Objective Dysregulated APRIL/BAFF signaling is implicated in the pathogenesis of multiple autoimmune diseases, including systemic lupus erythematosus and lupus nephritis. We undertook this study to develop and evaluate a high‐affinity APRIL/BAFF antagonist to overcome the clinical limitations of existing B cell inhibitors. Methods A variant of TACI‐Fc generated by directed evolution showed enhanced binding for both APRIL and BAFF and was designated povetacicept (ALPN‐303). Povetacicept was compared to wild‐type (WT) TACI‐Fc and related molecules in vitro and in vivo. Results Povetacicept inhibited APRIL and BAFF more effectively than all evaluated forms of WT TACI‐Fc and selective APRIL and BAFF inhibitors in cell‐based reporter assays and primary human B cell assays, mediating potent suppression of B cell proliferation, differentiation, and immunoglobulin (Ig) secretion. In mouse immunization models, povetacicept significantly reduced serum immunoglobulin titers and antibody‐secreting cells more effectively than anti‐CD20 monoclonal antibodies, WT TACI‐Fc, or APRIL and BAFF inhibitors. In the NZB × NZW mouse lupus nephritis model, povetacicept significantly enhanced survival and suppressed proteinuria, anti–double‐stranded DNA antibody titers, blood urea nitrogen, glomerulonephritis, and renal immunoglobulin deposition. In the bm12 mouse lupus model, povetacicept significantly reduced splenic plasmablasts, follicular helper T cells, and germinal center B cells. In non‐human primates, povetacicept was well tolerated, exhibited high serum exposure, and significantly decreased serum IgM, IgA, and IgG levels after a single dose. Conclusion Enhanced APRIL and BAFF inhibition by povetacicept led to greater inhibition of B cell populations critical for autoantibody production compared to WT TACI‐Fc and CD20‐, APRIL‐, or BAFF‐selective inhibitors. Potent, dual inhibition by povetacicept has the potential to significantly improve clinical outcomes in autoantibody‐related autoimmune diseases. image

Background:BAFF and APRIL are TNF superfamily members that form homo- and heteromultimers that bind TACI and BCMA on B cells; BAFF also binds BAFF-R. BAFF and APRIL support B cell development, differentiation, and survival, particularly for plasmablasts and plasma cells, and play critical roles in the pathogenesis of B cell-related autoimmune diseases. In nonclinical models, inhibition of either BAFF or APRIL alone mediates relatively modest effects, whereas their co-neutralization dramatically reduces B cell function, including antibody production. Fc fusions of wild-type (WT) TACI (e.g. atacicept and telitacicept) target both BAFF and APRIL and have demonstrated promising clinical potential in e.g. systemic lupus erythematosus (SLE) and IgA nephropathy but have not yet clearly exhibited long-term and/or complete disease remissions.Objectives:To generate a dual BAFF/APRIL antagonist with inhibitory activity superior to WT TACI and BCMA and with the potential to improve clinical outcomes in B cell-mediated diseases.Methods:Our directed evolution platform was used to identify a potent variant TNFR domain (vTD) of TACI that exhibits significantly enhanced affinity for BAFF and APRIL as compared to WT TACI; this TACI vTD domain was fused to a human IgG Fc to generate the therapeutic candidate ALPN-303. ALPN-303 was evaluated for functional activity in: 1) human lymphocyte assays, 2) the NOD.Aec1Aec2 spontaneous model of Sjogren’s syndrome (SjS), 3) the bm12-induced mouse model of lupus, 4) the (NZB/NZW)F1 spontaneous model of lupus, and 5) preclinical rodent and cynomolgus monkey pharmacokinetic/pharmacodynamic studies.Results:ALPN-303 inhibited BAFF- and APRIL-mediated signaling in vitro in human lymphocyte assays, with significantly lower IC50 values than WT TACI-Fc and belimumab comparators. In all mouse models evaluated, administration of ALPN-303 rapidly and significantly reduced key lymphocyte subsets including plasma cells, germinal center B cells, and follicular T helper cells. ALPN-303 significantly reduced autoantibodies and sialadenitis in the spontaneous SjS model, inhibited glomerular IgG deposition in the bm12-induced model of lupus, and potently suppressed anti-dsDNA autoAbs, blood urea nitrogen levels, proteinuria, sialadenitis, kidney lesions, and renal immune complex deposition in the NZB/W lupus model. As compared to WT TACI-Fc, ALPN-303 exhibited higher serum exposure and significantly and persistently decreased titers of serum IgM, IgG, and IgA antibodies in mice and cynomolgus monkeys (Figure 1).Figure 1.ALPN-303 induces more potent suppression, as compared to WT TACI-Fc, of serum immunoglobulins following a single 9 mg/kg IV infusion (on Day 0; arrows) in female cynomolgus monkeys.Conclusion:ALPN-303 is a potent BAFF/APRIL antagonist derived from our directed evolution platform that consistently demonstrates encouraging immunomodulatory activity and efficacy in vitro and in vivo, superior in preclinical studies to anti-BAFF antibody and WT TACI-Fc. This novel Fc fusion molecule demonstrates favorable preliminary developability characteristics, including higher serum exposures and more potent immunosuppressive activities, which may enable lower clinical doses and/or longer dosing intervals than WT TACI-Fc therapeutics. ALPN-303 may thus be an attractive development candidate for the treatment of multiple autoimmune and inflammatory diseases, particularly B cell-related diseases such as SLE, SjS, and other connective tissue diseases. Preclinical development is underway to enable the initiation of clinical trials later this year.Disclosure of Interests:Stacey R. Dillon Shareholder of: Alpine Immune Sciences, Bristol Myers Squibb, Employee of: Alpine Immune Sciences, Bristol Myers Squibb, Lawrence S. Evans Shareholder of: Alpine Immune Sciences, Employee of: Alpine Immune Sciences, Katherine E. Lewis Shareholder of: Alpine Immune Sciences, Employee of: Alpine Immune Sciences, Jing Yang Shareholder of: Alpine Immune Sciences, Employee of: Alpine Immune Sciences, Mark W. Rixon Shareholder of: Alpine Immune Sciences, Employee of: Alpine Immune Sciences, Joe Kuijper Shareholder of: Alpine Immune Sciences, Employee of: Alpine Immune Sciences, Dan Demonte Shareholder of: Alpine Immune Sciences, Employee of: Alpine Immune Sciences, Janhavi Bhandari Shareholder of: Alpine Immune Sciences, Employee of: Alpine Immune Sciences, Steve Levin Shareholder of: Alpine Immune Sciences, Employee of: Alpine Immune Sciences, Kayla Kleist Shareholder of: Alpine Immune Sciences, Employee of: Alpine Immune Sciences, Sherri Mudri Shareholder of: Alpine Immune Sciences, Employee of: Alpine Immune Sciences, Susan Bort Shareholder of: Alpine Immune Sciences, Employee of: Alpine Immune Sciences, Daniel Ardourel Shareholder of: Alpine Immune Sciences, Employee of: Alpine Immune Sciences, Michelle A. Seaberg Shareholder of: Alpine Immune Sciences, Employee of: Alpine Immune Sciences, Rachel Wang Shareholder of: Alpine Immune Sciences, Employee of: Alpine Immune Sciences, Chelsea Gudgeon Shareholder of: Alpine Immune Sciences, Employee of: Alpine Immune Sciences, Russell Sanderson Shareholder of: Alpine Immune Sciences, Employee of: Alpine Immune Sciences, Martin F. Wolfson Shareholder of: Alpine Immune Sciences, Employee of: Alpine Immune Sciences, Jan Hillson Shareholder of: Alpine Immune Sciences, Employee of: Alpine Immune Sciences, Stanford L. Peng Shareholder of: Alpine Immune Sciences, Employee of: Alpine Immune Sciences

Abstract Introduction: Checkpoint inhibition (CPI) has been shown to be an effective anti-tumor therapy, but CPI alone is frequently insufficient to control tumor growth, and costimulatory signals may also be required to produce clinically significant anti-tumor responses. PD1-PDL1 are established CPI targets and TMIGD2 is an inhibitory receptor expressed on T cells that is engaged by its cognate ligand HHLA2 on tumor cells, leading to inhibition of T cell responses. Novel biologics combining CD28 costimulation and CPI may yield promising tumor antigen-specific therapeutic candidates. Methods: Variants of CD86 with increased CD28 affinity were engineered using our directed evolution platform. PD1 and TMIGD2 variants were also engineered for increased affinity to PDL1 and HHLA2, respectively. Fusion proteins were generated including either PD1 or TMIGD2 domains, an effectorless Fc domain, and an engineered CD86 domain to generate proteins to provide target-dependent costimulation (TDC) and evaluated in multiple in vitro T cell stimulation assays. Costimulatory effects were confirmed by evaluating T cell proliferation, cytokine release, and tumor cell killing. Fusion proteins were also tested in vivo in a mouse tumor model using MC38 cells expressing human PDL1 or HHLA2, and in a humanized tumor model where an HPV+ squamous cell carcinoma cell line expressing either PDL1 or HHLA2 was implanted into NSG mice. Tumor-bearing NSG mice were given primary human T cells expressing an HPV peptide-specific TCR, with or without the corresponding fusion protein, and tumor growth was measured over time. Results: PD1-CD86 and TMIGD2-CD86 TDC proteins enhanced T cell costimulation in multiple in vitro T cell response assays, and costimulation was dependent on target cell lines expressing PDL1 or HHLA2, respectively. There was no effect on T cell responses if the target cells did not express the relevant target protein. PD1 and TMIGD2 domains alone had little effect on T cell responses, indicating these TDC proteins were driving enhanced responses. PD1-CD86 and TMIGD2-CD86 TDC proteins enhanced anti-tumor responses in vivo in a syngeneic MC38 implantation model only when MC38 cells expressed human PDL1 or HHLA2, respectively. Both proteins also enhanced antitumor responses in a humanized tumor implantation system using an HPV+ tumor cell line expressing the corresponding target antigen in the presence of human T cells expressing an HPV-specific TCR, but failed to control tumor growth when tumor cells lacked expression of the target protein. Conclusions: Tumor antigen-specific antitumor therapy can be achieved with fusion proteins that combine engineered CPI and CD28 costimulatory domains. Such novel biologics may provide promising approaches to enhancing the efficacy of CPI monotherapies and to address checkpoint inhibitor-resistant tumors. Citation Format: Steven D. Levin, Mark F. Maurer, Chelsea Gudgeon, Siddarth Chandrasekaran, Daniel Ardourel, Daniel Demonte, Joseph Kuijper, Martin Wolfson, Logan Garrett, Kayla N. Kleist, Sherri Mudri, Hieu Nguyen, Michelle Seaberg, Rachel Wang, Jing Yang, Katherine E. Lewis, Stacey R. Dillon, Mark Rixon, Stanford L. Peng. Engineered variant domain fusion proteins provide checkpoint inhibition and tumor antigen dependent CD28 costimulation resulting in potent anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1740.

Background: BAFF and APRIL are TNF superfamily members that bind both TACI and BCMA on B cells; BAFF also binds BAFF-R. Together, BAFF and APRIL support B cell development, differentiation, and survival. Their co-neutralization dramatically reduces B cell function, including antibody production, whereas inhibition of either BAFF or APRIL alone mediates relatively modest effects. Objectives: While CTLA-4-based therapeutics that block T cell costimulation provide safe and moderately effective T cell inhibition in many disease settings, and while B cell targeting therapies have demonstrated promising therapeutic potential, we postulate that improved, combined BAFF and APRIL inhibition, either alone or coupled with inhibition of T cell costimulation, will provide more effective and durable relief from severe B cell-related autoimmune diseases like SLE. Methods: We used our directed evolution platform to identify variant domains of the TNF family receptors TACI or BCMA that exhibit enhanced affinity for BAFF and APRIL as compared to their wild-type (WT) counterparts. These variant TACI or BCMA domains (vTD), alone or together with platform-derived CTLA-4 domains (vIgD), were fused to a modified human IgG1 Fc lacking effector function, yielding a panel of immunomodulatory molecules: TACI vTD-Fc, BCMA vTD-Fc, TACI vTD/CTLA-4 vIgD-Fc, & BCMA vTD/CTLA-4 vIgD-Fc. All were evaluated for functional activity: 1) in vitro in primary human B cell & MLR assays and in a Jurkat/NF-kB reporter cell line expressing TACI, and 2) in vivo in standard immunization models, and in the bm12-induced and NZB/NZW spontaneous mouse models of lupus. Results: The novel engineered TACI vTD-Fc or BCMA vTD-Fc fusion proteins significantly inhibited BAFF- and APRIL-mediated signaling in vitro in TACI + Jurkat cells. TACI (or BCMA) vTD/CTLA-4 vIgD-Fc proteins also attenuated T cell activation in primary human lymphocyte assays. When administered to mice, these molecules rapidly and potently reduced key B and T cell subsets, including plasma cells, follicular T helper cells, germinal center cells, & memory T cells. Treatment with TACI vTD-Fc or TACI vTD/CTLA-4 vIgD-Fc proteins also significantly reduced titers of antigen-specific antibodies in immunized mice more so than abatacept or WT TACI-Fc, and potently suppressed anti-dsDNA autoantibodies, blood urea nitrogen levels, proteinuria, and renal immune complex deposition in the bm12 & NZB/W lupus models. Conclusion: Directed evolution of TNFR and IgSF domains has successfully facilitated the development of Fc fusion proteins containing TACI or BCMA vTDs, with or without fusion to CTLA-4 vIgDs. These novel immunomodulators consistently demonstrate potent immunosuppressive activity and efficacy in vitro and in vivo , appearing superior to existing and/or approved immunomodulators like belimumab, abatacept, or atacicept. Such biologics may therefore be attractive candidates for the treatment of serious autoimmune diseases, particularly B cell-related diseases such as SLE, Sjogren’s syndrome, etc. Disclosure of Interests: : Stacey R. Dillon Shareholder of: Shareholder of Alpine Immune Sciences, Inc., Employee of: Employee of Alpine Immune Sciences, Inc., Lawrence S. Evans Shareholder of: Shareholder of Alpine Immune Sciences, Inc., Employee of: Employee of Alpine Immune Sciences, Inc., Mark W. Rixon Shareholder of: Shareholder of Alpine Immune Sciences, Inc., Employee of: Employee of Alpine Immune Sciences, Inc., Joe Kuijper Shareholder of: Shareholder of Alpine Immune Sciences, Inc., Employee of: Employee of Alpine Immune Sciences, Inc., Dan Demonte Shareholder of: Shareholder of Alpine Immune Sciences, Inc., Employee of: Employee of Alpine Immune Sciences, Inc., Katherine E. Lewis Shareholder of: Shareholder of Alpine Immune Sciences, Inc., Employee of: Employee of Alpine Immune Sciences, Inc., Steve Levin Shareholder of: Shareholder of Alpine Immune Sciences, Inc., Employee of: Employee of Alpine Immune Sciences, Inc., Kayla Kleist Shareholder of: Shareholder of Alpine Immune Sciences, Inc., Employee of: Employee of Alpine Immune Sciences, Inc., Sherri Mudri Shareholder of: Shareholder of Alpine Immune Sciences, Inc., Employee of: Employee of Alpine Immune Sciences, Inc., Susan Bort Shareholder of: Shareholder of Alpine Immune Sciences, Inc., Employee of: Employee of Alpine Immune Sciences, Inc., Janhavi Bhandari Shareholder of: Shareholder of Alpine Immune Sciences, Inc., Employee of: Employee of Alpine Immune Sciences, Inc., Fariha Ahmed-Qadri Shareholder of: Shareholder of Alpine Immune Sciences, Inc., Employee of: Employee of Alpine Immune Sciences, Inc., Jing Yang Shareholder of: Alpine Immune Sciences, Inc., Employee of: Alpine Immune Sciences, Inc., Michelle A. Seaberg Shareholder of: Shareholder of Alpine Immune Sciences, Inc., Employee of: Employee of Alpine Immune Sciences, Inc., Rachel Wang Shareholder of: Shareholder of Alpine Immune Sciences, Inc., Employee of: Employee of Alpine Immune Sciences, Inc., Russell Sanderson Shareholder of: Alpine Immune Sciences, Inc., Employee of: Alpine Immune Sciences, Inc., Martin F. Wolfson Shareholder of: Shareholder of Alpine Immune Sciences, Inc., Employee of: Employee of Alpine Immune Sciences, Inc., Jan Hillson Shareholder of: Alpine Immune Sciences, Inc., Employee of: Alpine Immune Sciences, Inc., Stanford L. Peng Shareholder of: Alpine Immune Sciences, Inc., Employee of: CMO and President of Alpine Immune Sciences, Inc., Kristine M. Swiderek Shareholder of: Shareholder of Alpine Immune Sciences, Inc., Employee of: Employee of Alpine Immune Sciences, Inc.

BAFF and APRIL are TNF superfamily members that bind both TACI and BCMA on B cells; BAFF also binds BAFF-R. Together, BAFF and APRIL support B cell development, differentiation, and survival. Their co-neutralization dramatically reduces B cell function, including antibody production, whereas inhibition of either BAFF or APRIL alone mediates relatively modest effects.While CTLA-4-based therapeutics that block T cell costimulation provide safe and moderately effective T cell inhibition in many disease settings, and while B cell targeting therapies have demonstrated promising therapeutic potential, we postulate that improved, combined BAFF and APRIL inhibition, either alone or coupled with inhibition of T cell costimulation, will provide more effective and durable relief from severe B cell-related autoimmune diseases like SLE.We used our directed evolution platform to identify variant domains of the TNF family receptors TACI or BCMA that exhibit enhanced affinity for BAFF and APRIL as compared to their wild-type (WT) counterparts. These variant TACI or BCMA domains (vTD), alone or together with platform-derived CTLA-4 domains (vIgD), were fused to a modified human IgG1 Fc lacking effector function, yielding a panel of immunomodulatory molecules: TACI vTD-Fc, BCMA vTD-Fc, TACI vTD/CTLA-4 vIgD-Fc, & BCMA vTD/CTLA-4 vIgD-Fc. All were evaluated for functional activity: 1)in vitroin primary human B cell & MLR assays and in a Jurkat/NF-kB reporter cell line expressing TACI, and 2)in vivoin standard immunization models, and in the bm12-induced and NZB/NZW spontaneous mouse models of lupus.The novel engineered TACI vTD-Fc or BCMA vTD-Fc fusion proteins significantly inhibited BAFF- and APRIL-mediated signalingin vitroin TACI+Jurkat cells. TACI (or BCMA) vTD/CTLA-4 vIgD-Fc proteins also attenuated T cell activation in primary human lymphocyte assays. When administered to mice, these molecules rapidly and potently reduced key B and T cell subsets, including plasma cells, follicular T helper cells, germinal center cells, & memory T cells. Treatment with TACI vTD-Fc or TACI vTD/CTLA-4 vIgD-Fc proteins also significantly reduced titers of antigen-specific antibodies in immunized mice more so than abatacept or WT TACI-Fc, and potently suppressed anti-dsDNA autoantibodies, blood urea nitrogen levels, proteinuria, and renal immune complex deposition in the bm12 & NZB/W lupus models.Directed evolution of TNFR and IgSF domains has successfully facilitated the development of Fc fusion proteins containing TACI or BCMA vTDs, with or without fusion to CTLA-4 vIgDs. These novel immunomodulators consistently demonstrate potent immunosuppressive activity and efficacyin vitroandin vivo, appearing superior to existing and/or approved immunomodulators like belimumab, abatacept, or atacicept. Such biologics may therefore be attractive candidates for the treatment of serious autoimmune diseases, particularly B cell-related diseases such as SLE, Sjogren’s syndrome, etc.Stacey R. Dillon Shareholder of: Shareholder of Alpine Immune Sciences, Inc., Employee of: Employee of Alpine Immune Sciences, Inc., Lawrence S. Evans Shareholder of: Shareholder of Alpine Immune Sciences, Inc., Employee of: Employee of Alpine Immune Sciences, Inc., Mark W. Rixon Shareholder of: Shareholder of Alpine Immune Sciences, Inc., Employee of: Employee of Alpine Immune Sciences, Inc., Joe Kuijper Shareholder of: Shareholder of Alpine Immune Sciences, Inc., Employee of: Employee of Alpine Immune Sciences, Inc., Dan Demonte Shareholder of: Shareholder of Alpine Immune Sciences, Inc., Employee of: Employee of Alpine Immune Sciences, Inc., Katherine E. Lewis Shareholder of: Shareholder of Alpine Immune Sciences, Inc., Employee of: Employee of Alpine Immune Sciences, Inc., Steve Levin Shareholder of: Shareholder of Alpine Immune Sciences, Inc., Employee of: Employee of Alpine Immune Sciences, Inc., Kayla Kleist Shareholder of: Shareholder of Alpine Immune Sciences, Inc., Employee of: Employee of Alpine Immune Sciences, Inc., Sherri Mudri Shareholder of: Shareholder of Alpine Immune Sciences, Inc., Employee of: Employee of Alpine Immune Sciences, Inc., Susan Bort Shareholder of: Shareholder of Alpine Immune Sciences, Inc., Employee of: Employee of Alpine Immune Sciences, Inc., Janhavi Bhandari Shareholder of: Shareholder of Alpine Immune Sciences, Inc., Employee of: Employee of Alpine Immune Sciences, Inc., Fariha Ahmed-Qadri Shareholder of: Shareholder of Alpine Immune Sciences, Inc., Employee of: Employee of Alpine Immune Sciences, Inc., Jing Yang Shareholder of: Alpine Immune Sciences, Inc., Employee of: Alpine Immune Sciences, Inc., Michelle A. Seaberg Shareholder of: Shareholder of Alpine Immune Sciences, Inc., Employee of: Employee of Alpine Immune Sciences, Inc., Rachel Wang Shareholder of: Shareholder of Alpine Immune Sciences, Inc., Employee of: Employee of Alpine Immune Sciences, Inc., Russell Sanderson Shareholder of: Alpine Immune Sciences, Inc., Employee of: Alpine Immune Sciences, Inc., Martin F. Wolfson Shareholder of: Shareholder of Alpine Immune Sciences, Inc., Employee of: Employee of Alpine Immune Sciences, Inc., Jan Hillson Shareholder of: Alpine Immune Sciences, Inc., Employee of: Alpine Immune Sciences, Inc., Stanford L. Peng Shareholder of: Alpine Immune Sciences, Inc., Employee of: CMO and President of Alpine Immune Sciences, Inc., Kristine M. Swiderek Shareholder of: Shareholder of Alpine Immune Sciences, Inc., Employee of: Employee of Alpine Immune Sciences, Inc.