Preclinical toxicology and pharmacology for the 4-1BB/HER2 bispecific PRS-343: A first-in-class costimulatory T cell engager Marlon J. Hinner, Rachida-Siham Bel Aiba, Thomas Jaquin, Sven Berger, Manuela D rr, Corinna Sch

Preclinical toxicology and pharmacology for the 4-1BB/HER2 bispecific PRS-343:

A first-in-class costimulatory T cell engager

Marlon J. Hinner, Rachida-Siham Bel Aiba, Thomas

Jaquin, Sven Berger, Manuela D rr,

Corinna Schlosser, Andrea Allersdorfer, Christine Rothe, Louis A. Matis, Shane A. Olwill

Pieris Pharmaceuticals, Inc., 255 State Street, Boston, Massachusetts

AACR Annual Meeting 2017

Abstract 3673 pieris PHARMACEUTICALS Background 4-1BB (CD137) is a key costimulatory immunoreceptor and a highly promising therapeutic target in cancer. To overcome toxicity and efficacy limitations of current 4-1BB-targeting antibodies, we have

developed PRS-343, a 4-1BB/HER2 bispecific based on Anticalin technology. We have previously reported on the generation and characterization of PRS-343 with regard to preclinical proof-of-concept and basic drug-like properties (1). Here, we

describe the preclinical dataset supporting initiation of a first-in-patient trial. The pharmacology of PRS-343 is investigated by ex vivo assays based on mixed culture of human PBMC and tumor cell lines. The assays are used to determine the

cytokine profile of T cells costimulated by PRS-343-induced 4-1BB clustering. Using a set of immortal cancer cell lines and primary cells spanning a range of HER2 surface copy numbers, we identify the threshold required to elicit a costimulatory

response, and a lower threshold below which costimulation can be reliably excluded. The risk of PRS-343-mediated, systemic 4-1BB activation and concomitant toxicity is investigated in a cytokine release assay and in a mouse toxicology model of human

PBMC-induced xenograft-vs-host disease (xGvHD). HER2-mediated toxicity is studied in a GLP-compliant, repeat-dose toxicology study in cynomolgus monkeys.

combined dataset provides an overview on the pharmacology, mode of action and safety profile of PRS-343.

Concept: tumor-specific and tumor-localized costimulatory

activation of T cells

Figure 1. Concept of costimulatory T cell engagement by PRS-343: Within a patient s tumor, tumor-specific T cells are bridged with

tumor cells by the costimulatory bispecific PRS-343 which simultaneously binds the tumor target HER2 and the immune receptor 4-1BB. The resulting clustering of 4-1BB provides a local co-activatory signal to the T cell, further enhancing its T cell

receptor (TCR)-mediated activity and leading to tumor destruction. Toxic side effects are expected to be manageable, as PRS-343 does not induce clustering and activation of 4-1BB in the absence of target-positive cells, and healthy tissue is spared

by tumor-costimulated T cells due to the absence of a primary, TCR-mediated signal.

Tumor cell T cell costimulation in tumor

Costimulatory bispecific Tumor target HER2 MHC- peptide T cell receptor Clustering Costimulatory receptor 4-1BB Tumor-specific T Cell Signal 2 Signal 1 Activation PRS-343 design,

target binding and activity in reporter and T cell costimulation assay A Anti-4-1bb Anticalin TM=74C (DSC) KD= 2.3nM (SPR) EC50 (FACS) = 5.9nM B PRS-343 Design a-HER2 a-4-1BB Ac PRS-343 C PRS-343 Design (DNA) Heavy rain VH anti-HER2 (transtuzumab)

CH1 IgG4 S228P FALA CH2 CH3 linker Anticalin anti-4-1BB Light chain VL anti-HER2 (trastuzumab) CL kappa D HER2 ELISA RFU trastuzumab PRS-343 50000 40000 30000 20000 10000 0 0.001 0.01 0.1 1 10 100 1000 conc [nM] E 4-1BB ELISA CD137-specific

Anticalin PRS-343 25000 20000 15000 10000 5000 0 0.001 0.01 0.1 1 10 100 1000 conc [nM] F Dual binding ELISA 30000 20000 10000 0 0.001 0.01 0.1 1 10 100 1000 conc [nM] G Cross-reactivity ELISA PRS-343 / human 4-1BB PRS-343 / cyno 4-1BB RFU

(normalized) 1.0 0.5 0.0 0.01 0.1 1 10 100 1000 conc [nM] H SPR Affinity target sample kon [M-1s-1] koff [s-1] KD [nM] HER2 HER2 4-1BB 4-1BB (high conc) 4-1BB PRS-343 trastuzumab PRS-343 PRS-343 4-1BB specific anticalin 3.1E+05 4.6E+05 1.9E+04

2.2E+04 (avidity) 3.9E+04 9.3E-05 9.4E-05 9.6E-05 3.3E-05 (avidity) 7.3E-05 0.30 0.20 5.03 1.51 (avidity) 1.87 I Reporte Assay 5000 4000 3000 2000 1000 0 10-6 10-4 0.01 0.1 1 10 100 1000 conc [nM] J T cell costimulation IL-2[pg/mL] 2000 1500 1000

500 0 0.001 0.01 0.1 1 10 conc [nM] Figure 2. PRS-343 Design, target binding and cell-based activity. (A) 4-1BB binding Anticalin. (B,C) Design. (D) HER2 ELISA shows similar potency of PRS-343 compared to trastuzumab. (E) 4-1BB ELISA shows similar

potency of PRS-343 compared to 4-1BB-specific Anticalin. (F) Dual binding: 4-1BB/HER2 bispecifics are capable of binding both targets at the same time according to Sandwich ELISA. (G) Cross-reactivity: PRS-343 displays reduced cross-reactivity to

4-1BB from cynomolgus monkey. (H) On-rate, off-rate and KD of binding to targets HER2 and 4-1BB for PRS-343 and reference molecules trastuzumab and 4-1BB specific Anticalin. 4-1BB binding affinity was determined using different target coating

concentrations minimizing or favoring avidity effects, respectively. (I) PRS-343 induces 4-1BB clustering and downstream signaling in a Jurkat Nf-kB reporter cell line in the presence of HER2-positive NCI-N87 cells with a potency of 50pM. (J)

PRS-343 induces IL-2 production in a costimulatory T cell activation assay in the presence of HER2-positive SKBR-3 cells, with a potency of 35pM. In both types of cell-based assays, the response is bell-shaped as expected for ternary complex

formation between PRS-343 and target cells (2). PRS-343-costimulated T cells express IL-2, GM-CSF, IFN and TNF T cells were co-incubated with HER2high

NCI-N87 cells and PRS-343. Supernatant concentrations were determined for a panel of cytokines Cytokines prominently induced by PRS-343-mediated costimulation were GM-CSF, IL-2, IFN and TNF These cytokines may serve as pharmacodynamic biomarkers in clinical studies GM-CSF IL-2 IFN- GM-CSF [pg/mL] 6000 4000 2000 0 0.01 0.1 1 10 100 1000 PRS-343 [nM]

IL-2 [pg/ml] 6000 4000 2000 0 0.01 0.1 1 10 100 1000 PRS-343 [nM] IFN- 150000 100000 50000 0 0.01 0.1 1 10 100 1000 PRS-343 [nM] TNF- TNF-alpha [pg/mL] 2500

2000 1500 1000 500 0 0.01 0.1 1 10 100 1000 PRS - 343[nM] IL-4 IL-4 [pg/mL] 80 60 40 20 0 0.01 0.1 1 10 100 1000 PRS-343 [nM] IL-6 IL-6 [pg/mL] 300 200 100 0 0.01 0.1 1 10 100 1000 PRS-343 [nM] IL-8 IL-8 [pg/mL] 6000 4000 2000 0 0.01 0.1 1 10 100

1000 PRS - 343[nM] Il-10 Il-10 [pg/mL] 600 400 200 0 0.01 0.1 1 10 100 1000 PRS-343 [nM] IL-1 IL-1 [pg/mL] 300 200 100 0 0.01 0.1 1 10 100 1000 PRS-343 [nM]

Figure 3. Cytokines induced by human T cells co-stimulated by PRS-343 in the presence of HER2-positive NCI-N87 cells in a T cell co-stimulation assay. Cytokine levels in the culture supernatants were measured by an electrochemiluminescence (ECL)

immunoassay. PRS-343-induced cytokine release in the absence of T cell receptor stimulation is negligible A cytokine release assay (3) was performed in the absence of T cell receptor (TCR) stimulation and presenting PRS-343 to PBMC in

solution, wet-coated and air-dried PRS-343 shows negligible cytokine induction activity compared to the positive anti-CD3 control OKT3 independent of presentation strategy The data confirms that 4-1BB is a costimulatory receptor that requires a

primary TCR signal; the risk of systemic cytokine release syndrome in clinical studies appears low GM-CSF GM-CSF [pg/mL] 100000 10000 1000 100 10 1 0.1 0.01 Air-dried Coating Solution Wet Coating IFN-

IFN- [pg/ml] 100000 10000 1000 100 10 1 0.1 0.01 Air-dried Coating Solution Wet Coating IL-4 IL-4 [pg-mL] 100000 10000 1000 100 10 1 0.1 0.01 Air-dried Coating Solution Wet Coating IL-8 IL-8 [pg/mL]

100000 10000 1000 100 10 1 0.1 0.01 Air-dried Coating Solution Wet Coating IL-2 IL-2 [pg/mL] 100000 10000 1000 100 10 1 0.1 0.01 Air-dried Coating Solution Wet Coating TNF- TNF- [pg/mL] 100000 10000 1000 100 10 1 0.1 0.01 Air-dried Coating Solution Wet Coating IL-6 IL-6 [pg/mL] 100000 10000 1000 100 10 1 0.1 0.01

Air-dried Coating Solution Wet Coating

IL-10 IL-10 [pg/mL] 100000 10000 1000 100 10 1 0.1 0.01

Air-dried Coating Solution Wet Coating hlgG4 1 g/mL OKT 3 0.1 g/mL OKT3 1 g/mL OKT3 10 g/mL PRS-343 0.1 g/mL PRS-343 1 g/mL PRS-343 10 g/mL PRS-343 100 g/mL Figure 4. Cytokine release assay with

PRS-343. PBMC were isolated from the blood of twelve healthy donors and incubated for 72 hours with PRS-343 either air dried, in soluble form, or wet coated. Four concentrations of PRS-343 in a volume of 50 l were tested in each setting as

indicated in the figure. The anti-CD3 monoclonal antibody OKT3 at three different concentrations served as the positive control, and an IgG4 isotype antibody was the negative control. Supernatant levels of ten cytokines (IL-1 , IL-2, IL-4,

IL-6, IL-8, IL-10, IL-12p70, GM-CSF, IFN- and TNF- ) were analyzed. The figure shows the average response for the ten donors that displayed a significant response to OKT3, and for a selection of the most relevant cytokines.

PRS-343-mediated T cell costimulation requires supraphysiological HER2 levels The costimulatory T cell activation assay was performed for a series of tumor cell lines and primary cells covering a wide range of HER2 positivity An anti-4-1BB benchmark

mAb was used as a positive control Response specificity was controlled by competition with an excess of trastuzumab The series of experiments shows

costimulation above HER2 levels corresponding to 14% of SKBR-3 (HER2 2+)

(ii) no costimulation in the physiological HER2 expression range (<2% of SKBR-3)

(iii) variable donor-dependent results in the intermediate range (2%-11%)

Costimulatory activity was observed in SUM225 and JIMT-1 cell lines described as resistant to conventional HER2-targeted therapy (4-6) A Co-stimulatory T cell

activation IL-2 [pg/mL] SKBR-3 (HER2high) 10000 8000 6000 4000 2000 0 0.001 0.01 0.1 1 10 100 PRS-343 [nM] +anti- HER2 PRS-343 25nM PRS-343 10nM isotype 25nM trastuzumab 25nM vehicle PRS-343 + tras. anti-4-1BB mAb 25 nM IL-2 [pg/mL] MDA-MB-231

(HER2low) 10000 8000 6000 4000 2000 0.001 0.01 0.1 1 10 100 PRS-343 [nM] PRS-343 25nM PRS-343 10nM isotype 25nM trastuzumab 25nM vehicle PRS-343 + tras. anti-4-1BB mAb 25 nM ns ns B HER2 surface expression dependency Tumor cell lines Primary cells

Best response (statistical significance) *** ** * ns NCI-N87 SKBR-3 SUM225 SK-OV-3 JIMT-1 ZR-75-1 MKN-7 LoVo MCF-7 MKN-45 HAT-29 A549 BxPC-3 A431 MDA-MB-231 *** ** * ns HUVEC HBSMC HAoSMC HPF HCF NHEK HCM HDMEC

Normalized HER2 surface expression level (log scale)

NCI-N87 SKBR-3 SUM225 SK-OV-3 JIMT-1 ZR-75-7 MKN-7 LoVo MCF-7 MKN-45 HAT-29 A549 BxPC-3 A431 MDA-MB-231

HUVEC HBSMC HAoSMC HPF HCF NHEK HCM HDMEC

Figure 5. PRS-343 costimulation dependence on target cell HER2 level. Tumor or primary cells of different HER2 positivity were subjected to a T cell costimulatory activation assay

using IL-2 supernatant levels as the readout. Experiments for each cell line were performed with at least two different donors. (A) Exemplar results. PRS-343 at various concentrations, target cells and healthy donor T cells were co-incubated in the

presence of coated anti-CD3 antibody. Negative controls used were IgG4 isotype, trastuzumab or vehicle. Anti-4-1BB benchmark mAb was the positive control. The experiment was performed also in the presence of an excess of trastuzumab to inhibit the

binding of PRS-343 to the SKBR3 cells. (B) Top: The best statistical significance obtained for any donor vs. control levels is reported for tumor cell lines and primary cells (p<0.001 (***), p<0.01 (**) or p<0.05 (*). Values of p>0.05

were considered not statistically significant (ns)). Bottom: relative cell surface HER2 levels (normalized against SKBR3 expression levels) are plotted for each tumor cell line and primary cell type on a logarithmic scale.

PRS-343 leads to TGI and tumor-localized increase in hCD45(+) cells in tumor in humanized mice

Immuno-compromised mice engrafted with HER2-positive tumor cells (SK-OV-3) were injected with human PBMC and treated over 3 weeks with PRS-343 at four dose levels

Control molecules were IgG4 isotype, an anti-4-1BB benchmark antibody and trastuzumab with an IgG4 backbone (Tras-IgG4)

Tumor IHC staining for human CD45 shows a dose-dependent increase in the frequency of human TIL for PRS-343 vs controls, suggesting tumor-localized T cell activation

PRS-343 showed dose-dependent tumor growth inhibition (TGI) comparable to Tras-IgG4, indicating that TGI is dominated by HER2 antagonism in this model

A Tumor growth (Median)

Avg tumor volume [mm3]

300 250 200 150 100 50 0 4 7 11 14 18 20

Days after start of treatment no PBMC PBMC only

Anti-CD137 100 g Isotype ctrl 100 g PRS-343 4ug PRS-343 20 g Tras-IgG4 80 g PRS-343 100 g RPS-343 100 g

Incomplete group due to

B TIL frequency (hCD45) by IHC

DAB-positive area of anti-hcD45 [%]

40 35 30 25 20 15 10 5 0

PBMC only no PBMC Isotype ctrl 200 g PRS-343

100 g PRS-343 20 g PRS-343 4 g PRS-343 80 g Tras-IgG4 100 g anti-CD137

Tras-IgG4 80 g IHC PRS-343 100 g IHC

Figure 6. PRS-343 activity in NOG mice engrafted with HER2-positive SK-OV-3 cell line and human PBMC. (A) Median of tumor growth. (B) Frequency of CD45+ cells determined by

immunohistochemistry of tumors after study end. Examples for sections of formalin-fixed and paraffin-embedded tumors stained for human CD45 are provided on the right. See reference (1) for further experimental details.

Humanized Mouse Toxicology: PRS-343 avoids systemic 4-1BB activation in contrast to benchmark

Immuno-compromised, tumor-free mice were injected with human PBMC and treated over 3 weeks with PRS-343 or controls (IgG4 isotype or anti-4-1BB benchmark mAb)

PRS-343 showed unchanged dynamics of xenograft-versus-host disease compared to isotype control, while anti-4-1BB benchmark significantly accelerated mortality

The results support a potentially improved safety profile of PRS-343 over benchmark by lack of systemic activation and concomitant toxicity A Survival Percent

survival 100 50 0 100 g PRS-343 100 g anti-4-1BB mAb 100 lgG4 isotype ctrl 0 10 20 30 40 treatment Days after start of treatment B Body weight changes Relative Body Weight [%] 115 110 105 100 95 90 85 80 100 g PRS-343

100 g anti-4-1BB mAb 100 lgG4 isotype ctrl 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 treatment Days after start of treatment Figure 7. Immuno-compromised female NOG mice were engrafted with 7 106 fresh human PBMC,

followed by weekly i.p. treatment with PRS-343, a 4-1BB benchmark agonist or isotype control at 100 g/dose (i.p.) for 3 weeks. Mice (n=15 per group) remained on the study until spontaneous death or if ethical sacrifice was required. (A)

Survival plot. (B) Relative median body weight of surviving animals. PRS-343 is Well Tolerated in Repeat-Dose Cynomolgus Monkey Toxicology Study The safety of PRS-343 was investigated in a GLP-compliant cynomolgus monkey study PRS-343 was given in

weekly doses of 0, 10 and 120mg/kg over 4 weeks as an intravenous infusion of 120 min duration (see Table 1 for study design) Delayed onset or reversibility of toxicity was studied in recovery groups (0 and 120 mg/kg) PRS-343 was well tolerated at

both doses tested, with no significant findings TK analysis demonstrated full, dose-proportional exposure at both dose levels, with a terminal half-life of 5-6 days Table 1. Study Design. Number of Animals Group Dose Level Toxicity Recovery Group

Description (mg/kg/week) Male Female Male Female 1 Control 0 3 3 2 2 2 Low 10 3 3 - - 3 High 120 3 3 2 2 Conclusion PRS-343 is a 4-1BB/HER2 bispecific based on the genetic fusion of a high- affinity 4-1BB-binding Anticalin and modified trastuzumab

The presented preclinical pharmacology and toxicology studies confirm previous results (1) and support that PRS-343 elicits its costimulatory effects strictly on T cells also receiving a primary TCR signal and strictly localized to HER2-positive

tumors: PRS-343-mediated 4-1BB activation requires supraphysiological HER2 levels PRS-343 costimulation leads to increased production of multiple pro- inflammatory cytokines associated with anti-tumor immune response The risk of systemic 4-1BB

activation is low based on negligible cytokine release in the absence of primary T cell receptor stimulation This is supported by a humanized mouse toxicology study, where PRS- 343 avoids the systemic peripheral activation of CD8+ T cells observed

with a benchmark 4-1BB antibody A GLP-compliant cynomolgus monkey toxicology study demonstrates that the benign toxicity profile of trastuzumab is retained in PRS-343 with regard to HER2 targeting The reported data support evaluation of PRS-343 in a