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The contribution of Fc-mediated effector functions to the therapeutic efficacy of some monoclonal antibodies has motivated efforts to enhance interactions with Fcγ receptors (FcγR). Although an early goal has been enhanced FcγRIIIa binding and natural killer (NK) cell antibody-dependent cell-mediated cytotoxicity (ADCC), other relevant cell types such as macrophages are dependent on additional activating receptors such as FcγRIIa. Here, we describe a set of engineered Fc variants with diverse FcγR affinities, including a novel substitution G236A that provides selectively enhanced binding to FcγRIIa relative to FcγRIIb. Variants containing this substitution have up to 70-fold greater FcγRIIa affinity and 15-fold improvement in FcγRIIa/FcγRIIb ratio and mediate enhanced phagocytosis of antibody-coated target cells by macrophages. Specific double and triple combination variants with this substitution are simultaneously capable of exhibiting high NK-mediated ADCC and high macrophage phagocytosis. In addition, we have used this unique set of variants to quantitatively probe the relative contributions of individual FcγR to effector functions mediated by NK cells and macrophages. These experiments show that FcγRIIa plays the most influential role for macrophages and, surprisingly, that the inhibitory receptor FcγRIIb has little effect on effector function. The enhancements in phagocytosis described here provide the potential to improve the performance of therapeutic antibodies targeting cancers. [Mol Cancer Ther 2008;7(8):2517–27]

Supplementary Fig. S1 from Optimization of antibody binding to FcγRIIa enhances macrophage phagocytosis of tumor cells

Supplementary Figure 2 from Potent In vitro and In vivo Activity of an Fc-Engineered Anti-CD19 Monoclonal Antibody against Lymphoma and Leukemia

Supplementary Figure 1 from Potent In vitro and In vivo Activity of an Fc-Engineered Anti-CD19 Monoclonal Antibody against Lymphoma and Leukemia

Supplementary Figure Legends 1-3 from Potent In vitro and In vivo Activity of an Fc-Engineered Anti-CD19 Monoclonal Antibody against Lymphoma and Leukemia

Exercise after breast cancer diagnosis and treatment improves cancer-related outcomes, although the mechanisms involved are not clear. This study evaluated the impact of exercise on body composition, strength, endurance, quality of life (QOL), fatigue, and endocrine and inflammatory biomarkers in breast cancer survivors participating in a highly monitored, clinically supervised, moderate-intensity exercise program. The association of hormonal and inflammatory biomarkers with the observed physiological changes was assessed. Female breast cancer survivors (BCS; n = 46) who engaged in a goal-oriented 14-week triathlon exercise training program were compared to an untrained control group of female BCS (n = 16). Psychosocial metrics, QOL, cancer-related fatigue, and exercise self-efficacy were evaluated via pre- and post-exercise intervention questionnaires. Serum estradiol and inflammatory biomarkers (C-reactive protein (CRP), sTNFR1a, estradiol, leptin, and adiponectin) were measured prior to the exercise training program start and after the completion of the goal triathlon. After exercise training, the exercise group had lower BMI and arm circumferences. Greater positive change was noted in the trained group for QOL, fatigue, and self-efficacy questionnaires. Functional endurance improved in the trained but not the control group. Knee and elbow strength were not different between the groups, except that knee flexion at 180 degrees∙sec−1 was higher in trained. The only significantly different biomarker was adiponectin, which decreased in the trained group. Group triathlon exercise training may be beneficial to BCS by significantly improving their psychosocial measures, functional endurance, and BMI.

Upregulation of CD47, the "don't eat me" signal, on the surface of tumors to evade immune surveillance is a common escape mechanism utilized during hematological malignancy and solid tumor development, progression, and relapse. We recently reported that AO-176, a clinical stage humanized anti-CD47 IgG2 antibody, possesses differentiated characteristics such as preferential binding of tumor cells compared to normal cells, negligible binding to red blood cells, non-ADCC direct tumor killing and elicits immunogenic cell death and DAMP induction, all in addition to single-agent phagocytosis. In vivo, AO-176 has exhibited broad anti-tumor activity in preclinical xenograft models of multiple myeloma (MM), acute myeloid leukemia, T cell acute lymphoblastic leukemia, and Burkitt lymphoma. In this study, the anti-tumor activity of AO-176 in an expanded set of preclinical models of B cell neoplasms was evaluated. We assessed In vivo anti-tumor activity in a diffuse large B cell lymphoma (DLBCL) preclinical xenograft model by inoculating Toledo cells into NSG mice and treating once weekly with either 25 mg/kg AO-176 or human IgG2 isotype control. Treatment with AO-176 resulted in profound tumor shrinkage, achieved complete responses in 8/10 mice, and extended survival for all treated mice through the 46 day dosing period, compared to all isotype control treated tumors reaching endpoint by day 21. Having previously observed significant tumor shrinkage and extension of survival in subcutaneous xenograft models of MM, we sought to evaluate anti-tumor activity of AO-176 in an orthotopic model of MM. Luciferase expressing RPMI-8226 cells were inoculated via intratibial injection into NOD-SCID mice and treated with either 25 mg/kg of AO-176 or human IgG2 isotype control once weekly. AO-176-treated mice showed significant reductions of bioluminescence on study days 7, 21, 35, and 41, and serum paraprotein at study end. Evaluation of bone lesions by x-ray showed significantly reduced average bone lysis score in the AO-176 treatment group at study day 41. We then compared the anti-tumor activity of AO-176 against the second generation proteosome inhibitor carfilzomib in a myeloma xenograft model. AO-176 dosed at 25 mg/kg once weekly achieved 72% TGI, compared to 47% and 27% TGI for tumors treated with 5 mg/kg and 2.5 mg/kg carfilzomib at day 29 post treatment. To elucidate the pathways and processes that may be underpinning the anti-tumor activity of AO-176, we performed bulk RNA sequencing on AO-176 or isotype control-treated tumors harvested at multiple time points from a MM xenograft model we previously reported as exhibiting profound sensitivity to AO-176 in vivo. Murine transcripts from harvested tumor RNA were evaluated to assess differences in immune infiltrate resulting from AO-176 treatment. Days 3 and 7 post treatment with AO-176 showed the greatest number of differentially expressed genes compared to control treated tumors. The top enriched pathway on day 3 was microglia phagocytosis. Principal component analysis of gene expression indicated partitioning of day 3 post AO-176 treatment from the rest of the groups. Furthermore, deconvolution of abundances of infiltrating immune cells using CIBERSORT via TIMER analytical tool showed an enrichment of macrophages relative to other cell types on day 3 post treatment. To extend our RNA sequencing findings, we then sought to evaluate intratumoral immune cell populations after AO-176 treatment in a subcutaneous MM xenograft model. MM cells were inoculated into NOD-SCID mice, then treated with 25 mg/kg AO-176 or human IgG2 isotype control. At 48 hours post treatment, tumors were harvested, and we observed an increase of macrophages in the AO-176 treated tumors, confirming our previous results. In summary, the robust preclinical data in DLBCL and MM warrants further development of AO-176 for treatment of hematological malignancies. AO-176 is being evaluated in phase 1/2 clinical trials for the treatment of patients with solid tumors (NCT03834948) and with MM (NCT04445701). Disclosures Wilson: Arch Oncology: Current Employment. Richards: Arch Oncology: Current Employment. Andrejeva: Arch Oncology: Current Employment. Capoccia: Arch Oncology: Current Employment. Puro: Arch Oncology: Current Employment. Donio: Arch Oncology: Current Employment. Hiebsch: Arch Oncology: Current Employment. Kashyap: Arch Oncology: Current Employment. Pereira: Arch Oncology: Current Employment.

Purpose Common reasons for hospitalization and death in patients with multiple myeloma (MM) are infections. As patients with MM are living longer and are treated with immunomodulatory drugs, there is a need to immunize against vaccine-preventable diseases and ultimately determine the efficacy of these vaccines. We evaluated vaccination practice patterns in MM patients at our health system using electronic medical records and data analytics. Methods This institutional review board-approved study retrospectively reviewed patients with MM who visited the health system from May 2012 to May 2014. Data collected included demographics, influenza vaccination (FV) and pneumonia vaccination (PV) history, hospitalization episodes and associated costs, and duration of survival. Patients were considered PV-positive if vaccinated within 5 years prior to study. FV was defined as optimal (two FV in 2012-2014), suboptimal (one FV in 2012-2014) or none (in 2012-2014). Results Of 411 MM patients, 55% were male and 85% Caucasian. Nearly 58% received PV in the past 5 years. FV was 15% optimal, 52% suboptimal and 33% none. A total of 444 hospitalizations involving 204 patients were observed over 2-year follow-up. More than $23 million was incurred from hospitalizations in the 2-year study period. There was no statistically significant difference in all-cause hospitalization and overall survival by FV and PV status. Conclusions Despite recommendations of vaccination in multiple myeloma, our cohort had low rates of influenza and pneumonia vaccination. FV and PV status did not show any significant association with additional hospitalization or overall survival in this pilot study. Future prospective studies are needed to ascertain the immunological and clinical efficacy and effectiveness of these vaccines in immunosuppressed patients.

Upregulation of tumor CD47, the "don't eat me" signal, to evade immune surveillance is a common escape mechanism that evolves during cancer development, progression, and relapse. Previous studies have shown multiple myeloma (MM) cells leverage this mechanism through broad upregulation of CD47 compared to non-malignant plasma cells, making CD47 an attractive therapeutic target for this disease. We recently reported that AO-176, a clinical stage humanized anti-CD47 IgG2 antibody, possesses differentiated characteristics such as preferential binding of tumor cells compared to normal cells, a lack of binding to red blood cells, non-ADCC direct tumor killing and elicits immunogenic cell death with DAMP induction, all in addition to single-agent phagocytosis. In this study, AO-176's anti-tumor activity in MM was evaluated. Immunohistochemical analyses of MM patient tumors with upregulated CD47 expression showed infiltration of innate immune cells such as macrophages and dendritic cells, both previously shown to be involved in anti-CD47 antibody mechanisms of action. AO-176 binding was confirmed on human cell lines frequently used in MM xenograft models. AO-176 exerted substantial single agent in vivo anti-tumor activity in multiple MM xenograft models when dosed at 25 mg/kg, including significant tumor growth inhibition of RPMI-8226 xenografted mice, and complete responses (CRs) in (10/10) NCI-H929 xenografted mice. These CRs were durable, with treated mice tumor-free up to 120 days post antibody dosing. Immunohistochemical analysis of AO-176 treated tumors from both models showed increased numbers of macrophages and dendritic cells compared to controls. An AO-176 dose response study resulted in CRs and increases in overall survival down to 10 mg/kg, with CRs observed as low as 3 mg/kg during dosing. In addition, we found that large NCI-H929 tumors (up to 1600mm3) showed pronounced regression after AO-176 treatment. The anti-tumor activity of AO-176 was also evaluated in combination with several standard of care MM therapies. When combined with the proteasome inhibitor bortezomib, AO-176 treatment at both 10 mg/kg and 25 mg/kg resulted in profound RPMI-8226 xenograft growth inhibition, near-total CRs (19/20 mice), and extended survival at both doses. Combining AO-176 and the anti-CD38 antibody daratumumab or immunomodulatory drugs (lenalidomide/pomalidomide) both produced significant enhancement of anti-tumor activity in xenograft models. The combined regimen of AO-176 with daratumumab led to significant MM.1S tumor growth inhibition compared to AO-176 or daratumumab alone. Both lenalidomide and pomalidomide combined with AO-176 resulted in significantly increased MM.1S tumor growth inhibition and extended survival compared to AO-176 alone, with an increased number of CRs observed in the combination groups compared to monotherapy groups. In summary, the pre-clinical potent single agent activity and enhanced activity when combined with standard of care anti-MM agents, warrants further development of AO-176 in MM treatment. AO-176 is being evaluated in phase 1 clinical trials for the treatment of patients with solid tumors (NCT03834948) and with MM (NCT04445701). Disclosures Wilson: Arch Oncology: Current Employment, Current equity holder in private company. Richards:Arch Oncology: Current Employment, Current equity holder in private company. Puro:Arch Oncology: Current Employment, Current equity holder in private company. Andrejeva:Arch Oncology: Current Employment, Current equity holder in private company. Capoccia:Arch Oncology: Current Employment, Current equity holder in private company. Donio:Arch Oncology: Current Employment, Current equity holder in private company. Hiebsch:Arch Oncology: Current Employment, Current equity holder in private company. Chakraborty:Arch Oncology: Current Employment, Current equity holder in private company. Sung:Arch Oncology: Current Employment, Current equity holder in private company. Pereira:Arch Oncology: Current Employment, Current equity holder in private company.

Multiple myeloma is a cancer of the immune system. Infection is a major cause of morbidity and mortality in patients with multiple myeloma. Some of these infections are preventable by vaccines available to the general population. However, little is known about the clinical effectiveness of these vaccines in patients with multiple myeloma, and the cellular and humoral immune response to vaccination has not been well characterized, especially in conjunction with modern myeloma therapies. The present report reviews the basics of multiple myeloma and the immune system, the available evidence on the immunologic response of patients with multiple myeloma after vaccination, and current practice recommendations regarding specific vaccines. Understanding the immune response to vaccines could help us understand how immuno-oncology-based therapies work in multiple myeloma and provide future directions for research.

Tumor-targeting antibodies have been successful in the treatment of various types of cancers. Antibodies engage the immune system with their Fc, stimulating immune cell effector function. In the clinic, FcγRIIIa polymorphisms with higher affinity for the Fc of antibodies were shown to improve response rates and overall survival. Efforts have been made to modify the Fc to enhance affinity to Fc receptors and thereby improve effector function. An alternative for improving immune effector function may be to increase the level of tumor antigen expression. In this study, tamoxifen was used to increase HER2/neu protein level to determine whether increased tumor antigen expression could enhance NK cell-mediated antibody-dependent cytotoxicity (ADCC). Tamoxifen was found to increase HER2/neu 1.5-fold to threefold in breast cancer cell lines that were HER2/neu non-amplified. Using flow cytometry to simultaneously evaluate NK cell degranulation and tumor cell death, the increase in HER2/neu enhanced NK cell-mediated ADCC. However, in cells that had HER2/neu gene amplification and estrogen receptor expression, tamoxifen elevated HER2/neu but failed to improve NK cell function. The quantity of HER2/neu on the tumor cell surface was approximately double that of the number of Fc receptors found on NK cells. This appears to reflect a ceiling at which increasing antigen expression fails to improve NK cell effector function. This has clinical implications as trying to increase antigen expression to enhance NK cell function may be useful for patients with antigen-low tumors, but not in those whose tumors have gene amplification or high levels of antigen expression.

The effector functions elicited by the fragment crystallizable (Fc) region of immunoglobulin G (IgG) antibodies are subject to variation by the presence of terminal sialic acid (Sia) residues at asparagine-297 (Asn-297). We have previously shown that the sialic acid-containing (Sia+) fraction of intravenous immune globulin (IVIG) influences cell surface marker expression and cytokine/ chemokine secretion during the differentiation and maturation of human dendritic cells (DC). The present study examined the effects of Sia+ IgG on human peripheral blood mononuclear cell (PBMC)-derived monocyte and macrophage surface marker expression and cytokine/chemokine secretion. Sia+ IgG induced increased expression of CD80 and dendritic cell immunoreceptor (DCIR) on monocytes, whereas the expression of HLA-DR was decreased. In addition, the production of IL-6, TNFα, IL-1β, and CXCL1 by monocytes was profoundly increased by treatment with Sia+ IgG. Sia+ IgG also increased the expression of cell surface markers associated with macrophage polarization (e.g. CD40 and CD206) on monocytes. In macrophage-colony stimulating factor (MCSF) generated macrophages, Sia+ IgG induced increased production of numerous cytokines/ chemokines including IL-6, TNFα, CXCL1, and IL-10, and the expression of the macrophage surface marker CD163. Our data extended prior observations of Sia+ IgG on DC function and showed that Sia+ IgG was able to differentially modulate multiple pathways in monocytes and macrophages. Our data indicate that the Sia+ fraction of IVIG possesses the ability to influence inflammatory processes in multiple immune cell types and induces novel signatures in cell surface marker expression and cytokine/chemokine production.

The monocyte network is important for therapeutic efficacy of antibody therapies against cancer. One mechanism which monocytes/macrophages use to kill cancer cells is phagocytosis. Using trastuzumab and human breast cancer cell lines as a model, we used flow cytometry to evaluate the importance of avidity, antigen density, Fcγ receptor (FcγR) expression, and FcγR polymorphisms in human monocyte phagocytosis. By increasing avidity for the tumor through the addition of pertuzumab to trastuzumab, there was a two-to-threefold increase in phagocytosis potency against the HCC1419 cell line compared to antibodies alone, while NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) failed to increase tumor cell death. Consistent with increasing the avidity through multiple antibodies, antigen density significantly enhanced phagocytosis with breast cancer cell lines that were HER2 gene-amplified compared to non-amplified tumor cells. Confirmation that high antigen density enhanced phagocytosis was obtained when HER2 was overexpressed in HER2 non-amplified cell lines. In contrast, NK cell ADCC failed to distinguish differences in tumor cell death when comparing gene-amplified and non-amplified breast cancer cell lines. The level of phagocytosis was influenced by FcγRIIa and FcγRIIIa expression. Most monocytes are FcγRIIIa−, and the induction of the receptor significantly enhances antibody-dependent phagocytosis. Although both receptors are involved, when blocked FcγRIIIa had a greater influence on phagocytosis. Furthermore, the polymorphism FcγRIIIa 158V significantly enhanced phagocytosis; whereas FcγRIIa 131H polymorphism appeared to improve phagocytosis but was not statistically significant. Targeting of monocytes for enhanced phagocytosis may improve the effectiveness of therapeutic antibodies to improve clinical outcomes.

Purpose: We tested the hypothesis that different glycoforms of antibodies from patients with metastatic melanoma have different functional effects on human dendritic cell differentiation and maturation. Methods: Antibodies to the cancer antigen NY-ESO-1 were affinity-purified from patients with melanoma and further fractionated into different glycoforms by lectin chromatography. Sialic acid-rich and sialic acid-poor fractions of these immunoglobulin G antibodies (IgG) were added to dendritic cell cultures during both differentiation and maturation, and the resulting cellular messenger RNA (mRNA) and culture supernatants were tested by microarray and enzyme-linked immunoassay for molecules related to inflammatory pathways. Results: We identified unique mRNA and secreted protein signatures that were induced by different glycoforms of IgG during dendritic cell differentiation and maturation. Among the variety of mRNA and proteins induced by the sialic acidrich IgG fraction, we found a dramatic increase in levels of the melanoma growth factor CXCL1. Conclusions: Our findings support the concept that alternate glycoforms of IgG induce differing functional programs of dendritic cell differentiation and maturation. The data also support the concept that the functional phenotype of IgG is related to glycosylation. Thus, subtle changes in glycan structure can change the effector function of IgG from an inflammatory to an anti-inflammatory program. This work highlights the importance of the interface between tumors and the immune system, revealing a potential explanation as to why tumors persist and progress despite potent immune responses against them. (J Patient-Centered Res Rev. 2014;1:171-187.)

AO-176 is a highly differentiated, humanized anti-CD47 IgG2 antibody that is unique among agents in this class of checkpoint inhibitors. AO-176 works by blocking the "don't eat me" signal, the standard mechanism of anti-CD47 antibodies, but also by directly killing tumor cells. Importantly, AO-176 binds preferentially to tumor cells, compared to normal cells, and binds even more potently to tumors in their acidic microenvironment (low pH). Hematological neoplasms are the fourth most frequently diagnosed cancers in both men and women and account for approximately 10% of all cancers. Here we describe AO-176, a highly differentiated anti-CD47 antibody that potently targets hematologic cancers in vitro and in vivo. As a single agent, AO-176 not only promotes phagocytosis (15-45%, EC50 = 0.33-4.1 µg/ml) of hematologic tumor cell lines (acute myeloid leukemia, non-Hodgkin's lymphoma, multiple myeloma, and T cell leukemia) but also directly targets and kills tumor cells (18-46% Annexin V positivity, EC50 = 0.63-10 µg/ml) in a non-ADCC manner. In combination with agents targeting CD20 (rituximab) or CD38 (daratumumab), AO-176 mediates enhanced phagocytosis of lymphoma and multiple myeloma cell lines, respectively. In vivo, AO-176 mediates potent monotherapy tumor growth inhibition of hematologic tumors including Raji B cell lymphoma and RPMI-8226 multiple myeloma xenograft models in a dose-dependent manner. Concomitant with tumor growth inhibition, immune cell infiltrates were observed with elevated numbers of macrophage and dendritic cells, along with increased pro-inflammatory cytokine levels in AO-176 treated animals. When combined with bortezomib, AO-176 was able to elicit complete tumor regression (100% CR in 10/10 animals treated with either 10 or 25 mg/kg AO-176 + 1 mg/kg bortezomib) with no detectable tumor out to 100 days at study termination. Overall survival was also greatly improved following combination therapy compared to animals treated with bortezomib or AO-176 alone. These data show that AO-176 exhibits promising monotherapy and combination therapy activity, both in vitro and in vivo, against hematologic cancers. These findings also add to the previously reported anti-tumor efficacy exhibited by AO-176 in solid tumor xenografts representing ovarian, gastric and breast cancer. With AO-176's highly differentiated MOA and binding characteristics, it may have the potential to improve upon the safety and efficacy profiles relative to other agents in this class. AO-176 is currently being evaluated in a Phase 1 clinical trial (NCT03834948) for the treatment of patients with select solid tumors. Disclosures Richards: Arch Oncology Inc.: Employment, Equity Ownership, Other: Salary. Bouchlaka:Arch Oncology Inc.: Consultancy, Equity Ownership. Puro:Arch Oncology Inc.: Employment, Equity Ownership. Capoccia:Arch Oncology Inc.: Employment, Equity Ownership. Hiebsch:Arch Oncology Inc.: Employment, Equity Ownership. Donio:Arch Oncology Inc.: Employment, Equity Ownership. Wilson:Arch Oncology Inc.: Employment, Equity Ownership. Chakraborty:Arch Oncology Inc.: Employment, Equity Ownership. Sung:Arch Oncology Inc.: Employment, Equity Ownership. Pereira:Arch Oncology Inc.: Employment, Equity Ownership.

Tumor-targeting antibodies have been successful in the treatment of various types of cancers. Antibodies engage the immune system with their Fc, stimulating immune cell effector function. In the clinic, FcγRIIIa polymorphisms with higher affinity for the Fc of antibodies were shown to improve response rates and overall survival. Efforts have been made to modify the Fc to enhance affinity to Fc receptors and thereby improve effector function. An alternative for improving immune effector function may be to increase the level of tumor antigen expression. In this study, tamoxifen was used to increase HER2/neu protein level to determine whether increased tumor antigen expression could enhance NK cell-mediated antibody-dependent cytotoxicity (ADCC). Tamoxifen was found to increase HER2/neu 1.5-fold to threefold in breast cancer cell lines that were HER2/neu non-amplified. Using flow cytometry to simultaneously evaluate NK cell degranulation and tumor cell death, the increase in HER2/neu enhanced NK cell-mediated ADCC. However, in cells that had HER2/neu gene amplification and estrogen receptor expression, tamoxifen elevated HER2/neu but failed to improve NK cell function. The quantity of HER2/neu on the tumor cell surface was approximately double that of the number of Fc receptors found on NK cells. This appears to reflect a ceiling at which increasing antigen expression fails to improve NK cell effector function. This has clinical implications as trying to increase antigen expression to enhance NK cell function may be useful for patients with antigen-low tumors, but not in those whose tumors have gene amplification or high levels of antigen expression.

CD40 is highly expressed on various B-lineage malignancies and represents an attractive immunotherapy target for neoplastic disease. Previous work showed that engineering the Fc domain of an antibody for increased binding to Fcγ receptors (FcγRs) significantly enhanced Fc-mediated immune effector function and antitumor activity in vitro and in vivo. We developed a humanized anti-CD40 antibody similarly Fc-engineered for increased FcγR binding (XmAbCD40) and compared its efficacy with that of an anti-CD40 native IgG1 analog and the anti-CD20 antibody rituximab. XmAbCD40 increased antibody-dependent cell-mediated cytotoxicity (ADCC) up to 150-fold relative to anti-CD40 IgG1 against B-lymphoma, leukemia, and multiple myeloma cell lines, and significantly enhanced ADCC against primary tumors. XmAbCD40 was also superior to rituximab in enhancing ADCC (both in cell lines and primary tumors) and in augmenting antibody-dependent cellular phagocytosis. XmAbCD40 significantly inhibited lymphoma growth in disseminated and established mouse xenografts and was more effective than the IgG1 analog or rituximab. An anti-CD40 antibody constructed to abrogate FcγR binding showed no reduction of tumor growth, indicating that the in vivo antitumor activity of XmAbCD40 is primarily mediated via FcγR-dependent mechanisms. These data demonstrate that XmAbCD40 displays potent antitumor efficacy and merits further evaluation for the treatment of CD40+ malignancies.

CD19 is a pan B-cell surface receptor expressed from pro–B-cell development until its down-regulation during terminal differentiation into plasma cells. CD19 represents an attractive immunotherapy target for cancers of lymphoid origin due to its high expression levels on the vast majority of non–Hodgkin's lymphomas and some leukemias. A humanized anti-CD19 antibody with an engineered Fc domain (XmAb5574) was generated to increase binding to Fcγ receptors on immune cells and thus increase Fc-mediated effector functions. In vitro, XmAb5574 enhanced antibody-dependent cell-mediated cytotoxicity 100-fold to 1,000-fold relative to an anti-CD19 IgG1 analogue against a broad range of B-lymphoma and leukemia cell lines. Furthermore, XmAb5574 conferred antibody-dependent cell-mediated cytotoxicity against patient-derived acute lymphoblastic leukemia and mantle cell lymphoma cells, whereas the IgG1 analogue was inactive. XmAb5574 also increased antibody-dependent cellular phagocytosis and apoptosis. In vivo, XmAb5574 significantly inhibited lymphoma growth in prophylactic and established mouse xenograft models, and showed more potent antitumor activity than its IgG1 analogue. Comparisons with a variant incapable of Fcγ receptor binding showed that engagement of these receptors is critical for optimal antitumor efficacy. These results suggest that XmAb5574 exhibits potent tumor cytotoxicity via direct and indirect effector functions and thus warrants clinical evaluation as an immunotherapeutic for CD19+ hematologic malignancies. [Cancer Res 2008;68(19):8049–57]

Objectivs Cytokine-dependent activation of fibroblasts to myofibroblasts, a key event in fibrosis, is accompanied by phenotypic changes with increased secretory and contractile properties dependent on increased energy utilization, yet changes in the energetic profile of these cells are not fully described. We hypothesize that the TGF-β1-mediated transformation of myofibroblasts is associated with an increase in mitochondrial content and function when compared to naive fibroblasts. Methods Cultured NIH/3T3 mouse fibroblasts treated with TGF-β1, a profibrotic cytokine, or vehicle were assessed for transformation to myofibroblasts (appearance of α-smooth muscle actin [α-SMA] stress fibers) and associated changes in mitochondrial content and functions using laser confocal microscopy, Seahorse respirometry, multi-well plate reader and biochemical protocols. Expression of mitochondrial-specific proteins was determined using western blotting, and the mitochondrial DNA quantified using Mitochondrial DNA isolation kit. Results Treatment with TGF-β1 (5 ng/mL) induced transformation of naive fibroblasts into myofibroblasts with a threefold increase in the expression of α-SMA (6.85 ± 0.27 RU) compared to cells not treated with TGF-β1 (2.52 ± 0.11 RU). TGF-β1 exposure increased the number of mitochondria in the cells, as monitored by membrane potential sensitive dye tetramethylrhodamine, and expression of mitochondria-specific proteins; voltage-dependent anion channels (0.54 ± 0.05 vs. 0.23 ± 0.05 RU) and adenine nucleotide transporter (0.61 ± 0.11 vs. 0.22 ± 0.05 RU), as well as mitochondrial DNA content (530 ± 12 μg DNA/106 cells vs. 307 ± 9 μg DNA/106 cells in control). TGF-β1 treatment was associated with an increase in mitochondrial function with a twofold increase in baseline oxygen consumption rate (2.25 ± 0.03 vs. 1.13 ± 0.1 nmol O2/min/106 cells) and FCCP-induced mitochondrial respiration (2.87 ± 0.03 vs. 1.46 ± 0.15 nmol O2/min/106 cells). Conclusions TGF-β1 induced differentiation of fibroblasts is accompanied by energetic remodeling of myofibroblasts with an increase in mitochondrial respiration and mitochondrial content.

Coccidioidomycosis, an infection endemic to the southwestern United States, is caused by the fungus Coccidioides immitis. Coccidioidal infection is overcome by the development of cell-mediated immunity. This study evaluated the role of dendritic cells (DCs) in the initiation of coccidioidal immunity in nonimmune individuals. It was demonstrated that DCs pulsed with the coccidioidal antigen preparation, toluene spherule lysate (TSL), induce DC maturation, autologous lymphocyte proliferation, and antigen-specific lymphocyte responses from nonimmune donors. Furthermore, TSL-primed lymphocytes secreted interferon-g after restimulation with TSL or antigen 2/proline-rich antigen, a subcomponent of TSL, but they did not do so when restimulated with ovalbumin or unpulsed DCs. The results demonstrate that DCs generated from individuals not exposed to C. immitis can specifically prime lymphocytes for coccidioidal antigens and that the response generated by the lymphocytes is characteristic of a cellular immune response. Coccidioides immitis is a pathogenic fungus that is endemic to the southwestern United States and is the causative agent of coccidioidomycosis, commonly known as valley fever [1]. The fungus infects ∼100,000 individuals each year [1]. Although most persons who become infected are asymptomatic or have mild influenza-like symptoms, 1%‐10% of those infected develop either chronic pulmonary disease or infection that disseminates beyond the thoracic cavity [2]. Previous studies have shown that individuals who overcome coccidioidal infection develop cell-mediated immunity against the fungus, as measured by lymphocyte proliferation and secretion of interferon (IFN)‐g from peripheral blood mononuclear cells (PBMC) cultured with coccidioidal antigens [3]. PBMC from nonimmune individuals or patients with disseminated coccidioidomycosis do not proliferate or secrete IFN-g. Although the general immunology of coccidioidomycosis is known, little is understood about the initiation of the immune response against this fungus.

Background Multiple myeloma (MM) is a cancer of the immune system. Infection is a major cause of morbidity and mortality in MM. Vaccinations (vax) are recommended in MM, but clinical efficacy endpoints have not been demonstrated and surrogate markers of efficacy have limited data. This pilot study evaluated sequential immunologic markers after standard vax for pneumococcal pneumonia (PV) and influenza (FV). Methods MM patients and non-MM control (Ctrl) participants signed informed consent to this IRB approved study. The MM patients were not selected for treatment or disease status. Vax was standard PV (PCV13 or PPV23) and/or FV (IIV4) with research laboratory testing at baseline and at 2, 4, 12, 24 weeks after vax. If PV, then PCV13 preferred followed by PPV23 suggested at >= 26 weeks. IgG antibodies (Ab) to influenza and pneumococcal antigens were detected by ELISA. Measurement of Ab to influenza was performed as previously described (Oaks et al. 2013OncoImmunology) and used Flulaval Quadrivalent 2015/2016 form (GlaxoSmithKline) as the coating antigen. Anti-pneumococcal IgG levels were determined with The Binding Site VaccZymeAnti-PCP IgG EIA Kit (#MK012-U) according to the manufacturer's instructions. Results Ten MM (5 M, 5 F) and 9 Ctrl (4 M, 5 F) were enrolled. MM median age was 69.8y (range: 59-78) and Ctrl was 63.5y (range: 56-73). Over the total time course 5/10 MM and 4/9 Ctrl had no missing samples. Vax included: 5 (2 MM, 3 Ctrl) FV only; 10 (6 MM, 4 Ctrl) PV (8 PCV13, 2 PPV23) only; and 4 (2 MM, 2 Ctrl) both FV and PV (3 PCV13, 1 PPV23). Tetanus Ab response was unchanged, which served as a negative control. FV results included 2/4 MM showed at least a doubling of anti-flu titer, although the other 2 showed no measurable response. All 5/5 Ctrl had at least a 2-fold increase in anti-flu titers at some point. PV humoral response varied considerably for both MM and Ctrl. All 4 Ctrl responded with at least a 2-fold increase in Ab titer, only 2 Ctrl had a sustained increase in titer at the closing visit. Six of 10 MM had at least a 2-fold Ab increase at some point during the course of the study. Only 2 MM patients showed a sustained increase of anti-PV Ab. Response rate differences were not statistically significant and there was no relationship between responsiveness to FV or PV and initial serum IgG concentration at entry into the study (data not shown). Discussion Study limitations include small sample size, sample dropouts, and heterogeneous population. We are unaware of prior MM vax immunology response time courses. This may be because of the difficulties including PV studies with PCV13->PPV23 recommendations and the potential long follow up. FV response may be more amenable to short term follow up. In 2013 Hahn and colleagues showed proof of principle with "Boost vax improves influenza humoral immune response in MM" (ow.ly/sEBEO), but without a time course or clinical outcomes data. The 2015Branagan et al. (http://ow.ly/W4tLS) pilot study showed a 2 dose FV strategy had lower flu infections compared to historical controls. Karlsson et al. (http://ow.ly/YUumy) showed that Ab response does not correlate with functional assays. We found no relationship with total IgG level. This is consistent with Beers et al. findings that IgG isotypes may be of more importance than the total IgG level. (http://ow.ly/Z5g2A) Conclusion We found that FV and PV Ab response was low and not sustained in most MM patients. Infection in MM remains an important concern. Future vax studies should address the following: 1) Homogeneous population - eg therapy trial or autologous stem cell transplant with a standardized re-vax and sequential testing, 2) validate relevant immunologic surrogates - eg Ab response twice baseline, 4) fresh assay vs frozen (batched), 5) correlation of metric with functional outcomes such as hospitalization, cost, and death, 6) and epidemiologic data may complement translational approaches. Funding Funding was provided by a Vince Lombardi Cancer Foundation Aurora Cancer Research Award. Responses to FV Table Table. Disclosures Thompson: Celgene: Membership on an entity's Board of Directors or advisory committees, Other: MDS/AML Registry; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; AIM Specialty Health: Membership on an entity's Board of Directors or advisory committees; VIA Oncology: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees, Other: Multiple Myeloma International Registry; Doximity: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Adoptive transfer of tumor-infiltrating lymphocytes (TIL) is in development for the treatment of metastatic melanoma. In phase II clinical trials, patients with metastatic melanoma that received TIL after preconditioning had a 50-70% clinical response rate. The current approach to generate TIL is to culture melanoma enzyme digests in the presence of IL-2 for a 10- to 20-day period followed by 2 weeks of rapid expansion (REP). Prior to administration, cell therapies are characterized and tested for purity. TIL are characterized by CD3 surface marker expression, and purity is assessed by the amount of tumor remaining in culture. Evaluating TIL purity has traditionally been done by immunohistochemistry, which is often considered semiquantitative. To generate a quantitative assay, we used multiparameter flow cytometry to evaluate the presence of viable tumor cells by staining TIL populations with a viability dye and an antibody cocktail that detects intracellular tumor-antigens gp100, Mart-1, tyrosinase, S100, and surface tumor-antigen melanoma chondroitin sulfate proteoglycan (MCSP), and CD3 on T cells. Tumors were identified by gating on the viable CD3(-) population. Antigens in tumors were initially optimized with individual antibodies using both immunohistochemistry and flow cytometry. When eight different tumor cell lines were spiked into an activated T cell culture, flow cytometry was able to distinguish lymphocytes from tumors in all samples tested. Most importantly, the assay was able to detect melanoma cells in all enzyme digests (9/9) from patient samples. After IL-2-induced TIL expansion, there was a significant decrease in tumor cells; tumor cells were detected in only 2 of 12 samples. In eight IL-2-induced TIL samples that were further expanded in REP, no tumor cells were detected. We have demonstrated that flow cytometry is an alternative to immunohistochemistry for defining the purity of a TIL population.

The contribution of Fc-mediated effector functions to the therapeutic efficacy of some monoclonal antibodies has motivated efforts to enhance interactions with Fcγ receptors (FcγR). Although an early goal has been enhanced FcγRIIIa binding and natural killer (NK) cell antibody-dependent cell-mediated cytotoxicity (ADCC), other relevant cell types such as macrophages are dependent on additional activating receptors such as FcγRIIa. Here, we describe a set of engineered Fc variants with diverse FcγR affinities, including a novel substitution G236A that provides selectively enhanced binding to FcγRIIa relative to FcγRIIb. Variants containing this substitution have up to 70-fold greater FcγRIIa affinity and 15-fold improvement in FcγRIIa/FcγRIIb ratio and mediate enhanced phagocytosis of antibody-coated target cells by macrophages. Specific double and triple combination variants with this substitution are simultaneously capable of exhibiting high NK-mediated ADCC and high macrophage phagocytosis. In addition, we have used this unique set of variants to quantitatively probe the relative contributions of individual FcγR to effector functions mediated by NK cells and macrophages. These experiments show that FcγRIIa plays the most influential role for macrophages and, surprisingly, that the inhibitory receptor FcγRIIb has little effect on effector function. The enhancements in phagocytosis described here provide the potential to improve the performance of therapeutic antibodies targeting cancers. [Mol Cancer Ther 2008;7(8):2517–27]

Cytokine-induced killer (CIK) cells are ex vivo, expanded T cells with proven anticancer activity in vitro and in vivo. However, their functional properties with the exception of their cancer cell-killing activity are largely unclear. Here, we show that CIK T cells recognize dendritic cells (DC), and although mature DC (mDC) induce CIK T cells to produce IFN-γ, immature DC (iDC) are killed selectively by them. Moreover, CIK T cell activation by mDC and their destruction of iDC are independent of the TCR. The cytotoxicity of CIK T cells to iDC is perforin-dependent. Our data have revealed an important regulatory role of CIK cells.

Natural-killer (NK)-cell dysfunction and IFN-γ deficiencies have been associated with increased incidence of both malignancy and infection. The immunologic basis of NK-cell defects in cancer-bearing hosts has not been extensively studied. Here, we demonstrate that multiple lineages of tumors, including thymoma, breast cancer, colon cancer, and melanoma cell lines, interrupt functional maturation during NK-cell development in the bone marrow. The immature NK cells in the periphery of tumor-bearing mice had impaired IFN-γ production but seemingly normal cytotoxicity. T cells are not involved in this NK maturation arrest, because T-cell depletion did not restore NK-cell development. Moreover, the extent of tumor-cell infiltration into the bone marrow does not correlate with defective NK maturation. Interestingly, the defect was associated with a significant reduction in the IL-15Rα+ cells in the non-T, non-NK compartment of bone marrow cells and restored by overexpression of IL-15. Our data demonstrate that tumor growth can impede functional maturation of NK cells, most likely by interrupting the requisite IL-15 signaling pathway. (Blood. 2006;108:246-252)

Coccidioides immitis is a pathogenic, dimorphic fungus found in the southwestern United States and is the causative agent of coccidioidomycosis. Extrathoracic dissemination of coccidioidomycosis is associated with a lack of cellular immunity. Dendritic cells (DCs) have been shown to initiate and modulate cellular immune responses. To determine whether DCs could modulate or initiate the immune response in this disease, monocyte-derived DCs were generated from coccidioidal Ag nonresponsive patients with disseminated coccidioidomycosis and healthy nonimmune individuals. DCs generated from both groups demonstrated phenotypes characteristic of DCs and stimulated strong allogeneic MLR. DCs from patients and healthy nonimmune individuals pulsed with the coccidioidal Ag preparation T27K induced lymphocyte proliferation. Mature DCs were much more efficient than immature DCs in these stimulations. Furthermore, restimulation of T27K-primed PBMC with Ag-pulsed DCs generated a C. immitis-specific cellular immune response in PBMC from patients with disseminated coccidioidomycosis as well as healthy nonimmune individuals. These results show that 1) DCs have the capacity to stimulate specific cellular immune responses from patients with disseminated coccidioidomycosis who are nonresponsive to coccidioidal Ag and healthy nonimmune individuals in vitro; 2) DCs can be used to screen coccidioidal Ags as candidates for human vaccine development; and 3) DC therapy may be useful in the treatment of disseminated coccidioidomycosis.

Background We have a poor understanding of the vaccination immune response and outcomes in multiple myeloma (MM). As MM patients (Pts) are living longer and therapies are immunomodulatory there is an unmet medical need to further characterize the role of the immune system. A common reason for hospitalization or death in MM Pts is infection. As an initial step in MM Cancer Care Delivery Research (CCDR), we evaluated the current vaccination practice patterns in MM Pts at Aurora Health Care using the EMR and data analytics. Methods An IRB approved study reviewed MM Pts from 5/15/2012 to 5/15/2014. Data collected included demographics, influenza (FV) and pneumonia vaccination (PV) history, hospitalization episodes, cost associated with hospitalization, and admission and discharge diagnoses. Pts were considered PV positive if vaccinated within 5 years prior to study with any PV type. FV was none (no FV in 2012-2014), optimal [FV in 2012 and (2013 or 2014)], or suboptimal [FV in 2012 or (2013 or 2014)]. Data was analyzed using SAS and STATA 12. Results A total of 1131 MM Pts were identified. Race included 70% white, 13% black, and 17% mixed, other or information not available. MM median age at diagnosis was 71 and only 4% (47) had prior autologous stem cell transplantation. PV rate was 30%. FV was 55% none, 24% suboptimal and 20% optimal. There was no statistically significant difference in the rate of PV and FV when stratified vs age, gender, and race. Over two years there were a total of 662 hospitalization events involving 317 MM Pts. The total cost of hospitalization was approx $35M. The average charge per hospitalized patient was $110K (range: $2K -1.3M) with an average $52K per hospitalization encounter (range: 2K – 648K). The rate of PV and FV vaccination among Pts with index hospitalization is significantly higher than non-hospitalized patients. There was no difference in hospitalization cost based on vaccination status. (See Table 1) Discussion Vaccination rates were low and did not correlate with hospital outcomes. This may be explained as a limitation for a retrospective EMR analysis without accounting for temporal relationship of vaccines – i.e. possible vaccination after admission. Alternatively, this may indicate that our current methods of vaccination in MM are not effective. Other limitations include need for a more granular review of treatment regimens and infectious complications. Additional surrogate markers are needed to understand the effect of vaccines and the immune system on health care outcomes such as hospitalizations, cost, and survival. This will be addressed in prospective registries and immunologic studies at our center and may be queried at other health systems. | Vaccination Status | % | Hospitalization Events, % | Hospitalization Charge, $ | | ------------------ | --- | ------------------------- | ------------------------- | | PV – No | 70% | 20% | $16M | | PV – Yes | 30% | 52% | $18M | | | | | | | FV – None | 55% | 16% | $9M | | FV – Suboptimal | 24% | 42% | $13M | | FV - Optimal | 20% | 43% | $12M | Table 1 – Vaccination Status and Hospitalizations Disclosures No relevant conflicts of interest to declare.

CD40, a transmembrane glycoprotein belonging to the tumor necrosis factor receptor family, is an attractive target for cancers of lymphoid origin since it is expressed on most mature B-cell malignancies, some early B-cell acute lymphocytic leukemias, and multiple myeloma. Finding efficient therapies for multiple myeloma (MM), chronic lymphocytic leukemia (CLL) and rituximab-refractory Non-Hodgkin Lymphoma (NHL) represents an unmet need. Several anti-CD40 antibodies, both agonistic and antagonistic, have demonstrated objective responses in early clinical NHL trials and thus validated this antigen as a target for lymphoproliferative diseases. Here we present the characterization of a novel Fc-engineered and humanized anti-CD40 antibody, XmAb®5485, that was generated using our XmAb antibody engineering technology. This antibody is highly cytotoxic against lymphoma, leukemia and multiple myeloma cell lines as well as primary cancer cells. XmAb5485 is characterized by: i) increased affinity for Fc gamma receptors (FcgR), ii) improved effector function, and iii) significantly increased antitumor potency. We investigated several direct and indirect (Fc-mediated) mechanisms of antibody-mediated cytotoxicity in vitro. The potency (EC50) of XmAb5485 in antibody-dependent cell-mediated cytotoxicity (ADCC) increased up to 150-fold relative to the native non Fc-engineered version (anti-CD40 IgG1) of the antibody in a screen of Burkitt’s lymphoma [BL], CLL and MM-derived cell lines. In the same cell lines, ADCC potency and maximal efficacy (% lysis) of XmAb5485 were also superior to that of rituximab: 74- and 1.3-fold higher in CLL, 12.5- and 1.4-fold higher in BL, and 190- and 1.9-fold higher in MM. In a MM cell line with low density of CD40 expression (~3500 per cell) XmAb5485 facilitated efficient ADCC whereas anti-CD40 IgG1 was virtually ineffective. Furthermore, using a BL cell line (Ramos) XmAb5485 displayed antibody-dependent cellular phagocytosis (ADCP) with potency and efficacy increased relative to rituximab (15- and 1.6-fold) and anti-CD40 IgG1 (5- and 1.2-fold). XmAb5485 also exhibited anti-proliferative apoptotic activity that was similar to that of rituximab. Ex vivo, XmAb5485 mediated potent ADCC of multiple primary patient-derived CLL, MCL, and plasma cell leukemia (PCL, an aggressive form of MM) cells, with substantially increased potency and efficacy relative to rituximab; in contrast, anti-CD40 IgG1 displayed minimal or no activity in these primary tumor cells. In vivo, in an established large (210–350 mm3) sc Ramos tumor xenograft model, 6 mg/kg XmAb5485 cured 80% of mice of detectable tumors and displayed statistically significant superiority over anti-CD40 IgG1. In contrast, only 7% of animals in the rituximab cohort were cured. In summary, our data suggest that XmAb5485, an anti-CD40 Fc variant antibody engineered for increased effector function, is a promising next-generation immunotherapeutic for leukemias, lymphomas, and multiple myeloma.