CONCLUSION We generated and functionally characterized a novel humanized APRIL neutralizing antibody, designated BION-1301. The mechanism-of-action and anti-tumor activity described for the parental antibody hAPRIL.01A in vitro and in vivo strongly support the development of BION-1301 as a single agent or in combination with lenalidomide or bortezomib, and suggest a rationale for combination with checkpoint inhibitors. BION-1301 is expected to enter clinical development in 2017.

INTRODUCTION A PRoliferation Inducing Ligand (APRIL, TNFSF13), is a ligand for the receptors BCMA and TACI. APRIL serum levels are enhanced in patients diagnosed with Multiple Myeloma (MM), Chronic Lymphocytic Leukemia (CLL), and Colorectal Carcinoma correlated with poor prognosis. Our anti-APRIL hAPRIL.01A parental antibody blocked CLL survival and inhibited mouse B1 hyperplasia and IgA production in vivo (Guadagnoli et al., 2011). APRIL is produced by cells in the bone marrow niche, including myeloid-derived cells, osteoclasts and plasmacytoid dendritic cells. APRIL critically triggers BCMA in vitro and in vivo to drive proliferation and survival of human MM (Tai et al., 2016). Importantly, APRIL induces resistance to lenalidomide, bortezomib and other standard-of-care drugs. Furthermore, APRIL drives expression of PD-L1, IL-10, VEGF and TGFβ forcing an immunosuppressive phenotype on BCMA+ cells. As MM and B-cell survival, resistance to treatment and the immunosuppressive phenotype can be blocked by neutralizing APRIL (Tai et al., 2016), development of an antibody blocking APRIL provides a novel avenue for the treatment of MM and possibly CLL.

Fig. 4 Blocking of APRIL-induced B-cell proliferation and IgA production in vitro and in vivo by BION-1301. Fig. 1 Unique epitope mapped to receptor binding sites, no other fully anti-APRIL blocking antibodies known.

Fig.2 Binding BION-1301 to APRIL and blocking of APRIL to BCMA or TACI.

BCMA TACI Binding site BION-1301

A) B) C)

-●- hAPRIL.01A -●- BION-1301

Fig.3 Complete blockade of APRIL-induced cytotoxicity of BCMA-Fas and TACI-Fas Jurkat transfectants by BION-1301.

Fig. 5 hAPRIL.01A inhibits MM cell proliferation and survival induced by osteoclasts, macrophages, pDC.

Fig. 7 hAPRIL.01A blocks APRIL enhanced TGFß, IL-10 and PD-L1 expression.

Fig. 8 hAPRIL.01A inhibits tumor growth in a humanized MM model. Human bone chips with INA-6 MM cells were implanted into SCID mice. Mice were treated with hAPRIL.01A (mouse anti-human APRIL) or with isotype control at 20 mg/kg 5 days per week starting at day 2 (n=4 per group). Tumor growth was measured weekly by serum human sIL-6. P<0.001 at day 32 (Two way ANOVA). Tai et al., 2016.

Fig. 9. hAPRIL.01A blocks B1 B-cell expansion Upon aging the human APRIL transgenic mice developed a CLL-like phenotype characterized by expansion of peritoneal CD5+ B1 B-cells, infiltrating spleen, kidney and liver. Peritoneal exudates cells of mice were stained for IgD (FITC) and CD43 (PE) to discriminate B1 and B2 B cells. In contrast to mouse IgG1 treatment, weekly injection of 100 µg hAPRIL.01A for three months fully inhibited the B1 B-cell hyper proliferation, restoring the B1:B2 cell ratio in the peritoneal cavity to wild type levels. NS= non significant. *P=<0.05. **P=<0.01, 1-way ANOVA. Guadagnoli et al., 2011.

titer

A) B) C)

day

Anti-NP-IgM Anti-NP-IgG Anti-NP-IgA

B) Bortezomib

A)  Lenalidomide

A)

DevelopmentofafirstinclassAPRILFullyBlockingAn=bodyBION-1301fortheTreatmentofMul=pleMyeloma JohnDulos1,LilianDriessen1,MarcoGuadagnoli1,AstridBertens1,DavidLutjeHulsik1,Yu-TzuTai2,KennnethC.Anderson2,JPMedema3,HansvanEenennaam1andAndreavanElsas1

1.AduroBiotechEurope,Oss,TheNetherlands|2.Dana-FarberCancerInsPtute,HarvardMedicalSchool,Boston,Medicine,USA|3.UniversityofAmsterdam,Amsterdam,TheNetherlands.

Fig. 6 hAPRIL.01A enhances IMID and PI mediated MM cytotoxicity. MM cells were incubated with APRIL in the presence or absence of Lenalidomide or Bortezomib, with or without anti-APRIL hAPRIL.01A (10 µg/ml), and assayed by [3H]thymidine uptake at d3. hAPRIL.01A selectively blocks APRIL-induced protection in MM cells, alone and in cocultures with OCs, an effect which is further enhanced by lenalidomide (A) or bortezomib (B). Tai et al., 2016.

References: Guadagnoli M, Kimberley FC, Phan U, Cameron K, Vink PM, Rodermond H, Eldering E, Kater AP, van Eenennaam H, Medema JP. Development and characterization of APRIL antagonistic monoclonal antibodies for treatment of B-cell lymphomas. Blood. 2011 Jun 23;117(25):6856-65

Tai YT, Acharya C, An G, Moschetta M, Zhong MY, Feng X, Cea M, Cagnetta A, Wen K, van Eenennaam H, van Elsas A, Qiu L, Richardson P, Munshi N, Anderson KC. APRIL and BCMA promote human multiple myeloma growth and immunosuppression in the bone marrow microenvironment. Blood. 2016 Jun 23;127(25):3225-36

IC50 ~3.1 nM

A) B) IC50 ~0.9 nM

BION-1301 (blue) shares a binding site similar as TACI (purple) and BCMA (orange) to APRIL.

Mouse B-cells were stimulated with recombinant FLAG-APRIL, proliferation and IgA secretion was determined after 6 days of incubation. The mouse parental anti-APRIL antibody hAPRIL.01A inhibits APRIL-mediated B-cell proliferation (A) and IgA production (B). Human APRIL transgenic (Tg) or wild type (WT) mice were immunized with NP-Ficoll and humoral immune response was determined by detection of IgM, IgG and IgA antibody titers against NP-Ficoll antigen (C). Mice were treated with PBS, hAPRIL.01A (hA.01A), hAPRIL.03A (hA0.03A) or mouse IgG1 isotype-matched control. ***= p<0.001, **= p<0.01

H929 or MM1S MM cells were incubated overnight with or without APRIL in the presence or absence of hAPRIL.01A. Levels of downstream target genes were assayed using real-time qRT-PCR, normalized to 18S or GAPDH internal controls. Fold changes to control media are shown. Tai et al., 2016.

FLAG-APRIL was captured using anti-FLAG antibodies and binding of BION-1301 was detected. EC50 was calculated in which the average value of 3 independent experiments was 0.29 ± 0.05 (A). ELISA plates were coated with BCMA or TACI and BION-1301 was preincubated with recombinant FLAG-APRIL and added to the plates. Binding of FLAG-APRIL was detected using anti-FLAG antibodies. Blocking potency (IC50) was 1.61 ± 0.78 nM BCMA (B) and 1.29 ± 0.89 nM TACI (C) respectively.

MM cell lines (n=5) (A) and patient MM cells (n=3) (B) were cultured with osteoclasts (OC), with or without anti-APRIL, followed by assayed by [3H]thymidine uptake. MM cells were cultured with or without macrophages (Mφ), in the presence or absence of hAPRIL.01A (20 µg/ml) for 1d (C). In addition, MM cells were cultured with plasmacytoid dendritic cells (pDCs), in the presence of APRIL and anti-APRIL for 3d and viability was measured (D). **=P<0.01 (ANOVA). Tai et al., 2016.

A) B) D) C)

BCMA and TACI are fused to the intracellular domains of the FAS-receptor on Jurkat cells. Activation of BCMA (A) or TACI (B) was determined by detection of apoptosis induction. hAPRIL.01A dose-dependently reduced induction of BCMA-FAS (A) or TACI-FAS (B) apoptosis.