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Purpose: VEGF-targeted therapies have modest efficacy in cancer patients, but acquired resistance is common. The mechanisms underlying such resistance are poorly understood.Experimental Design: To evaluate the potential role of immune cells in the development of resistance to VEGF blockade, we first established a preclinical model of adaptive resistance to anti-VEGF therapy. Additional in vitro and in vivo studies were carried out to characterize the role of macrophages in such resistance.Results: Using murine cancer models of adaptive resistance to anti-VEGF antibody (AVA), we found a previously unrecognized role of macrophages in such resistance. Macrophages were actively recruited to the tumor microenvironment and were responsible for the emergence of AVA resistance. Depletion of macrophages following emergence of resistance halted tumor growth and prolonged survival of tumor-bearing mice. In a macrophage-deficient mouse model, resistance to AVA failed to develop, but could be induced by injection of macrophages. Downregulation of macrophage VEGFR-1 and VEGFR-3 expression accompanied upregulation of alternative angiogenic pathways, facilitating escape from anti-VEGF therapy.Conclusions: These findings provide a new understanding of the mechanisms underlying the modest efficacy of current antiangiogenesis therapies and identify new opportunities for combination approaches for ovarian and other cancers. Clin Cancer Res; 23(22); 7034–46. ©2017 AACR.

S Figure 1 (A) C57BL/6 mice that received intra-peritoneal injections of IG10 cells were randomly assigned to one of two groups (control or B20 treatment). S Figure 2 (A) Bar graph represents the CD11b+/F4/80+ macrophages in tumor tissue by FACS analysis. S Figure 3 Anti-VEGF therapyââ,¬â€œresistant macrophages have heightened viability, migration, and invasion. S Figure 4 (A) Bar graph represents the percentage of viability of B20 treated macrophages. S Figure 5. VEGFR1-positive macrophages increase the effectiveness of anti-VEGF therapy.

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Background: Acute myeloid leukemia (AML) is characterized by clonal proliferation of malignant myeloid blasts in the bone marrow. We have recently reported that blocking B7-H3 using a novel monoclonal antibody (mAB), T-1A5 enhances natural killer (NK) cell mediated cytotoxicity against AML cells. IL-15 is an essential component for NK cell activity. Recombinant human IL-15 (rIL-15), however, degrades rapidly and therefore may provide a suboptimal stimulus to NK cells. Studies have shown that BCL2 inhibitor, venetoclax (ABT-199), enhances NK cell induced antibody dependent cellular cytotoxicity (ADCC) against cancer cells. However, the combined effect of B7-H3 blocking and BCL2 inhibition remains unexplored in AML. We hypothesized that blocking B7-H3 using T-1A5 in the presence of novel investigational polymer-engineered IL-15 receptor agonist, NKTR-255, and the BCL2 inhibitor synergistically induces NK cell mediated ADCC in AML cells. Methods: CD16 and NKG2D activation markers measured by flow cytometry in AML patient and healthy donor derived NK cells. The NKTR-255 was used to generate NK cells, and their cytotoxicity was compared to that of NK cells generated with rIL-15 using IncuCyte. To investigate these NK cells’ cytotoxic activity against AML cells, we treated B7-H3+ AML cell lines (OCI-AML3 and MV4-11) with anti-B7-H3 chimeric mAb ChT-1A5 in the presence and absence of NK cells generated using NKTR-255 or rIL-15. To assess the potential synergy of BCL2 and B7-H3 in NK cell mediated anti-leukemic activity against AML cells, we treated B7-H3+ cells with T-1A5 with or without ABT-199in the presence or absence of NK cells. Results: The expression of NK cell activation markers were significantly lower in NK cells derived from AML patients than healthy donors. To assess the effect of NKTR-255 on NK cell activity, we generated NK cells using NKTR-255 and compared them with NK cells generated using rIL-15 using the standard 14-day culture protocol. We observed similar proliferation and NK cell numbers between NKTR-255 and rIL-15. However, NK cells stimulated with NKTR-255 showed significantly enhanced (more than 2-fold) cytolytic activity against B7-H3+ AML cell lines compared with NK cells stimulated with rIL-15. Moreover, when combined with ChT-1A5, NKTR-255 significantly enhanced NK cell mediated ADCC activity in B7-H3+ AML cells compared to rIL-15. Interestingly, we observed that the combination of T-1A5 and venetoclax synergistically induced an anti-leukemic effect in B7-H3+ AML cell lines when compared with either drug alone or isotype IgG1 in a dose-dependent manner. Conclusion: Our data demonstrate that blockade of the immune checkpoint protein, B7-H3 with the IL-15 receptor agonist, NKTR-255, or the BCL2 inhibitor, ABT-199, synergistically induces NK cell mediated cytotoxicity against B7-H3+ AML cells. Citation Format: Anudishi Tyagi, A. Mario Marcondes, Willem Overwijk, Venkata Lokesh Battula. B7-H3 blockade in combination with natural killer cell activity enhancers including NKTR-255 and venetoclax synergistically induces cytotoxicity in B7-H3+ AML cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5098.

Background: Breast cancer stem-like cells (BCSCs) are reported to be a major contributing factor for tumor growth and chemotherapy resistance in triple-negative breast cancer (TNBC). We reported that the disialoganglioside GD2 is highly expressed on BCSCs and that targeting GD2 with dinutuximab (chimeric anti-GD2 antibody) in combination with NK cells synergistically reduced tumor volumes in TNBC animal models. Here, we hypothesize that naxitamab targets GD2+ BCSCs and inhibits breast cancer (BC) growth by enhancing macrophage mediated phagocytosis and T cell mediated cytotoxicity. In addition, we explore whether addition of an investigational polymer-engineered IL-15 receptor-agonist, NKTR-255 treated NK cells, can further enhance this anti-GD2 mediated anti-cancer activity. Methods: Naxitamab is a US FDA approved fully humanized anti-GD2 (hu3F8) monoclonal antibody for the treatment of pediatric neuroblastoma. We evaluated the effects of naxitamab treatment on macrophage mediated phagocytosis of BC cells using the live cell imaging system, IncuCyte®. Next, we compared the effects of NKTR-255 treated NK cells with recombinant human IL-15 (rIL-15) treated NK cells in naxitamab induced ADCC in GD2+ BC cells. We also performed immunohistochemistry (IHC) to examine GD2 expression and correlate it with T cell infiltration in primary TNBC patient samples with high and low GD2 levels. The effect of naxitamab treatment on T cell mediated cytotoxicity in BC cells was also observed. Results: When compared to control, naxitamab treatment promoted macrophage mediated phagocytosis of Hs578T and HCC1395 cells in a dose dependent manner (p Conclusion: TNBC with high GD2 expression inhibits immune cell infiltration and naxitamab can inhibit BCSC growth by targeting GD2. Combination of naxitamab with macrophages induced macrophage mediated phagocytosis TNBC cells. NK cells were more stable and cytotoxic when generated with NKTR-255 vs. native IL-15, and naxitamab further enhanced NK cell mediated ADCC of GD2+ BCSCs. The increased T cell mediated killing of breast cancer cells treated with naxitamab further supports the immunomodulatory role of GD2. Citation Format: Vivek Anand, Venkatesh Hegde, Maryam Siddiqui, Anudishi Tyagi, Bolutyfe Oderinde, Mario Marcondes, Willem Overwijk, Venkata Lokesh Battula. Novel humanized anti-GD2 antibody inhibits GD2-mediated immunosuppression by targeting GD2+ breast cancer stem-like cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5115.

Background Multiple myeloma (MM) is characterized by an immunosuppressive microenvironment that enables tumor development. One of the mechanisms of immune evasion used by MM cells is the inhibition of NK cell effector functions; thus, the restoration of NK cell antitumor activity represents a key goal for new immunotherapeutic approaches, increasing tumor cell recognition, avoiding tumor escape and potentially enhancing the effect of other drugs. Methods Here we investigate the potential of NKTR-255, a novel polymer-conjugated human IL-15 to engage the IL-15 pathway and overcome the inhibitory status observed in NK cells from MM patients. For this purpose, we have analyzed ex vivo and in vivo effects of NKTR-255 on phenotypic features, effector functions and cytotoxicity of NK cells against MM cells. Results We observed that incubation with NKTR-255 was able to tilt the balance towards an activated phenotype in NK cells isolated from peripheral blood mononuclear cells of MM patients, with increased expression of activating receptors (NKG2D, NKp46, NKp30, DNAM-1, CD69, TRAIL) on the surface of treated NK cells. This resulted in an enhanced degranulation, cytokine release and anti-tumor cytotoxicity when the NK cells were exposed to both MM cell lines and primary MM cells. For a more accurate assessment of the effect of NKTR-255 on NK cell activity in an autologous setting in the presence of the bone marrow (BM) milieu, we cultured whole BM samples from non-treated newly-diagnosed MM patients with increasing doses of NKTR-255 for 5 days. NK cells experienced a dose-dependent induction of proliferation and activation (as shown by increased expression of CD69 and NKG2D), which translated in a reduced viability of CD138+ MM cells in the presence of NKTR-255. We further evaluated the in vivo effect of NKTR-255 in fully humanized immunocompetent mice subcutaneously engrafted with H929 MM cells. Compared to placebo, weekly injection of the mice with NKTR-255 increased the number of circulating NK cells in peripheral blood and delayed tumor growth. Finally, we also tested in vitro and in vivo efficacy of a combination of NKTR-255 with daratumumab. We observed a more efficient antibody-dependent cellular cytotoxicity against MM cells in vitro and decreased tumor growth in vivo, where NKTR-255 rescued CD38+ NK cell levels from depletion by daratumumab. Conclusions Taken together, these results support the restoration and expansion of NK cell activity in MM with NKTR-255, providing rationale for its clinical use as a novel immunotherapeutic approach for MM patients alone or in combination with monoclonal antibodies or other immunomodulatory drugs.

Type I Interferons (IFN) are potent inducers of IL-15 transcription and cell surface expression. Previous studies have shown that sIL-15 complexes, which act as IL-15 agonists, are generated by TLR stimulation and after non-myeloablative lymphodepletion. Since TLR stimulation induces type I IFNs, we investigated the role of type I IFNs in generating sIL-15 complexes. We report that treatment with a plasmid inducing IFN-α production potently increased serum sIL-15 complexes levels in mice. Production of sIL-15 complexes by bone marrow derived dendritic cells (BMDC) also increased after treatment with IFN-α in an IFNAR-dependent manner. Increases in sIL-15 complexes after total body irradiation coincided with appearance of serum IFN-α and were significantly impaired in the absence of Type I IFN signaling. In addition, we found that IFN-α induced activity of the metalloprotease, ADAM17 in BMDCs, which was critical as induction of sIL-15 complexes by IFN-α was abolished after deleting ADAM17. We also determined whether sIL-15 complexes are generated during a Vesicular stomatitis virus (VSV) infection as VSV induces Type I IFNs. One day after infection, sIL-15 complexes are transiently induced, which was surprisingly unabated in the absence of Type I IFN signaling. Overall, our findings show that Type I IFNs induce production of sIL-15 complexes by activating ADAM17-mediated cleavage, thus identifying a new mechanism regulating sIL-15 complexes during inflammation.