Alison L. Tutt

CD40 antibody evokes a cytotoxic T-cell response that eradicates lymphoma and bypasses T-cell help

CD40 is essential in enabling antigen-presenting cells to process and present antigen effectively to T cells. We demonstrate here that when antibody against CD40 is used to treat mice with syngeneic lymphoma, a rapid cytotoxic T-cell response independent of T-helper cells occurs, with tenfold expansion of CD8+ T cells over a period of 5 days. This response eradicates the lymphoma and provides protection against tumor rechallenge without further antibody treatment. Thus, it seems that by treating mice with monoclonal antibody against CD40, we are immunizing against syngeneic tumors. The phenomenon proved reproducible with two antibodies against CD40 (3/23 and FGK-45) in three CD40+ lymphomas (A20, A31 and BCL1) and gave partial protection in one of two CD40– lymphomas (EL4 and Ten1). Although the nature of the target antigens on these lymphomas is unknown, CD8+ T cells recovered from responding mice showed powerful cytotoxic activity against the target B-cell lymphoma in vitro.

Novel type II anti-CD20 monoclonal antibody (GA101) evokes homotypic adhesion and actin-dependent, lysosome-mediated cell death in B-cell malignancies

The anti-CD20 mAb rituximab has substantially improved the clinical outcome of patients with a wide range of B-cell malignancies. However, many patients relapse or fail to respond to rituximab, and thus there is intense investigation into the development of novel anti-CD20 mAbs with improved therapeutic efficacy. Although Fc-FcγR interactions appear to underlie much of the therapeutic success with rituximab, certain type II anti-CD20 mAbs efficiently induce programmed cell death (PCD), whereas rituximab-like type I anti-CD20 mAbs do not. Here, we show that the humanized, glycoengineered anti-CD20 mAb GA101 and derivatives harboring non-glycoengineered Fc regions are type II mAb that trigger nonapoptotic PCD in a range of B-lymphoma cell lines and primary B-cell malignancies. We demonstrate that GA101-induced cell death is dependent on actin reorganization, can be abrogated by inhibitors of actin polymerization, and is independent of BCL-2 overexpression and caspase activation. GA101-induced PCD is executed by lysosomes which disperse their contents into the cytoplasm and surrounding environment. Taken together, these findings reveal that GA101 is able to potently elicit actin-dependent, lysosomal cell death, which may potentially lead to improved clearance of B-cell malignancies in vivo.

Interaction with FcγRIIB Is Critical for the Agonistic Activity of Anti-CD40 Monoclonal Antibody

A high activatory/inhibitory FcγR binding ratio is critical for the activity of mAb such as rituximab and alemtuzumab that attack cancer cells directly and eliminate them by recruiting immune effectors. Optimal FcγR binding profiles of other anti-cancer mAb, such as immunostimulatory mAb that stimulate or block immune receptors, are less clear. In this study, we analyzed the importance of isotype and FcγR interactions in controlling the agonistic activity of the anti-mouse CD40 mAb 3/23. Mouse IgG1 (m1) and IgG2a (m2a) variants of the parental 3/23 (rat IgG2a) were engineered and used to promote humoral and cellular responses against OVA. The mouse IgG1 3/23 was highly agonistic and outperformed the parental Ab when promoting Ab (10–100-fold) and T cell (OTI and OTII) responses (2- to >10-fold). In contrast, m2a was almost completely inactive. Studies in FcγR knockout mice demonstrated a critical role for the inhibitory FcγRIIB in 3/23 activity, whereas activatory FcγR (FcγRI, -III, and -IV) was dispensable. In vitro experiments established that the stimulatory effect of FcγRIIB was mediated through Ab cross-linking delivered in trans between neighboring cells and did not require intracellular signaling. Intriguingly, activatory FcγR provided effective cross-linking of 3/23 m2a in vitro, suggesting the critical role of FcγRIIB in vivo reflects its cellular distribution and bioavailability as much as its affinity for a particular Ab isotype. In conclusion, we demonstrate an essential cross-linking role for the inhibitory FcγRIIB in anti-CD40 immunostimulatory activity and suggest that isotype will be an important issue when optimizing reagents for clinical use.

Expression and costimulatory effects of the TNF receptor superfamily members CD134 (OX40) and CD137 (4-1BB), and their role in the generation of anti-tumor immune responses

This study addresses the relative importance of CD134 (OX40) and CD137 (4‐1BB) in the costimulation of CD4+ and CD8+ T cells under comparable conditions of antigenic stimulation. We demonstrate that CD134 is capable of directly costimulating CD8+ T cells. However, costimulation of CD8+ T cells by CD134 is less potent than that triggered by CD137. The higher costimulatory activity of CD137, when compared with CD134, correlates well with its faster expression kinetics and higher levels on CD8+ T cells. Furthermore, induction of CD137 expression on CD8+ T cells is highly sensitive to low levels of TCR stimulation, which is in contrast with CD134. Conversely, CD134 is more effective than CD137 in costimulating CD4+ T cells. This, however, could not be attributed to differential expression. We also demonstrate that the transient nature of CD134 and CD137 expression on activated CD4+ T cells is the resultof proteolytic shedding. Consistent with the greater ability of CD137 to costimulate CD8+ T cells, stimulation of CD137 in vivo is considerably more effective than CD134 in augmenting anti‐tumor immune responses. Therefore, agents that stimulate signaling via CD137 are likely to be more useful in clinical conditions where highly effective CD8+ CTL responses are required.

Triggering FCα-Receptor I (CD89) Recruits Neutrophils as Effector Cells for CD20-Directed Antibody Therapy

CD20 Abs induce clinical responses in lymphoma patients, but there are considerable differences between individual patients. In 51Cr release assays with whole blood as effector source, RAJI cells were effectively killed by a mouse/human chimeric IgG1 construct of CD20 Ab 1F5, whereas ARH-77 proved resistant to killing by this Ab. When whole blood was fractionated into plasma, mononuclear cells, or granulocytic effector cells, RAJI cells were effectively killed in the presence of complement-containing plasma, whereas the mature B cell line ARH-77 proved complement resistant. However, with a bispecific Ab (BsAb) against the myeloid receptor for IgA (CD89; FcαRI) and CD20, a broad range of B cell lines were effectively killed. FcαRI is expressed on monocytes/macrophages, neutrophils, and eosinophils. As the numbers of these effector cells and their functional activity can be enhanced by application of G-CSF or GM-CSF, lysis via (FcαRI × CD20) BsAb was significantly enhanced in blood from patients during therapy with these myeloid growth factors. Interestingly, the major effector cell population for this BsAb were polymorphonuclear neutrophils, which proved ineffective in killing malignant B cells with murine, chimeric IgG1, or FcγRI- or FcγRIII-directed BsAbs against CD20. Experiments with blood from human FcαRI/FcγRI double-transgenic mice showed corresponding results, allowing the establishment of relevant syngenic animal models in these mice. In conclusion, the combination of myeloid growth factors and an (FcαRI × CD20) BsAb may represent a promising approach to improve effector cell recruitment for CD20-directed lymphoma therapy.

Systemic Inflammation Modulates Fc Receptor Expression on Microglia during Chronic Neurodegeneration

Chronic neurodegeneration is a major worldwide health problem, and it has been suggested that systemic inflammation can accelerate the onset and progression of clinical symptoms. A possible explanation is that systemic inflammation “switches” the phenotype of microglia from a relatively benign to a highly aggressive and tissue-damaging phenotype. The current study investigated the molecular mechanism underlying this microglia phenotype “switching.” We show in mice with chronic neurodegeneration (ME7 prion model) that there is increased expression of receptors that have a key role in macrophage activation and associated signaling pathways, including TREM-2, Siglec-F, CD200R, and FcγRs. Systemic inflammation induced by LPS further increased protein levels of the activating FcγRIII and FcγRIV, but not of other microglial receptors, including the inhibitory FcγRII. In addition to these changes in receptor expression, IgG levels in the brain parenchyma were increased during chronic neurodegeneration, and these IgG levels further increased after systemic inflammation. γ-Chain–deficient mice show modified proinflammatory cytokine expression in the brain after systemic inflammation. We conclude that systemic inflammation during chronic neurodegeneration increases the expression levels of activating FcγR on microglia and thereby lowers the signaling threshold for Ab-mediated cell activation. At the same time, IgG influx into the brain could provide a cross-linking ligand resulting in excessive microglia activation that is detrimental to neurons already under threat by misfolded protein.

T Cell Immunity to Lymphoma Following Treatment with Anti-CD40 Monoclonal Antibody

In this study we demonstrate that treatment with anti-CD40 mAb eradicates a range of mouse lymphomas (BCL1, A31, A20, and EL4), but only when used against i.v. tumor doses in excess of 107 cells. Only partial protection was seen against smaller tumor loads. We saw no evidence that anti-CD40 mAb changed the phenotype of the lymphomas or inhibited their growth in the initial period following treatment, but it did result in a rapid expansion of cytotoxic CD8+ cells that was able to clear the neoplastic disease and provide long-term protection against tumor rechallenge. The CTL responses were blocked by mAb against a range of coreceptors and cytokines, including CD8, B7-1, B7-2, LFA-1, and IFN-γ, but not CD4 or CTLA-4, indicating the presence of a conventional cellular Th1 response. Furthermore, we found evidence of cross-recognition between lymphomas (BCL1 and A20) as measured by cytotoxicity and IFN-γ responses in vitro and using tumor rechallenge experiments, suggesting common target Ags. Finally, although anti-CD40 was shown to stimulate NK cell killing, we could find no role for these cells in controlling tumor growth. These data underline the ability of anti-CD40 mAb to potentiate CTL responses and the potency of cellular immunity in eradicating large quantities of syngeneic tumor.

CD40 ligation for immunotherapy of solid tumours

Tumour vaccines provide an important focus of current cancer research and are often based on the premise that although T-cells do respond naturally to certain tumours, this is usually weak and therefore ineffective at controlling disease. An integral and necessary part of a T-cell immune response involves triggering of CD40 on antigen-presenting cells (APC) by its ligand, CD154, on responding T helper (Th) cells. Furthermore, cytotoxic responses to tumours may fail because the Th-cell response is inadequate and unable to provide CD40 stimulation of APC. Growing evidence shows that stimulating APC with soluble CD40L or an agonistic anti-CD40 mAb can, at least in part, replace the need for Th cells and generate APC that are capable of priming cytotoxic T lymphocytes (CTL). The aim of this study was to investigate whether a range of solid tumours (CD40(-)) could be treated with anti-CD40 mAb. It was found that this treatment was effective, and correlated with the intrinsic immunogenicity and aggressiveness of the tumours. The mAb could be delivered locally or at a distal site, but increased antigen load provided by irradiated tumour cells added little to the effectiveness of the treatment. T-cells were required since cytokine (interferon-gamma) and CTL activity were demonstrated following treatment and the therapeutic efficacy was lost in nude mice. In addition, depletion of CD8(+) cells abrogated protection whilst depletion of CD4(+) cells had no effect. This study demonstrates that solid CD40(-) tumours are sensitive to anti-CD40 mAb therapy and that the response bypasses the need for Th cells.

Control of established melanoma by CD27 stimulation is associated with enhanced effector function and persistence, and reduced PD-1 expression, of tumor infiltrating CD8+ T cells

The immune response to the tumor can be enhanced by targeting costimulatory molecules on T cells. As the CD70-CD27 costimulatory axis plays an important role in the activation, survival, and differentiation of lymphocytes, we have examined the efficacy of agonistic anti-CD27 antibodies as monotherapies for established melanoma in a murine model. We show that this approach leads to a substantial reduction in the outgrowth of both experimental lung metastases and subcutaneous tumors. Anti-CD27 treatment supports the maintenance of tumor-specific CD8+ T cells within the tumor, reduces the frequency of FoxP3-expressing CD4+ T cells within tumors, and potentiates the ability of NK1.1+ and CD8+ tumor infiltrating cells to secrete IFNγ upon coculture with tumor cells. The enhanced effector function correlated with lower levels of PD-1 expression on CD8+ T cells from anti-CD27–treated mice. Despite the modulating effect of anti-CD27 on multiple cell types, only CD8+ T cells were absolutely required for tumor control. The CD4+ T cells were dispensable, whereas NK1.1+ cells were needed during early stages of tumor growth but not for the effectiveness of anti-CD27. Thus, CD27-mediated costimulation provides a potent boost to multiple aspects of the endogenous responses to tumor, and may be exploited to enhance tumor immunity.

Agonist Anti-Human CD27 Monoclonal Antibody Induces T Cell Activation and Tumor Immunity in Human CD27–Transgenic Mice

The CD70/CD27 pathway plays a significant role in the control of immunity and tolerance, and previous studies demonstrated that targeting murine CD27 (mCD27) with agonist mAbs can mediate antitumor efficacy. We sought to exploit the potential of this pathway for immunotherapy by developing 1F5, a fully human IgG1 mAb to human CD27 (hCD27) with agonist activity. We developed transgenic mice expressing hCD27 under control of its native promoter for in vivo testing of the Ab. The expression and regulation of hCD27 in hCD27-transgenic (hCD27-Tg) mice were consistent with the understood biology of CD27 in humans. In vitro, 1F5 effectively induced proliferation and cytokine production from hCD27-Tg–derived T cells when combined with TCR stimulation. Administration of 1F5 to hCD27-Tg mice enhanced Ag-specific CD8+ T cell responses to protein vaccination comparably to an agonist anti-mCD27 mAb. In syngeneic mouse tumor models, 1F5 showed potent antitumor efficacy and induction of protective immunity, which was dependent on CD4+ and CD8+ T cells. The requirement of FcR engagement for the agonistic and antitumor activities of 1F5 was demonstrated using an aglycosylated version of the 1F5 mAb. These data with regard to the targeting of hCD27 are consistent with previous reports on targeting mCD27 and provide a rationale for the clinical development of the 1F5 mAb, for which studies in advanced cancer patients have been initiated under the name CDX-1127.