Vaccination with CD47 deficient tumor cells elicits an antitumor immune response in mice.

Cancer cells are poorly immunogenic and have a wide range of mutations, which makes them unsuitable for use in vaccination treatment. Here, we show that elimination of CD47, a ligand for the myeloid cell inhibitory receptor SIRPα, from tumor cells by genetic deletion or antibody blocking, significantly improves the effectiveness of the immune response to tumour cells. In both solid and hematopoietic mouse tumor models, vaccination with tumor cells or tumor antigen-expressing cells, that lack CD47 or were pre-coated with anti-CD47 antibodies, achieved an antitumor immune response. The efficacy of this approach was synergistically enhanced when used in combination with anti-PD-1 antibodies. The induction of antitumor responses depends on SIRPα+CD11c+ DCs, which exhibit rapid expansion following introduction of CD47-deficient tumor cells. Our results indicate that CD47-deficient whole tumor cells can induce antitumor responses.

Cancer immunotherapy targeting the CD47/SIRPα axis.

The success of cancer immunotherapy has generated tremendous interest in identifying new immunotherapeutic targets. To date, the majority of therapies have focussed on stimulating the adaptive immune system to attack cancer, including agents targeting CTLA-4 and the PD-1/PD-L1 axis. However, macrophages and other myeloid immune cells offer much promise as effectors of cancer immunotherapy. The CD47/signal regulatory protein alpha (SIRPα) axis is a critical regulator of myeloid cell activation and serves a broader role as a myeloid-specific immune checkpoint. CD47 is highly expressed on many different types of cancer, and it transduces inhibitory signals through SIRPα on macrophages and other myeloid cells. In a diverse range of preclinical models, therapies that block the CD47/SIRPα axis stimulate phagocytosis of cancer cells in vitro and anti-tumour immune responses in vivo. A number of therapeutics that target the CD47/SIRPα axis are under preclinical and clinical investigation. These include anti-CD47 antibodies, engineered receptor decoys, anti-SIRPα antibodies and bispecific agents. These therapeutics differ in their pharmacodynamic, pharmacokinetic and toxicological properties. Clinical trials are underway for both solid and haematologic malignancies using anti-CD47 antibodies and recombinant SIRPα proteins. Since the CD47/SIRPα axis also limits the efficacy of tumour-opsonising antibodies, additional trials will examine their potential synergy with agents such as rituximab, cetuximab and trastuzumab. Phagocytosis in response to CD47/SIRPα-blocking agents results in antigen uptake and presentation, thereby linking the innate and adaptive immune systems. CD47/SIRPα blocking therapies may therefore synergise with immune checkpoint inhibitors that target the adaptive immune system. As a critical regulator of macrophage phagocytosis and activation, the potential applications of CD47/SIRPα blocking therapies extend beyond human cancer. They may be useful for the treatment of infectious disease, conditioning for stem cell transplant, and many other clinical indications.

Induction of tumor regression by intratumoral STING agonists combined with anti-programmed death-L1 blocking antibody in a preclinical squamous cell carcinoma model.

BACKGROUND: Cyclic dinucleotides (CDNs) are bacterial intracellular messengers that have demonstrated antitumor activity in melanoma and breast tumors, although their role in immunotherapy of head and neck squamous cell cancers (HNSCCs) has not been well investigated. METHODS: We measured primary tumor growth rates, mechanism of antitumor activity, and efficacy of programmed death-L1 blockade combinatorial therapy in SCCFVII tumor-bearing C3H/HeOUJ mice undergoing intratumoral injections with RR-cyclic-di-guanine (synthetic CDG), CDG (natural cyclic-di-guanine), R848 (TLR 7/8 agonist), or phosphate buffered saline (PBS, control). RESULTS: Intratumoral CDN treatment groups showed decreased tumor size and enhanced splenocyte Th1 response when compared to the PBS treatment control group (p < .05). The RR-CDG tumor microenvironment showed upregulated interferon (IFN)-γ+CD8+ and programmed death-L1. Combining programmed death-L1 blocking antibody with RR-CDG induced regression of established tumors. CONCLUSION: This study demonstrates the antitumor effects of CDNs in a HNSCC cell line. These preclinical data strongly support the future clinical development of intratumoral CDN in patients with HNSCC. © 2017 Wiley Periodicals, Inc. Head Neck 39: 1086-1094, 2017.

Effects of polyvalent immunoglobulins in patients with recurrent pregnancy loss and antibodies to the choriocarcinoma cell line JEG-3.

OBJECTIVE: The benefit of polyvalent immunoglobulins (IVIG) for patients with recurrent pregnancy loss (RPL) is controversially discussed. Anti-trophoblast antibodies as an expression of immune pathology have been associated with RPL. We investigated whether the antibody activity against the choriocarcinoma cell line JEG-3 in RPL patients is influenced in vitro by IVIG. STUDY DESIGN: Sera of 110 unexplained RPL patients with positive anti-JEG-3 antibodies were coincubated with IVIG in different concentrations (10mg/ml, 20mg/ml, 40mg/ml). Coincubation with human albumin in identical concentrations served as control. Anti-JEG-3 reactivity was measured by using flow cytometry in comparisons with two in-house standards antibody probes of low and high reactivity as described before. Anti-JEG-3 reactivity above the 95% confidence interval of controls was defined as positive. RESULTS: Incubating RPL sera with 10mg/ml IVIG significantly decreased anti-JEG-3 activity (p<0.001). Increasing IVIG concentration to 40mg/ml resulted in a slightly additionally reduction (p=0.42). In contrast, coincubation with albumin in identically concentrations did not affect anti-JEG-3 activity (p>0.40). CONCLUSION: Coincubation with IVIG in vitro leads to a significant suppression of anti-JEG-3 activity in the sera of RPL patients.

Radioimmunoguided surgery (RIGS), PET/CT image-guided surgery, and fluorescence image-guided surgery: past, present, and future.

(125)I-labeled anti-TAG-72 antibodies were applied in radioimmunoguided surgery (RIGS) to remove gross and occult tumors. It is challenging to handle (125)I-labeled materials. PET/CT image-guided surgery utilizes (18)FDG to monitor the biochemical activity of the tumor and to integrate pre- and postoperative imaging for complete tumor removal. PET/CT image-guided surgery only detects later stage disease. Fluorescence image-guided surgery using anti-TAG-72 antibodies may provide opportunities for intraoperative cancer detection of both gross and occult tumors.

Standard treatments induce antigen-specific immune responses in prostate cancer.

PURPOSE: Prostate tumors express antigens that are recognized by the immune system in a significant proportion of patients; however, little is known about the effect of standard treatments on tumor-specific immunity. Radiation therapy induces expression of inflammatory and immune-stimulatory molecules, and neoadjuvant hormone therapy causes prominent T-cell infiltration of prostate tumors. We therefore hypothesized that radiation therapy and hormone therapy may initiate tumor-specific immune responses. EXPERIMENTAL DESIGN: Pretreatment and posttreatment serum samples from 73 men with nonmetastatic prostate cancer and 50 cancer-free controls were evaluated by Western blotting and SEREX (serological identification of antigens by recombinant cDNA expression cloning) antigen arrays to examine whether autoantibody responses to tumor proteins arose during the course of standard treatment. RESULTS: Western blotting revealed the development of treatment-associated autoantibody responses in patients undergoing neoadjuvant hormone therapy (7 of 24, 29.2%), external beam radiation therapy (4 of 29, 13.8%), and brachytherapy (5 of 20, 25%), compared with 0 of 14 patients undergoing radical prostatectomy and 2 of 36 (5.6%) controls. Responses were seen within 4 to 9 months of initiation of treatment and were equally prevalent across different disease risk groups. Similarly, in the murine Shionogi tumor model, hormone therapy induced tumor-associated autoantibody responses in 5 of 10 animals. In four patients, SEREX immunoscreening of a prostate cancer cDNA expression library identified several antigens recognized by treatment-associated autoantibodies, including PARP1, ZNF707 + PTMA, CEP78, SDCCAG1, and ODF2. CONCLUSION: We show for the first time that standard treatments induce antigen-specific immune responses in prostate cancer patients. Thus, immunologic mechanisms may contribute to clinical outcomes after hormone and radiation therapy, an effect that could potentially be exploited as a practical, personalized form of immunotherapy.

Alemtuzumab induces caspase-independent cell death in human chronic lymphocytic leukemia cells through a lipid raft-dependent mechanism.

Alemtuzumab is a humanized IgG1 kappa antibody directed against CD52, a glycosyl-phosphatidylinositol linked cell-membrane protein of unknown function. Herein, we demonstrate that alemtuzumab promotes rapid death of chronic lymphocytic leukemia (CLL) cells in vitro, in a complement and accessory cell free system. Using minimal detergent solubilization of CLL membranes, we found that CD52 colocalizes with ganglioside GM-1, a marker of membrane rafts. Fluorescence microscopy revealed that upon crosslinking CD52 with alemtuzumab+anti-Fc IgG, large patches, and in many cases caps, enriched in CD52 and GM-1 formed upon the CLL cell plasma membrane. Depletion of membrane cholesterol or inhibition of actin polymerization significantly diminished the formation of alemtuzumab-induced caps and reduced alemtuzumab-mediated CLL cell death. We compared alemtuzumab-induced direct cytotoxicity, effector cell-mediated toxicity and complement-mediated cytotoxicity of CLL cells to normal T cells. The direct cytotoxicity and observed capping was significantly greater for CLL cells as compared to normal T cells. Cell-mediated and complement-mediated cytotoxicity did not significantly differ between the two cell types. In summary, our data support the hypothesis that alemtuzumab can initiate CLL cell death by crosslinking CD52-enriched lipid rafts. Furthermore, the differential direct cytotoxic effect suggests that CD52 directed antibodies could possibly be engineered to more specifically target CLL cells.

Immune responses in advanced colorectal cancer following repeated intradermal vaccination with the anti-CEA murine monoclonal antibody, PR1A3: results of a phase I study.

BACKGROUND AND AIMS: The aim was to determine the toxicity, clinical and immune responses to the murine monoclonal anti-carcinoembryonic antigen (CEA) antibody, PR1A3, in patients with advanced colorectal cancer. MATERIALS AND METHODS: Fifteen patients with advanced colorectal cancer received either 0.5-, 1.0- or 5.0-mg doses of PR1A3 mixed with 10% w/v Alum adjuvant (Superfos Biosector, Denmark) intradermally at 4-week intervals for 3 months. Patient serum was assessed for anti-idiotypic (Ab2), anti-anti-idiotypic (Ab3) and human anti-mouse antibody (HAMA) reactivity. Peripheral blood mononuclear cell (PBMC) proliferation with phytohaemagglutinin (PHA), CEA and PR1A3, stimulated IL-2, IL-4 and IFN-gamma levels and PR1A3-stimulated IL-2 receptor expression during immunotherapy were determined. Comparisons were made with 16 age-matched controls without malignant disease. RESULTS: Hyperimmune sera from 12 of the 15 patients showed Ab2 reactivity with no detectable Ab3 responses. Strong HAMA reactivity was recorded in 7 of the 15 cases with no adverse clinical effect. Delayed-type hypersensitivity (DTH) responses developed in 12 of the 15 patients. Pre-treatment PBMC proliferation with PHA was subnormal in each patient compared with controls, becoming normal (or supranormal) in all patients during immunisation (P<0.001). PBMC proliferation with CEA and PR1A3 increased during immunotherapy (P<0.001) along with stimulated production of IL-2, IFN-gamma and IL-2 receptor expression. Progressive disease was observed in 14 of the 15 patients with minimal toxicity. CONCLUSION: PR1A3 generated limited idiotypic responses but robust DTH reactivity in most patients. In vitro PBMC proliferation with mitogens and recall antigens is greatly increased during the course of immunisation, with a shift in stimulated cytokine profile.

Treatment of multiple myeloma by antibody mediated immunotherapy and induction of myeloma selective antigens.

BACKGROUND: In view of the successful use of serotherapy in many B-cell malignancies, we and others have sought to identify tumor selective antigens for the serotherapy of plasma cell dyscrasias (PCD) including multiple myeloma (MM), and Waldenstrom's macroglobulinemia (WM). We recently identified Muc-1 core protein as a MM selective antigen. Though Muc-1 core protein is abundantly expressed on most MM plasma cells, expression of this antigen can be absent, or weak on some plasma cells which could potentially result in the selection of Muc-1 core protein negative clones following serotherapy of PCD. In addition to Muc-1 core protein, we have also been examining the use of CD20 directed serotherapy for PCD. DESIGN: As part of these efforts, we recently initiated a phase II clinical trial examining the use of Rituximab (Rituxan, MabThera) as a single agent in MM patients; as well several WM patients have been treated with Rituximab at our Institutions. RESULTS: In previous studies, we have shown that CD20 is abundantly expressed on the plasma cells of most WM patients; in contrast, CD20 is expressed on plasma cells from a minority of MM patients, and in these patients expression of CD20 can be weak or heterogeneous with both CD20+ and CD20- plasma cells present. As such, we have sought out clinically useful inducers of Muc-1 core protein, and of CD20 on malignant plasma cells. CONCLUSIONS: These efforts resulted in the identification of dexamethasone (Dex) as a potent inducer of Muc-1 core protein on MM plasma cells, and interferon-gamma (IFN-gamma) as a potent inducer of CD20 on MM plasma cells and B-cells. Importantly, these agents induced their respective antigens at pharmacologically achievable doses.

Enhanced tumour specificity of an anti-carcinoembrionic antigen Fab' fragment by poly(ethylene glycol) (PEG) modification.

Polyethylene glycol (PEG) modification of a chimeric Fab' fragment (F9) of A5B7 (alpha-CEA), using an improved coupling method, increases its specificity for subcutaneous LS174T tumours. PEGylation increased the area under the concentration-time curve (AUC0-144) in all tissues but there were significant differences (variance ratio test, F = 27.95, P < 0.001) between the proportional increases in AUC0-144, with the tumour showing the greatest increase. The increase in AUCtumour from F9 to PEG-F9 was similar to the reported increase from Fab' to F(ab')2 while the increase in AUCblood by PEGylation of F9 was only 21% of the reported increase from Fab' to whole IgG. A two sample t-test showed no significant differences between maximal tumour/tissue ratios for PEG-F9 and F9 while the tumour/tissue ratios for PEG-F9 remained high over a longer period, with tumour levels at least double those for F9. PEG-F9 emerges as a new generation antibody with potential advantages for both radioimmunotherapy and tumour imaging. Since there was a reduction in antigen binding, optimisation of PEGylation might further improve tumour specificity. The latter resulted from complex effects on both the entry into and exit rates from tumour and normal tissues in a tissue-specific fashion.

Anti-p53 antibodies in sera of workers occupationally exposed to vinyl chloride.

BACKGROUND: The p53 tumor suppressor gene (also known as TP53) is often mutated in a wide variety of cancers, including angiosarcoma of the liver (ASL). Anti-p53 antibodies have been detected in the sera of patients with leukemia, childhood lymphoma, or cancers such as those of the breast, lung, colon, esophagus, and liver (hepatocellular carcinoma). PURPOSE: The objective of this study was to determine the prevalence and time of appearance of serum anti-p53 antibodies during the pathogenesis of ASL associated with occupational exposure to vinyl chloride. METHODS: Enzyme-linked immunoassay (EIA) was used to detect anti-p53 antibodies in 148 serum samples from 92 individuals occupationally exposed (in France or in Kentucky) to vinyl chloride; 15 of these individuals (six from France and nine from Kentucky) had ASL. A subset of coded EIA-positive and EIA-negative sera was further analyzed for anti-p53 antibodies by immunoblotting and immunoprecipitation. Nucleotide sequence analysis of exons 5-8 of the p53 gene was conducted on ASL DNA from six patients. We tested sera from 31 men who had no occupational exposure to vinyl chloride; they made up the control group. Statistical analyses were done using the Kruskal-Wallis chi-squared approximation and the Wilcoxon two-sample test for normal approximation. All P values result from two-sided tests. RESULTS: Fourteen serum samples (from nine individuals) were positive in the EIA. Five of the 15 individuals with ASL were positive for anti-p53 antibodies by EIA, immunoblotting, and immunoprecipitation: one individual at 11.3 and 10.8 years before diagnosis, another at 4 months before and shortly after diagnosis, and three when diagnosed or shortly thereafter. Four of the 77 vinyl chloride-exposed workers without diagnosed ASL were positive for anti-p53 antibodies; two of the four had symptoms related to vinyl chloride toxicity. Tumors from three of the six vinyl chloride-exposed workers from which sufficient DNA for analysis was obtained had A:T to T:A missense mutations of the p53 gene. Anti-p53 antibodies were detected in two of these individuals. Among the control group, two of 15 serum samples from 15 lung cancer patients and zero of 15 serum samples from control subjects without cancer had anti-p53 antibodies as substantially lower levels than the nine (10%) of 92 vinyl chloride-exposed workers who were positive for anti-p53 antibodies. CONCLUSIONS AND IMPLICATIONS: Serum anti-p53 antibodies can predate clinical diagnosis of certain tumors, such as ASL, and may be useful in identifying individuals at high cancer risk, such as workers with occupational exposure to vinyl chloride.

Phorbol ester tumor promoter regulation of natural antitumor antibody binding depends on protein kinase C and an intact microfilament system.

Phorbol ester tumor promoter treatment produced a biphasic effect on the binding of polyclonal whole-serum natural antibody (NAb) by L5178Y-F9 murine lymphoma cells. In vitro tumor growth in 100 ng/ml 12-O-tetradecanoylphorbol-13-acetate (TPA) produced a rapid decrease followed by a reversible and unstable increase in NAb binding detected at 4 degrees C. The latter was associated with a functional decrease in NAb binding at 37 degrees C and increases in the tumorigenic and metastatic potentials in vivo. Colchicine, cytochalasin B and sodium azide inhibited the NAb binding of TPA-treated cells, while only colchicine reduced the binding of controls, suggesting the dependence of the TPA-induced increase in NAb binding on microfilament organization and active energy production. The non-tumor-promoting, non-PKC-activating TPA analogue 4-O-Me-TPA failed to alter NAb binding, arguing against nonspecific effects of TPA. The non-tumor-promoting, PKC-activating diacylglycerol, OAG, reproduced the initial decrease in NAb binding but was unable to mimic the subsequent TPA-induced increase. The PKC inhibitor H-7, but not HA1004, could block the TPA-induced increase in NAb binding. Together the data argue that PKC activation is required for both TPA-induced changes in NAb binding but that it is not sufficient to generate the energy- and microfilament-system-dependent, unstable high-NAb-binding phenotype associated with increased tumor progression.

Human-human monoclonal antibody directed against tumor surface antigen in the treatment of human malignancy. A pilot study.

Eighteen patients with a variety of refractory solid tumors received infusions of IgM class monoclonal antibody directed against tumor surface antigen and raised in a nonviral transformed human-human system. Three other patients with refractory hematologic or solid tumors had autologous marrow purged with antibody, regrown, and reinfused following high dose chemotherapy. A molecular weight of 200,000 was demonstrated for the whole antibody produced by the fusion line. Each antibody failed to react with nonspecific tumor tissue. No fetal antigen reactivity was noted. Antibody was cytotoxic. Complement dependence was not demonstrated. Chloroquine phosphate pretreatment was employed prior to each antibody infusion to block receptor recycling and host-antigen processing. Antibody dose was doubled with each treatment. Self-limiting chill and fever (with tachycardia) was noted in 12-hour infusions. Tumor biopsy following infusion demonstrated good binding for as long as 48 hours after dosing. Tumor regression was noted during infusions. Pulmonary metastases responded rapidly following infusion. Maximal shrinkage of skin metastases was noted at 4 days with some regrowth at 7 days. Reduction in size of liver metastases was the most durable response. Responses correlated with survival. There were no antibody-related deaths. Antibody used ex vivo to purge autologous bone marrow-cleared marrow of tumor. Marrow engraftment was noted between 7 and 21 days. Follow-up showed no antihuman antibody formation, alteration in renal or hepatic function, alteration in thyroid or adrenal function, or circulating antigen-antibody complexes. Natural killer cell activity and complement-activated clearing of immune complexes increased following infusion. The time for complete clearing of immune complexes from the circulation was 10 days. Mild anemia was seen after several courses of infusion. Reticulocytosis was unimpaired. Long-term follow-up showed no antihuman antibody formation or organ dysfunction.