Gentulizumab, a novel anti-CD47 antibody with potent antitumor activity and demonstrates a favorable safety profile.

BACKGROUND: Targeting CD47/SIRPα axis has emerged as a promising strategy in cancer immunotherapy. Despite the encouraging clinical efficacy observed in hematologic malignancies through CD47-SIRPα blockade, there are safety concerns related to the binding of anti-CD47 antibodies to CD47 on the membrane of peripheral blood cells. METHODS: In order to enhance the selectivity and therapeutic efficacy of the antibody, we developed a humanized anti-CD47 monoclonal antibody called Gentulizumab (GenSci059). The binding capacity of GenSci059 to CD47 was evaluated using flow cytometry and surface plasmon resonance (SPR) methods, the inhibitory effect of GenSci059 on the CD47-SIRPα interaction was evaluated through competitive ELISA assays. The anti-tumor activity of GenSci059 was assessed using in vitro macrophage models and in vivo patient-derived xenograft (PDX) models. To evaluate the safety profile of GenSci059, binding assays were conducted using blood cells. Additionally, we investigated the underlying mechanisms contributing to the weaker binding of GenSci059 to erythrocytes. Finally, toxicity studies were performed in non-human primates to assess the potential risks associated with GenSci059. RESULTS: GenSci059 displayed strong binding to CD47 in both human and monkey, and effectively inhibited the CD47-SIRPα interaction. With doses ranging from 5 to 20 mg/kg, GenSci059 demonstrated potent inhibition of the growth of subcutaneous tumor with the inhibition rates ranged from 30.3% to complete regression. Combination of GenSci059 with 2.5 mg/kg Rituximab at a dose of 2.5 mg/kg showed enhanced tumor inhibition compared to monotherapy, exhibiting synergistic effects. GenSci059 exhibited minimal binding to hRBCs compared to Hu5F9-G4. The binding of GenSci059 to CD47 depended on the cyclization of N-terminal pyroglutamic acid and the spatial conformation of CD47, but was not affected by its glycosylation modifications. A maximum tolerated dose (MTD) of 450 mg/kg was observed for GenSci059, and no significant adverse effects were observed in repeated dosages up to 10 + 300 mg/kg, indicating a favorable safety profile. CONCLUSION: GenSci059 selectively binds to CD47, effectively blocks the CD47/SIRPα axis signaling pathway and enhances the phagocytosis effects of macrophages toward tumor cells. This monoclonal antibody demonstrates potent antitumor activity and exhibits a favorable safety profile, positioning it as a promising and effective therapeutic option for cancer.

CD47 masks pro-phagocytic ligands in cis on tumor cells to suppress antitumor immunity.

Cancer cells often overexpress CD47, which triggers the inhibitory receptor SIRPα expressed on macrophages, to elude phagocytosis and antitumor immunity. Pharmacological blockade of CD47 or SIRPα is showing promise as anticancer therapy, although CD47 blockade has been associated with hematological toxicities that may reflect its broad expression pattern on normal cells. Here we found that, in addition to triggering SIRPα, CD47 suppressed phagocytosis by a SIRPα-independent mechanism. This mechanism prevented phagocytosis initiated by the pro-phagocytic ligand, SLAMF7, on tumor cells, due to a cis interaction between CD47 and SLAMF7. The CD47-SLAMF7 interaction was disrupted by CD47 blockade and by a first-in-class agonist SLAMF7 antibody, but not by SIRPα blockade, thereby promoting antitumor immunity. Hence, CD47 suppresses phagocytosis not only by engaging SIRPα, but also by masking cell-intrinsic pro-phagocytic ligands on tumor cells and knowledge of this mechanism may influence the decision between CD47 blockade or SIRPα blockade for therapeutic purposes.

CD47/SIRPα axis: bridging innate and adaptive immunity.

Myeloid immune cells are frequently present in the tumor environment, and although they can positively contribute to tumor control they often negatively impact anticancer immune responses. One way of inhibiting the positive contributions of myeloid cells is by signaling through the cluster of differentiation 47 (CD47)/signal regulatory protein alpha (SIRPα) axis. The SIRPα receptor is expressed on myeloid cells and is an inhibitory immune receptor that, upon binding to CD47 protein, delivers a 'don't eat me' signal. As CD47 is often overexpressed on cancer cells, treatments targeting CD47/SIRPα have been under active investigation and are currently being tested in clinical settings. Interestingly, the CD47/SIRPα axis is also involved in T cell-mediated antitumor responses. In this perspective we provide an overview of recent studies showing how therapeutic blockade of the CD47/SIRPα axis improves the adaptive immune response. Furthermore, we discuss the interconnection between the myeloid CD47/SIRPα axis and adaptive T cell responses as well as the potential therapeutic role of the CD47/SIRPα axis in tumors with acquired resistance to the classic immunotherapy through major histocompatibility complex downregulation. Altogether this review provides a profound insight for the optimal exploitation of CD47/SIRPα immune checkpoint therapy.

Novel nanocarriers for silencing anti-phagocytosis CD47 marker in acute myeloid leukemia cells.

Acute myeloid leukemia (AML), a malignant disorder of Hematopoietic stem cells, can escape immunosurveillance by over expression of the cluster of differentiation 47 (CD47) marker, which functions as an inhibitory signal, suppressing phagocytosis by binding to signal regulatory protein α (SIRPα) on macrophages. AML is treated mainly by chemotherapy, which has drastic side effects and poor outcomes for the patients. Most AML patients develop drug resistance, so other methods to treat AML are highly required. Small interfering RNA (siRNA) is considered as an antitumor therapeutic due to its ability to silence genes associated with the overexpressed cancer markers and subsequently re-sensitize cancer cells. However, delivering siRNA into cells faces challenges, and the development of an effective delivery system is desired for successful silencing at the gene level. Herein, we report the usage of different formulations of graphene oxide (GO) as carriers for the delivery of CD47_siRNA (siRNA against CD47) into AML cells in vitro. The polyethylene glycol (PEG) and dendrimers (PAMAM) modified GO with small flake sizes achieved the highest silencing efficiency of the anti-phagocytosis marker CD47 gene, resulted CD47 protein down-regulation in AML cells. Moreover, the concentration at which the GO-based formulations was used has shown no cytotoxicity in AML cells or normal blood cells, which could be used to screen potential drugs for targeted gene therapy in AML.

Keratinocyte differentiation antigen-specific T cells in immune checkpoint inhibitor-treated NSCLC patients are associated with improved survival.

Immune checkpoint inhibitors (ICIs) have improved the survival of patients with non-small cell lung cancer (NSCLC) by reinvigorating tumor-specific T cell responses. However, the specificity of such T cells and the human leukocyte antigen (HLA)-associated epitopes recognized, remain elusive. In this study, we identified NSCLC T cell epitopes of recently described NSCLC-associated antigens, termed keratinocyte differentiation antigens. Epitopes of these antigens were presented by HLA-A 03:01 and HLA-C 04:01 and were associated with responses to ICI therapy. Patients with CD8+ T cell responses to these epitopes had improved overall and progression-free survival. T cells specific for such epitopes could eliminate HLA class I-matched NSCLC cells ex vivo and were enriched in patient lung tumors. The identification of novel lung cancer HLA-associated epitopes that correlate with improved ICI-dependent treatment outcomes suggests that keratinocyte-specific proteins are important tumor-associated antigens in NSCLC. These findings improve our understanding of the mechanisms of ICI therapy and may help support the development of vaccination strategies to improve ICI-based treatment of these tumors.

A single-valent long-acting human CD47 antagonist enhances antibody directed phagocytic activities.

Many cancer cells express CD47 as a 'don't eat me' signal to mask their presences from immune recognition and destruction. Such a signal is transmitted when CD47 binds to the signal regulatory protein-α (SIRPα) on macrophages to cut the phagocytic reaction. Most recent studies have focused on developing CD47 blocking agents with different affinities and avidities in order to optimize the therapeutic window between efficacy and toxicities involving normal cells expressing CD47. We described in this study a new design to fuse one CD47 binding domain of SIRPα with a pharmacokinetics modifying domain F8. The resulted single valent long-acting CD47 antagonist SIRPα-F8 was able to bind to CD47 and disrupt CD47-SIRPα axis. However, by itself it cannot trigger endocytosis and has no effect on tumor growth. Only when used in combination with the anti-CD20 mAbs, there were greatly improved phagocytic activities towards CD20 positive cancer cells. In vivo the combination also resulted in better tumor growth inhibition comparing to the vehicle control group. In addition, we showed that the F8 fusion bound to hFcRn only inside endosomes at pH 6.0, enabled hFcRn mediated recycling and thus greatly extended the circulation half-life in hFcRn knock-in mice. Taken together, the SIRPα-F8 design may suggest a new option to improve the therapeutic index of antibody treatment in clinical use towards tumors.

Anti-SIRPα antibodies as a potential new tool for cancer immunotherapy.

Tumor cells are thought to evade immune surveillance through interaction with immune cells. Much recent attention has focused on the modification of immune responses as a basis for new cancer treatments. SIRPα is an Ig superfamily protein that inhibits phagocytosis in macrophages upon interaction with its ligand CD47 expressed on the surface of target cells. Here, we show that SIRPα is highly expressed in human renal cell carcinoma and melanoma. Furthermore, an anti-SIRPα Ab that blocks the interaction with CD47 markedly suppressed tumor formation by renal cell carcinoma or melanoma cells in immunocompetent syngeneic mice. This inhibitory effect of the Ab appeared to be mediated by dual mechanisms: direct induction of Ab-dependent cellular phagocytosis of tumor cells by macrophages and blockade of CD47-SIRPα signaling that negatively regulates such phagocytosis. The antitumor effect of the Ab was greatly attenuated by selective depletion not only of macrophages but also of NK cells or CD8+ T cells. In addition, the anti-SIRPα Ab also enhances the inhibitory effects of Abs against CD20 and programmed cell death 1 (PD-1) on tumor formation in mice injected with SIRPα-nonexpressing tumor cells. Anti-SIRPα Abs thus warrant further study as a potential new therapy for a broad range of cancers.

Engineered SIRPα variants as immunotherapeutic adjuvants to anticancer antibodies.

CD47 is an antiphagocytic signal that cancer cells employ to inhibit macrophage-mediated destruction. Here, we modified the binding domain of human SIRPα, the receptor for CD47, for use as a CD47 antagonist. We engineered high-affinity SIRPα variants with about a 50,000-fold increased affinity for human CD47 relative to wild-type SIRPα. As high-affinity SIRPα monomers, they potently antagonized CD47 on cancer cells but did not induce macrophage phagocytosis on their own. Instead, they exhibited remarkable synergy with all tumor-specific monoclonal antibodies tested by increasing phagocytosis in vitro and enhancing antitumor responses in vivo. This "one-two punch" directs immune responses against tumor cells while lowering the threshold for macrophage activation, thereby providing a universal method for augmenting the efficacy of therapeutic anticancer antibodies.

IFITM1 is a tight junction protein that inhibits hepatitis C virus entry.

UNLABELLED: Type 1 interferon (IFN) continues to be the foundation for the current standard of care combination therapy for chronic hepatitis C virus (HCV) infection, yet the component interferon-stimulated genes (ISGs) that mediate the antiviral actions of IFN are not fully defined. Interferon-induced transmembrane protein 1 (IFITM1) is an ISG product that suppresses early stage infection by a number of viruses through an unknown mechanism of action. Moreover, the actions of IFITM1 on HCV infection are not fully elucidated. Here we identify IFITM1 as a hepatocyte tight junction protein and a potent anti-HCV effector molecule. IFITM1 expression is induced early during IFN treatment of hepatocytes and accumulates at hepatic tight junctions in HCV-infected human patient liver during IFN therapy. Additionally, we found that IFITM1 interacts with HCV coreceptors, including CD81 and occludin, to disrupt the process of viral entry. Thus, IFITM1 is an anti-HCV ISG whose actions impart control of HCV infection through interruption of viral coreceptor function. CONCLUSION: This study defines IFITM1 as an ISG effector with action against HCV entry. Design of therapy regimens to enhance IFITM1 expression should improve the virologic response among HCV patients undergoing treatment with type I IFN.

Procalcitocina en pacientes con neutropenia y fiebre secundarias a quimioterapia / Procalcitocina in patients with neutropenia and fever secondary to chemotherapy

Los pacientes con malignidades hematológicas frecuentemente se complican por infecciones. La procalcitonina (PCT) puede diferenciar la sepsis de la inflamación. Establecer la utilidad de PCT para determinar complicaciones infecciosas en adultos con malignidades hematológicas y neutropenia febril (NF) provenientes de la Ciudad Hospitalaria “Dr. Enrique Tejera” (CHET) y Centro Policlínico Valencia (CPV), Valencia, Venezuela. Estudio prospectivo que incluyó 30 pacientes neutropénicos febriles (H:M, 20:10), edad 49,4±21 años. En 5ml de sangre venosa, extraidos en las primeras 48 horas siguientes a la fiebre, se midió mediante ensayo semicuantitativo PCT-Q®. PCT<0.5ng/ml considerada negativa, >0.5 <2ng/ml infección sistémica, >2 <10ng/ml posible sepsis severay >10ng/ml: posible shock séptico. Se utilizó estadística descriptiva y probabilidad de asociación mediante test de Fisher, con p<0,05 considerada estadísticamente significativa. La asociación entre PCT y hallazgos clínicos de infección no fue estadísticamente significativa (p = 0,1); la asociación entre PCT y hemocultivos positivos fue estadísticamente significativa (p = 0,006) y también entre PCT y mortalidad (p = 0,034). La PCT elevada se asocia a bacteriemia y mortalidad en adultos neutropéniucos febriles, empeorando su pronóstico

Frequently patients with haematologic malignancies (HM) are complicated with infections. Procalcitonin (PCT) allows to differentiate sepsis from inflammation. To assess the usefulness of PCT in infectious complications in adult patients with HM and febrile neutropenia (FN). This prospective study, included 30 FN patients (M:F,20:10), age 49,4±21 years. A blood sample in the first 48 hours after the start of fever PCT was processed by semi-quantitative assay (PCT-Q®). Values of PCT<0.5ng / ml were considered negative, >0.5<2ng/ml suggested systemic infection, >2<10ng/ml, possible severe sepsis and >10ng/ml possible septic shock. Descriptive statistics and probability of association by fisher test were performed and p < 0,05 considered statistically significant. There was no stastical correlation between PCT and clinical findings of infection (p= 0,1) but between PCT and positive blood cultures it was statistically significant (p = 0,006) and also between PCT and mortality (p = 0,034). Elevated PCT is associated with bacteremia and mortality among adult FN patients with HM, worsening their prognosis

Tremelimumab (CP-675,206), a cytotoxic T lymphocyte associated antigen 4 blocking monoclonal antibody in clinical development for patients with cancer.

Tremelimumab (CP-675,206) is a fully human monoclonal antibody specific for human cytotoxic T lymphocyte-associated antigen 4 (CTLA-4, CD152) in clinical development for patients with cancer. Blocking the CTLA-4 negative costimulatory receptor with the antagonistic antibody tremelimumab results in immune activation. Administration of tremelimumab to patients with locally advanced and metastatic melanoma has resulted in a subset of patients with durable objective tumor regressions. Its IgG(2) isotype minimizes the possibility of cytotoxic effects on activated T lymphocytes and cytokine release syndrome. Preclinical testing in vitro and in large animal models predicted the target concentrations of circulating antibody in humans necessary for a pharmacodynamic effect. Phase I clinical trials provided evidence of dose- or exposure-related effects consistent with the anticipated mechanism of action. Further clinical development has led to two ongoing registration trials in patients with metastatic melanoma: a phase III randomized trial of tremelimumab versus dacarbazine or temozolomide in previously untreated patients with advanced melanoma and a phase II trial of tremelimumab in previously treated patients with advanced melanoma.

Update on anti-CTLA-4 antibodies in clinical trials.

Breaking immune tolerance against tumor self-antigens is presently an area of intense research in the design of cancer therapies. One possible method to enhance immune system activation against tumor antigens is by blocking the inhibitory co-stimulatory signals mediated by cytotoxic T lymphocyte antigen 4, (CTLA-4) expressed on activated T cells. The fully human monoclonal antibodies that are directed against human CTLA-4, ipilimumab (Medarex/Bristol-Myers Squibb) and CP-675,206 (Pfizer/Abgenix, now Amgen), have demonstrated activity against metastatic melanoma, hormone refractory prostate cancer and other malignancies. They have also uncovered unusual immune-related adverse events manifesting as self-limiting inflammatory reactions of the bowel, skin and pituitary. This article reviews preclinical development and data generated from Phase I, II and III studies with regard to the end points reported and immune-related adverse events.

CTLA-4: From conflict to clinic.

CTLA-4 was first identified in 1991 as a second receptor for the T cell costimulation ligand B7. Uncertainties about its biological function plagued the early years after its discovery until 1995, when it was confirmed to be an inhibitor of T cell responses. CTLA-4 has since scored in the clinic as a target for antitumor therapy and as a soluble inhibitor of autoimmunity.

Soluble PD-1 facilitates 4-1BBL-triggered antitumor immunity against murine H22 hepatocarcinoma in vivo.

PURPOSE: The use of costimulatory molecules targeting distinct T-cell signaling pathways has provided a means for triggering and enhancing antitumor immunity; however, it is still not fully understood what types of costimulatory molecules are suitable for the combination in tumor therapy. Our purpose in this study is to establish an effective antitumor immune approach by using costimulatory molecule 4-1BBL in combination with soluble PD-1. EXPERIMENTAL DESIGN: The murine H22 hepatocarcinoma served as an ectopic tumor model. Local gene transfer was done by injection with naked plasmid p4-1BBL and/or psPD-1. The synergistic mechanism of dual-gene therapy was elucidated by detecting the change of gene expression of immunoregulatory factors in tumor microenvironment. The effects of immunotherapy were evaluated by testing the function of tumor-specific T cells, measuring tumor weight or volume, survival of mice, and H&E staining of tissues. RESULTS: 4-1BBL expressed by normal nonimmune cells effectively enhanced antitumor immune response but up-regulated PD-L1 and did not reduce IL-10 and transforming growth factor-beta (TGF-beta). sPD-1 synergized with 4-1BBL to establish efficient antitumor immune environment, including down-regulation of IL-10 and TGF-beta, further up-regulation of interleukin (IL)-2 and IFN-gamma, and higher CD8(+) T-cell infiltration. The combined treatment by 4-1BBL/sPD-1 eradicated tumors from mice with small amounts of preexistent tumor cells or tumors from approximately 60% of individuals with larger amounts of preexistent tumor cells. CONCLUSIONS: Our findings in this report imply a great potential of 4-1BBL in combination with sPD-1 in tumor therapeutics with the in vivo existent tumor cells as antigens.

CTLA4 blockade and GM-CSF combination immunotherapy alters the intratumor balance of effector and regulatory T cells.

CTL-associated antigen 4 (CTLA4) blockade releases inhibitory controls on T cell activation and proliferation, inducing antitumor immunity in both preclinical and early clinical trials. We examined the mechanisms of action of anti-CTLA4 and a GM-CSF-transduced tumor cell vaccine (Gvax) and their impact on the balance of effector T cells (Teffs) and Tregs in an in vivo model of B16/BL6 melanoma. Tumor challenge increased the frequency of Tregs in lymph nodes, and untreated tumors became infiltrated by CD4+Foxp3- and CD4+Foxp3+ T cells but few CD8+ T cells. Anti-CTLA4 did not deplete Tregs or permanently impair their function but acted in a cell-intrinsic manner on both Tregs and Teffs, allowing them to expand, most likely in response to self antigen. While Gvax primed the tumor-reactive Teff compartment, inducing activation, tumor infiltration, and a delay in tumor growth, the combination with CTLA4 blockade induced greater infiltration and a striking change in the intratumor balance of Tregs and Teffs that directly correlated with tumor rejection. The data suggest that Tregs control both CD4+ and CD8+ T cell activity within the tumor, highlight the importance of the intratumor ratio of effectors to regulators, and demonstrate inversion of the ratio and correlation with tumor rejection during Gvax/anti-CTLA4 immunotherapy.

CTLA-4 gene polymorphisms and natural soluble CTLA-4 protein in psoriasis vulgaris.

CTLA-4 molecule is an important inhibitor of T-lymphocyte activation. Several single nucleotide polymorphisms (SNPs) in the CTLA-4 gene were found, and their associations with many human diseases were described. So far, however, such studies have not been performed in psoriasis vulgaris in Caucasoids. Therefore, we examined the distribution of three CTLA-4 SNPs: -1147C/T, -318C/T and +49 A/G in 116 patients with psoriasis vulgaris and 123 healthy blood donors using the polymerase chain reaction-restriction fragment length polymorphism method. For all three SNPs, the frequencies of alleles, genotypes and three-point haplotypes were very similar in patients and controls, suggesting no contribution of these genetic variants to psoriasis.

Unraveling the complex relationship between cancer immunity and autoimmunity: lessons from melanoma and vitiligo.

A relationship between melanoma and vitiligo, a skin disorder characterized by the loss of melanocytes, has been postulated for many decades. In some cases, vitiligo is almost certainly a manifestation of autoimmune-mediated destruction of melanocytes. Melanocytes and melanoma cells share melanocyte differentiation antigens. Based on a number of observations, de novo vitiligo developing in patients with melanoma has been regarded as a sign of good prognosis. The immune system tolerates or ignores differentiation antigens because these antigens are self-derived. Therefore, immune tolerance or ignorance must be overcome to prime naive T and B cells to induce cancer immunity and autoimmunity against melanocyte differentiation antigens. Mouse models of concurrent melanoma and autoimmune vitiligo have revealed strategies to overcome immune ignorance or tolerance to melanocyte differentiation antigens: immunization with self-antigens as altered self (e.g., orthologues or mutated versions), expression in viral vectors, passive immunization with antibodies or T cells, incorporating potent adjuvants into active immunization, and blockade or removal of a downregulatory mechanism. Extensive investigations into the mechanisms that lead to tumor immunity and autoimmunity elicited by certain differentiation antigens have further revealed a variety of distinct cellular and molecular requirements, which are redundant and alternative.