
gG
4 (
4 m
g/k
g)
an
t i-4
-1B
B (
5 m
g/k
g)
2 0
4 0
6 0
8 0
hC
D8
+ o
f C
D4
5+
PB
MC
at
stu
dy
en
d [
%]
A
0 4 7 1 1 1 4 1 8 2 0
5 0
1 0 0
1 5 0
2 0 0
2 5 0
3 0 0 P B M C o n ly
n o P B M C
Is o ty p e 5 m g /k g
P R S -3 4 3 1 0 m g /k g
P R S -3 4 3 5 m g /k g
P R S -3 4 3 1 m g /k g
P R S -3 4 3 0 .2 m g /k g
T ra s t-Ig G 4 4 m g /k g
a n ti-4 -1 B B 5 m g /k g
D a y s a fte r s ta r t o f tre a tm e n t
av
g t
um
or v
olu
me
[m
m3
]
C
Iso
typ
e c
trl
PR
S-3
43 (
5 m
g/k
g)
Tra
st-
IgG
4 (
4 m
g/k
g)
an
t i-4
-1B
B (
5 m
g/k
g)
2 0
4 0
6 0
8 0
hC
D4
5+
of
tota
l
PB
MC
at
stu
dy
en
d [
%]
% CD45+
% CD45+ and CD8+
B
E
Gr. 1 IgG4 isotype ctrl
Gr. 2 PRS-343
Gr. 3 anti-4-1BB-mAb
0 1 0 2 0 3 0 4 00
5 0
1 0 0
M P A L 3 9 5 1 0 0 µ g
M P A L 2 9 4 1 0 0 µ g
P L 2 5 5 9 1 0 0 µ g
T im e
Pe
rc
en
t s
urv
iv
al
T ra s t- Ig G 4 A n ti-4 -1 B B P R S -3 4 3
0
1
2
3
4
5
h C D 4 5
Fo
ld i
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re
as
e
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T ra s t- Ig G 4 A n ti-4 -1 B B P R S -3 4 3
0
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2
3
4
5
h C D 3
Fo
ld i
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re
as
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ty
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ntro
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T ra s t- Ig G 4 A n ti-4 -1 B B P R S -3 4 3
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3
4
5
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Fo
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T ra s t- Ig G 4 A n ti-4 -1 B B P R S -3 4 3
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3
4
5
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D
Figure 5
0 1 0 2 0 3 0 4 00
5 0
1 0 0
M P A L 3 9 5 1 0 0 µ g
M P A L 2 9 4 1 0 0 µ g
P L 2 5 5 9 1 0 0 µ g
T im e
Pe
rc
en
t s
urv
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Research. on May 18, 2021. © 2019 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on May 28, 2019; DOI: 10.1158/1078-0432.CCR-18-3654
Published OnlineFirst May 28, 2019.Clin Cancer Res Marlon J Hinner, Rachida Siham Bel Aiba, Thomas J Jaquin, et al. 4-1BB/HER2 bispecific antibody-Anticalin fusion PRS-343Tumor-localized costimulatory T cell engagement by the
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