Lung cancer is one of the leading causes of cancer-related deaths in the United States. A major hurdle for improved therapies is immune suppression mediated by the tumor and its microenvironment. The lung tumor microenvironment (TME) contains large numbers of tumor-associated macrophages (TAMs), which suppress the adaptive immune response, increase neo-vascularization of the tumor, and provide pro-tumor factors to promote tumor growth. CD11b is highly expressed on myeloid cells, including TAMs, where it forms a heterodimeric integrin receptor with CD18 (known as CD11b/CD18, Mac-1, CR3, and αMβ2), and plays an important role in recruitment and biological functions of these cells, and is a validated therapeutic target. Here, we describe our pre-clinical studies targeting CD11b in the context of lung cancer, using pharmacologic and genetic approaches that work via positive allosteric modulation of CD11b function. GB1275 is a novel small molecule modulator of CD11b that is currently in Phase 1/2 clinical development. We assess GB1275 treatment effects on tumor growth and immune infiltrates in the murine Lewis Lung Carcinoma (LLC) syngeneic tumor model. Additionally, as an orthogonal approach to determine mechanisms of action, we utilize our recently developed novel CD11b knock-in (KI) mouse that constitutively expresses CD11b containing an activating isoleucine to glycine substitution at residue 332 in the ligand binding CD11b A-domain (I332G) that acts as a positive allosteric modulator of CD11b activity. We report that pharmacologic modulation of CD11b with GB1275 significantly reduces LLC tumor growth. CD11b KI mice similarly show significant reduction in both the size and rate of LLC tumor growth, as compared to WT mice, mimicking our observed treatment effects with GB1275. Tumor profiling revealed a significant reduction in TAM infiltration in GB1275-treated and in CD11b KI mice, increase in the ratio of M1/M2-like TAMs, and concomitant increase in cytotoxic T cells. The profiling also showed a significant decrease in CCL2 levels and a concomitant reduction in Ly6C hi monocytes in circulation in both groups. These findings suggest that positive allosteric modulation of CD11b reduces TAM density and reprograms them to enhance the adaptive immune response and is a novel therapeutic strategy against lung cancer.

SUMMARY Spatial organization of molecules and cells in complex tissue microenvironments provides essential organizational cues in health and disease. A significant need exists for improved visualization of these spatial relationships. Here, we describe a multiplex immunofluorescence imaging method, termed SeqStain, that uses fluorescent-DNA-labeled antibodies for immunofluorescent staining and nuclease treatment for de-staining that allows selective enzymatic removal of the fluorescent signal. SeqStain can be used with primary antibodies, secondary antibodies, and antibody fragments to efficiently analyze complex cells and tissues. Additionally, incorporation of specific endonuclease restriction sites in antibody labels allows for selective removal of fluorescent signals while retaining other signals that can serve as marks for subsequent analyses. The application of SeqStain on human kidney tissue provided a spatialomic profile of the organization of >25 markers in the kidney, highlighting it as a versatile, easy-to-use, and gentle new technique for spatialomic analyses of complex microenvironments.

Current classifications of sporadic Creutzfeldt–Jakob disease (sCJD) identify five subtypes associated with different disease phenotypes. Most of these histopathological phenotypes (histotypes) co-distribute with distinct pairings of methionine (M)/valine (V) genotypes at codon 129 of the prion protein (PrP) gene and the type (1 or 2) of the disease-associated PrP (PrPD). Types 1 and 2 are defined by the molecular mass (~ 21 kDa and ~ 19 kDa, respectively) of the unglycosylated isoform of the proteinase K-resistant PrPD (resPrPD). We recently reported that the sCJDVV1 subtype (129VV homozygosity paired with PrPD type 1, T1) shows an electrophoretic profile where the resPrPD unglycosylated isoform is characterized by either one of two single bands of ~ 20 kDa (T120) and ~ 21 kDa (T121), or a doublet of ~ 21–20 kDa (T121−20). We also showed that T120 and T121 in sCJDVV have different conformational features but are associated with indistinguishable histotypes. The presence of three distinct molecular profiles of T1 is unique and raises the issue as to whether T120 and T121 represent distinct prion strains. To answer this question, brain homogenates from sCJDVV cases harboring each of the three resPrPD profiles, were inoculated to transgenic (Tg) mice expressing the human PrP-129M or PrP-129V genotypes. We found that T120 and T121 were faithfully replicated in Tg129V mice. Electrophoretic profile and incubation period of mice challenged with T121−20 resembled those of mice inoculated with T121 and T120, respectively. As in sCJDVV1, Tg129V mice challenged with T121 and T120 generated virtually undistinguishable histotypes. In Tg129M mice, T121 was not replicated while T120 and T121−20 generated a ~ 21–20  kDa doublet after lengthier incubation periods. On second passage, Tg129M mice incubation periods and regional PrP accumulation significantly differed in T120 and T121−20 challenged mice. Combined, these data indicate that T121 and T120 resPrPD represent distinct human prion strains associated with partially overlapping histotypes.

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Lung cancer is the leading cause of cancer-related deaths in the United States. With a 5-year survival rate at 19%, lung cancer patients are urgently in need of new therapeutic options. A major problem facing cancer therapy is the immune suppression by the tumor and its subsequent escape from the immune response, which is mediated by the tumor microenvironment. Tumors have large numbers of myeloid-derived suppressor cells (MDSCs), which suppress the adaptive immune response, increase neovascularization of the tumor, and promote tumor survival. Integrin heterodimer CD11b/CD18 (aka Mac-1 or αMβ2) is highly expressed on MDSCs and plays an important role in innate cell recruitment and other biologic functions. GB1275 (previously known as ADH503) is a novel small-molecule CD11b allosteric modulator. In experimental models of pancreatic cancer, GB1275 reduces MDSC infiltration and M2 macrophage polarization leading to tumor growth inhibition. GB1275 is currently in phase 1/2 clinical development in patients with pancreatic, esophageal, gastric/GEJ, triple-negative breast, castration-resistant prostate, or microsatellite stable colorectal cancer. To investigate the impact of CD11b modulation in the context of lung cancer, GB1275 treatment effects on tumor growth and immune infiltrates were assessed in the Lewis Lung Carcinoma (LLC) syngeneic tumor model, propagated in C57BL/6 wild-type (WT) mice. Additionally, we developed a transgenic CD11b knockin (KI) mouse that renders CD11b in a partially active state. CD11b KI mice were engineered to express partially active CD11b by introducing a point mutation at position 332 causing an isoleucine to glycine switch (I332G). We studied LLC tumor growth in CD11b KI mice as an orthogonal approach to test the mechanism of action. We will present data showing that pharmacologic modulation of CD11b with GB1275 significantly reduces LLC tumor growth. Concomitantly, we will present our studies with the CD11b KI mice that show a significant reduction in both the size and the rate of LLC tumor growth, as compared to the WT mice, mimicking treatment effects with GB1275. Our comprehensive pharmacologic and genetic studies show that CD11b activation reprograms MDSCs to enhance the adaptive immune response and is a novel strategy against lung cancer. This abstract is also being presented as Poster B71. Citation Format: Terese D. Geraghty, Anugraha Rajagopalan, Rabail Aslam, Vineet Gupta. Modulation of integrin CD11b as a novel therapeutic strategy against lung cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr PR4.