tumor-localized costimulatory t cell engagement by the 4 ... · 5/25/2019 · engrafted with...

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 [email protected].

Marlon J. Hinner; email [email protected].

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

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

mailto:[email protected]


http://clincancerres.aacrjournals.org/

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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




11

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




12

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),




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:




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.




15

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.




16

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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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tumor-localized costimulatory t cell engagement by the 4 ... · 5/25/2019 · engrafted with...

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