Blocking PD-1/PD-L1 by an ADCC enhanced anti-B7-H3/PD-1 fusion protein engages immune activation and cytotoxicity.

Antibody therapy based on PD-1/PD-L1 blocking or ADCC effector has produced significant clinical benefit for cancer patients. We generated a novel anti-B7-H3 antibody (07B) and engineered the Fc fragment to enhance ADCC. To improve efficacy and tumor selectivity, we developed anti-B7-H3/PD-1 bispecific fusion proteins that simultaneously engaged tumor associate marker B7-H3 and immune suppressing ligand PD-L1 as well as enhanced ADCC to promote potent and highly selective tumor killing. Fusion proteins were designed by fusing human PD-1 extra domain to 07B in four different formats and showed good binding capacity to both targets. Indeed, the affinity of fusion proteins to B7-H3 is over 10,000 fold higher compared to that of the analogous PD-L1 and the blocking of fusion proteins to PD-L1 was worse but it greatly enhanced when bound to B7-H3, thus achieving directly PD-L1-blockade to B7-H3-expressing tumor cells. Importantly, IL-2 production was enhanced by fusion proteins from staphylococcal enterotoxin B (SEB) stimulated PBMC. Similarly, cytokines induced by fusion proteins was enhanced when co-cultured with stimulated CD8+ T cells and B7-H3/PD-L1 transfected raji cells. Additionally, fusion proteins improved activation to CD16a by Fc modification and delivered selective cytotoxicity to B7-H3 expressing tumor cells. In conclusion, fusion proteins blocked the PD-1/PD-L1 signal pathway and significantly increased potency of ADCC in a B7-H3-directed manner, thereby selectively activating CD8+ T cells and enhancing natural killing towards tumor. This novel fusion protein with its unique targeting preference may be useful to enhance efficacy and safety of immunotherapy for B7-H3-overexpressing malignancies.

Xu Y ，Xiao Y ，Luo C ，Liu Q ，Wei A ，Yang Y ，Zhao L ，Wang Y ... -  《-》

Functional and mechanistic advantage of the use of a bifunctional anti-PD-L1/IL-15 superagonist.

Anti(α)-programmed cell death-1 (PD-1)/programmed death-ligand 1 (PD-L1) monotherapy fails to provide durable clinical benefit for most patients with carcinoma. Recent studies suggested that strategies to reduce immunosuppressive cells, promote systemic T-cell responses and lymphocyte trafficking to the tumor microenvironment (TME) may improve efficacy. N-809 is a first-in-class bifunctional agent comprising the interleukin (IL)-15 superagonist N-803 fused to two αPD-L1 domains. Thus, N-809 can potentially stimulate effector immune cells through IL-15 and block immunosuppressive PD-L1. Here, we examined the antitumor efficacy and immunomodulatory effects of N-809 versus N-803+αPD-L1 combination. The ability of N-809 to block PD-L1 and induce IL-15-dependent immune effects was examined in vitro and in vivo. Antitumor efficacy of N-809 or N-803+αPD-L1 was evaluated in two murine carcinoma models and an extensive analysis of immune correlates was performed in the tumor and tumor-draining lymph node (dLN). We demonstrate that N-809 blocks PD-L1 and induces IL-15-dependent immune effects. N-809 was well-tolerated and reduced 4T1 lung metastasis, decreased MC38 tumor burden and increased survival versus N-803+αPD-L1. Compared with N-803+αPD-L1, N-809 enhanced natural killer (NK) and CD8+ T-cell activation and function in the dLN and TME, relating to increased gene expression associated with interferon and cytokine signaling, lymphoid compartment, costimulation and cytotoxicity. The higher number of TME CD8+ T cells was attributed to enhanced infiltration, not in situ expansion. Increased TME NK and CD8+ T-cell numbers correlated with augmented chemokine ligands and receptors. Moreover, in contrast to N-803+αPD-L1, N-809 reduced immunosuppressive regulatory T cells (Treg), monocytic myeloid-derived suppressor cells (M-MDSC) and M2-like macrophages in the TME. Our results suggest that N-809 functions by a novel immune mechanism to promote antitumor efficacy. Foremost, N-809 enhances intratumoral lymphocyte numbers by increasing trafficking via altered chemokine levels in the TME and chemokine receptor expression on CD8+ T cells and NK cells. In addition, N-809 reduces immunosuppressive and pro-tumorigenic immune cells in the TME, including Treg, M2-like macrophages and M-MDSC. Overall, these novel effects of N-809 promote an inflamed TME, leading to lower tumor burden and increased survival. These results provide mechanistic insight and rationale supporting the potential clinical study of N-809 in patients with carcinoma.

Knudson KM ，Hicks KC ，Ozawa Y ，Schlom J ，Gameiro SR ... -  《Journal for ImmunoTherapy of Cancer》

B7-H3 Negatively Modulates CTL-Mediated Cancer Immunity.

Purpose: Anti-programmed-death-1 (PD-1) immunotherapy improves survival in non-small cell lung cancer (NSCLC), but some cases are refractory to treatment, thereby requiring alternative strategies. B7-H3, an immune-checkpoint molecule, is expressed in various malignancies. To our knowledge, this study is the first to evaluate B7-H3 expression in NSCLCs treated with anti-PD-1 therapy and the therapeutic potential of a combination of anti-PD-1 therapy and B7-H3 targeting.Experimental Design: B7-H3 expression was evaluated immunohistochemically in patients with NSCLC (n = 82), and its relationship with responsiveness to anti-PD-1 therapy and CD8+ tumor-infiltrating lymphocytes (TILs) was analyzed. The antitumor efficacy of dual anti-B7-H3 and anti-programmed death ligand-1 (PD-L1) antibody therapy was evaluated using a syngeneic murine cancer model. T-cell numbers and functions were analyzed by flow cytometry.Results: B7-H3 expression was evident in 74% of NSCLCs and was correlated critically with nonresponsiveness to anti-PD-1 immunotherapy. A small number of CD8+ TILs was observed as a subpopulation with PD-L1 tumor proportion score less than 50%, whereas CD8+ TILs were still abundant in tumors not expressing B7-H3. Anti-B7-H3 blockade showed antitumor efficacy accompanied with an increased number of CD8+ TILs and recovery of effector function. CD8+ T-cell depletion negated antitumor efficacy induced by B7-H3 blockade, indicating that improved antitumor immunity is mediated by CD8+ T cells. Compared with a single blocking antibody, dual blockade of B7-H3 and PD-L1 enhanced the antitumor reaction.Conclusions: B7-H3 expressed on tumor cells potentially circumvents CD8+-T-cell-mediated immune surveillance. Anti-B7-H3 immunotherapy combined with anti-PD-1/PD-L1 antibody therapy is a promising approach for B7-H3-expressing NSCLCs. Clin Cancer Res; 24(11); 2653-64. ©2018 AACR.

Yonesaka K ，Haratani K ，Takamura S ，Sakai H ，Kato R ，Takegawa N ，Takahama T ，Tanaka K ，Hayashi H ，Takeda M ，Kato S ，Maenishi O ，Sakai K ，Chiba Y ，Okabe T ，Kudo K ，Hasegawa Y ，Kaneda H ，Yamato M ，Hirotani K ，Miyazawa M ，Nishio K ，Nakagawa K ... -  《-》

Development of a novel anti-B7-H4 antibody enhances anti-tumor immune response of human T cells.

B7-H4 is a member of the B7 superfamily that is expressed on the surface of tumors and exhibits limited expression on normal tissue. B7-H4 negatively regulates tumor immunity by interacting with the B7-H4 receptor, which is expressed by activated CD8 + T cells. Hence, we sought to generate an immunomodulatory antibody that targets B7-H4 and blocks the immunosuppressive activity of B7-H4. Anti-B7-H4 antibodies were generated using the hybridoma technique and screened by a binding assay based on B7-H4-expressing tumor cells. The B7-H4 antagonistic antibodies were further screened based on their checkpoint blockade activity using a SEB-stimulated peripheral blood mononuclear cell (PBMC) assay, which comprised B7-H4-expressing antigen presenting cells (APCs) and activated T cells. To assess the immunomodulatory activity of anti-B7-H4 antibodies, activated human CD8+ T cells were cultured in B7-H4 protein-coated plates, and the production of IL-2 and the proliferation rate of CD8+ T cells were measured. In addition, we evaluated the ADCC effect of anti-B7-H4 antibodies against tumor cell lines. The in vivo antitumor efficacy of the anti-B7-H4 antibody was also evaluated in human T cell-engrafted NOG mice. A panel of anti-B7-H4 antibodies was generated. The top 23 antibodies were screened to identify antibodies that disabled B7-H4-mediated inhibition. Antibody 17 exhibited the greatest induction of the production of IL-2 and IFN-gamma in SEB-stimulated PBMCs. Antibody 17 was constructed as a chimeric antibody (CH17) with a human IgG1 constant domain. CH17 showed high affinity for human B7-H4 and fully cross-reacted with cynomolgus B7-H4. Additionally, CH17 mediated potent antibody-dependent cell cytotoxicity (ADCC) against different B7-H4-positive tumor cell lines. More importantly, CH17 relieved B7-H4-mediated T cell suppression by enhancing IL2 production and promoting T cell proliferation. In an MDA-MB-468-bearing mouse model in which human pan-T cells were engrafted, CH17 delayed tumor growth by engaging T cells and exerted a synergistic effect in combination with an anti-human PD-1 antibody. We successfully generated an immunomodulatory antibody targeting B7-H4 that possesses both T cell immune checkpoint inhibitory activity and ADCC activity in B7-H4-positive tumors. B7-H4-targeting antibodies might represent a promising immunotherapy for B7-H4-expressing tumors.

Miao G ，Sun X 《-》

Bispecific Targeting of PD-1 and PD-L1 Enhances T-cell Activation and Antitumor Immunity.

The programmed cell death protein 1 receptor (PD-1) and programmed death ligand 1 (PD-L1) coinhibitory pathway suppresses T-cell-mediated immunity. We hypothesized that cotargeting of PD-1 and PD-L1 with a bispecific antibody molecule could provide an alternative therapeutic approach, with enhanced antitumor activity, compared with monospecific PD-1 and PD-L1 antibodies. Here, we describe LY3434172, a bispecific IgG1 mAb with ablated Fc immune effector function that targets both human PD-1 and PD-L1. LY3434172 fully inhibited the major inhibitory receptor-ligand interactions in the PD-1 pathway. LY3434172 enhanced functional activation of T cells in vitro compared with the parent anti-PD-1 and anti-PD-L1 antibody combination or respective monotherapies. In mouse tumor models reconstituted with human immune cells, LY3434172 therapy induced dramatic and potent antitumor activity compared with each parent antibody or their combination. Collectively, these results demonstrated the enhanced immunomodulatory (immune blockade) properties of LY3434172, which improved antitumor immune response in preclinical studies, thus supporting its evaluation as a novel bispecific cancer immunotherapy.

Kotanides H ，Li Y ，Malabunga M ，Carpenito C ，Eastman SW ，Shen Y ，Wang G ，Inigo I ，Surguladze D ，Pennello AL ，Persaud K ，Hindi S ，Topper M ，Chen X ，Zhang Y ，Bulaon DK ，Bailey T ，Lao Y ，Han B ，Torgerson S ，Chin D ，Sonyi A ，Haidar JN ，Novosiadly RD ，Moxham CM ，Plowman GD ，Ludwig DL ，Kalos M ... -  《-》