Fibroblast activation (FAP)-selective delivery of CD40 agonistic DARPin® molecule for tumor-localized immune activation Poster 637 Nicolo’ Rigamonti1*, Anja Schlegel1, Sophie Barsin1, Jonas Schwestermann1, Susanne Mangold1, Yvonne Kaufmann1, Christof Zitt1, Niina Veitonmäki1, Victor Levitsky1, Simon Plyte1, and Clara Metz1

1Molecular Partners AG, Schlieren-Zurich, Switzerland. *Corresponding Author: [email protected]

Summary Human DARPin®: Dendritic cell assay Mouse DARPin®: B cell assay

CD40 is a co-stimulatory molecule belonging to the tumor necrosis factor receptor superfamily which can ® ® activate both innate and adaptive immune system, making it an interesting target for tumor immunotherapy. DARPin molecule activates dendritic cells (DC) in a FAP-specific Mouse DARPin molecule activates B cells in a FAP-specific manner Systemic activation of CD40 receptor, induced by administration of agonistic CD40 antibodies, has shown signs manner MARK MFI % of activity in cancer patients, but dose-limiting toxicities have impaired the efficacy. New therapeutic approaches ER CD69 + + are therefore needed to increase the therapeutic index of CD40-targeting molecules and achieve better clinical hCD40/FAP DARPin® with +FAP outcomes. Here, we report a novel approach based on systemic administration of a tumor-localized CD40 CD80 +++ +++ 4000 hCD40/FAP DARPin® with -FAP 4000 MARKER MFI % +FAP +FAP -FAP CD86 +++ ++ agonistic DARPin® molecule targeting human CD40 and fibroblast activation protein (FAP) alpha, a tumor- anti-human CD40 mAb -FAP MHCII + + associated antigen (TAA) abundantly expressed in many solid tumors, enabling CD40 pathway activation 1500 1500 +FAP 3000 LPS 3000 1500 mCD40/FAP DARPin® mCD40/FAP DARPin® exclusively in the presence of TAA-expressing cells. CD86 +++ + -FAP anti-mouse CD40 mAb anti-mouse CD40 mAb

MFI CD86 MFI 2000 2000 MFI CD86 MFI CD83 + ++ 1000 negative 1000 negative control DARPin® 1000 Using ribosome display technology, we generated a panel of bispecific CD40/FAP DARPin® molecules able to control

trigger specifically CD40 receptor and activate the NF-κB pathway in a reporter cell assay, in the presence of 1000 DARPin® MFI CD69 MFI

1000 CD69 MFI FAP-transfected Chinese hamster ovary (CHO) cells, but not in the presence of parental CHO cells. Selected CD80 +++ +++ CD69 MFI 500 500 500 bispecific DARPin® clones were then tested in more meaningful cell assays using human 1) primary B cells, 2) 10-3 10-2 10-1 100 101 102 monocyte-derived dendritic cells and 3) monocyte-derived macrophages from whole blood, in the presence of LPS anti-human hCD40/FAP FAP-positive or FAP-negative cells. In these immune cell populations, bispecific CD40/FAP DARPin® molecules concentration (nM) CD40 mAb DARPin® neg. control anti-mouse mCD40/FAP 10-3 10-2 10-1 100 101 102 103 10-3 10-2 10-1 100 101 102 103 confirmed a FAP-dependent activation of CD40 pathway inducing an upregulation of costimulatory molecules DARPin® CD40 mAb DARPin® and proinflammatory , such as CD86 and interleukin (IL)-12, only in the presence of FAP-expressing concentration (nM) concentration (nM) hCD40/FAP DARPin® with +FAP cells. In order to properly address the in vivo activity, a surrogate mouse-specific CD40/FAP DARPin® molecule hCD40/FAP DARPin® with -FAP ® was also generated and tested in different in vitro assays showing a FAP-dependent activation and similar Figure 7. Functional characterization of CD40/FAP bispecific DARPin molecules in a B cell activation assay. Purified 10000 anti-human CD40 mAb 10000 results as the human counterpart. In vivo pharmacokinetic and pharmacodynamic experiments, performed in +FAP splenic CD20+ B cells were cocultured with FAP-positive (+FAP) or FAP-negative (-FAP) CHO cells, as described in Fig.3 and LPS -FAP CD69, CD80, CD86 and MHC-II expression on CD20+ cells was analyzed by FACS as MFI and % (shown only in table). tumor-free mice, showed a comparable half-life between mouse-specific CD40/FAP DARPin® molecule and the Column graph highlights the values obtained for the highest dose of the titration graphs (red square). Crosslinked antibody, benchmark anti-mouse CD40 antibody (clone FGK45). However, DARPin® molecule, differently from the 5000 clone FGK45, (anti-mouse CD40 Ab) was used as +CTRL. Negative control DARPin® is the human-specific CD40/FAP

benchmark antibody FGK45, did not increase the serum level of IL-6, supporting a mode of action that is 5000 DARPin® molecule. Table summarizes the expression of the other markers: + = 10  x < 20, ++ = 20  x  30 and +++ = x >

12 (pg/ml) 12

12 (pg/ml) 12

- -

dependent on FAP-mediated crosslinking of CD40 receptor. IL 30. x = fold change. IL 0 ND In conclusion, we have generated bispecific agonist CD40/FAP DARPin® molecules able to activate the CD40 Mouse DARPin® molecule activates B cells in presence of pathway in cellular assays in a targeting-dependent manner, supporting the hypothesis that these DARPin® ND 0 +FAP MC-38 tumor cells molecules could lead to a tumor-localized immune activation in vivo. In vivo experiments in mouse tumor models 10-3 10-2 10-1 100 101 102 LPS anti-human hCD40/FAP CD40 mAb DARPin® to test this hypothesis are ongoing. In vivo studies were approved by Veterinary Authorities of the Canton Zurich, concentration (nM) 800 -FAP/MC-38 approval number ZH102/16 600

Figure 4. Functional characterization of human CD40/FAP bispecific DARPin® molecule in a DC activation assay. +FAP/MC-38 -FAP/MC-38 events 400

° +FAP/MC-38 N Human monocyte-derived DC were cocultured with irradiated FAP-positive (+FAP) or FAP-negative (-FAP) CHO cells as 200 described in Fig.3, and CD86, CD80 and CD83 expression on CD14- cells was analyzed by FACS as MFI and % (shown only 4000 mCD40/FAP DARPin® 4000 mCD40/FAP DARPin® in table). IL-12 production was measured by ELISA in the supernatant. Column graphs highlight the values obtained for the 102 103 104 105 106 highest dose of the titration graphs (red square). Antibody (anti-human CD40 Ab) was used as +CTRL and clinical comparator. anti-mouse CD40 mAb anti-mouse CD40 mAb FAP 3000 Mode of Action Lipopolysaccharide (LPS) was used as further +CTRL. Table summarizes the expression of the other markers: += 2

2000 2000 CD69 +++ ++

MFI CD69 MFI MFI CD69 MFI CD86 +++ ++ • DARPin® molecules are small engineered , derived from natural 1000 1000 CD80 +++ + ankyrin repeat proteins, able to bind to specific targets with high affinity MHCII + +

® -3 -2 -1 0 1 2 3 -3 -2 -1 0 1 2 3 ® 10 10 10 10 10 10 10 10 10 10 10 10 10 10 • Individual DARPin molecules can be linked together genetically in order to Human DARPin : Macrophage assay concentration (nM) concentration (nM) Fibroblast create multi-specific drug molecules FAP Figure 8. Bispecific CD40/FAP DARPin® molecule activates B cells when FAP is expressed by tumor cells. Purified FAP-CD40 • CD40 receptor-mediated signaling requires a high level of oligomerization of DARPin® splenic CD20+ B cells were cocultured either with irradiated FAP-transfected MC38 (+FAP/MC38)- or wild type MC38 (- CD40 the CD40 receptor ® FAP/MC38)- tumor cells. After 48h incubation, CD20+ B cells were analyzed by FACS for the upregulation, in terms of MFI DARPin molecule activates macrophages in a FAP-specific manner and % (shown only in the table), of CD69, CD80, CD86 and MHCII. Crosslinked antibody, clone FGK45, (anti-mouse CD40 NFkB • FAP is abundantly expressed in the stroma of many solid tumors, in contrast Ab) was used as +CTRL. Negative control DARPin® is the human-specific CD40/FAP DARPin® molecule. Table summarizes to healthy human tissues APC CCL17 the expression of the other markers: + = 10  x < 20, ++ = 20  x  30 and +++ = x > 30. x = fold change. FACS dot plot shows FAP expression on MC38 tumor cells. • Targeting-dependent mechanism of action: tumor-confined antigen presenting cell (APC) activation by binding to FAP-expressing cells Figure 1. Mechanism of action. 12000 +FAP 10000 -FAP 8000 In vivo 6000 4000 Assay description Pg/ml 2000 LPS DARPin® molecule shows an half-life comparable to the benchmark 500 PBS CD40 Ab, FGK45 Buffy coat from healthy blood donor Isolating/ differentiating Figure 2. Illustration depicting the in vitro cellular PBS anti-human hCD40/FAP anti-human hCD40/FAP effector cells LPS ® assay used to characterize the activity of human CD40 mAb DARPin® CD40 mAb DARPin® Differently from the FGK45, DARPin molecule does not induce a Ficoll CD40/FAP bispecific DARPin® molecule. CD40 All graphs of this poster show representative data of at systemic increase of IL-6 +LPS PBMCs Effector cells least two independent experiments. All graphs present (B-cells, DC or macrophages) means, and error bars represent standard deviations 1) PK 2) IL-6 ® in presence of FAP in absence of FAP (SD). Figure 5. Functional characterization of human CD40/FAP bispecific DARPin molecule in a macrophage activation assay. Human monocyte-derived CD68+ macrophages were cocultured with irradiated FAP-positive (+FAP) or FAP-negative 1 0 0 1000 activation marker (-FAP) CHO cells, as described in Fig.3 and CCL17 was measured in the supernatant by ELISA and array. Antibody (e.g CD86) Effector cells (anti-human CD40 Ab) was used as +CTRL and clinical comparator. LPS was used as further +CTRL.

Cytokine /ml) CD40 1 0 Effector cells FAP-CD40 DARPin pg release CD40 100 ( (e.g IL-12) mCD40/FAP DARPin® FAP-CD40 DARPin LLOQ FAP CHO or tumor HL= 18 h 1 CHO or tumor cells 10 anti-mouse FAP- cells CD40 mAb

+++ Concentration FAP Mouse DARPin®: Dendritic cell assay (nmol/l) Concentration HL= 15 h ND 1 0 . 1 0 50 100 Time (h)

® ® Human DARPin : B cell assay Mouse DARPin molecule activates DC in a FAP-specific manner Figure 9. Pharmacokinetic and pharmacodynamic characterization of CD40/FAP bispecific DARPin® molecules. Tumor free mice were injected with 1 mg/kg of the indicated drugs and the sera concentrations of 1) the drug (left graph) at different time points and 2) IL-6 after 22 h (right graph) were measured by ELISA. Three mice per group were used. ND= Non- MARK MFI % Detectable. LLOQ= Lower Limit of Quantification. HL= half life. ® ER DARPin molecule activates B cells in a FAP-specific manner CD69 + ++ CD80 + + +FAP -FAP CD86 +++ ++ +FAP -FAP MHCII ++ + 6000 MFI +++ +FAP 18000 mCD40/FAP DARPin® 18000 mCD40/FAP DARPin® 18000 +FAP 6000 hCD40/FAP DARPin® 6000 hCD40/FAP DARPin® -FAP % + Conclusions anti-mouse CD40 mAb anti-mouse CD40 mAb -FAP anti-human CD40 mAb anti-human CD40 mAb negative control DARPin® IgG2 ctrl 4000 12000 12000 negative control DARPin® 12000 4000 IgG2 ctrl 4000

• Active and potent human-specific and mouse-specific CD40/FAP DARPin® molecules were generated and

MFI CD86 MFI

MFI CD86 MFI

MFI CD86 MFI

MFI CD86 MFI MFI CD86 MFI 2000 CD86 MFI 6000 6000 6000 were able to activate different immune cell populations with a targeting (FAP)-dependent mechanism of 2000 2000 action in in vitro cellular assays.

® 10-2 10-1 100 101 102 103 10-2 10-1 100 101 102 103 neg. control anti-mouse mCD40/FAP • CD40/FAP DARPin molecule shows CD40-agonist activity also in presence of FAP-expressing MC38 tumor -3 -2 -1 0 1 2 -3 -2 -1 0 1 2 anti-human 10 10 10 10 10 10 10 10 10 10 10 10 IgG2 ctrl hCD40/FAP DARPin® CD40 mAb DARPin® CD40 mAb concentration (nM) concentration (nM) cells. concentration (nM) concentration (nM) DARPin®

® ® • Mouse-specific CD40/FAP DARPin molecules show a good PK profile in terms of half-life. Figure 3. Functional characterization of human CD40/FAP bispecific DARPin molecule in a B cell activation assay. Figure 6. Functional characterization of mouse CD40/FAP bispecific DARPin® molecule in a DC activation assay. Purified human CD19+ B cells were cocultured either with FAP-positive (+FAP) or FAP-negative (-FAP) CHO cells and Mouse bone marrow-derived DC were cocultured with irradiated FAP-positive (+FAP) or FAP-negative (-FAP) CHO cells, as ® treated with different concentration of reagents, as indicated in the titration graphs. After 24 h incubation, CD20+ B cells were described in Fig.3 and CD69, CD80, CD86 and MHC-II expression on CD45+CD11c+ double positive cells was analyzed by • As expected, injection in tumor-free mice of mouse-specific CD40/FAP DARPin molecule does not lead to analyzed by FACS for the upregulation of CD86. Mean fluorescence intensity (MFI) and percentage (%, shown only in the FACS as MFI and % (shown only in table). Column graph highlights the values obtained for the highest dose of the titration immune systemic activation measured by IL-6 production in the sera. table) were measured. Antibody (anti-human CD40 Ab) was used as positive control (+CTRL) and clinical comparator. Column graphs (red square). Crosslinked antibody, clone FGK45, (anti-mouse CD40 Ab) was used as +CTRL. Negative control graph highlights the values obtained for the highest dose of the titration graphs (red square). += 2 3. x = fold change.

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