anti-CTLA4 mAb Research Articles

Fcγ receptor dependency of agonistic CD40 antibody in lymphoma therapy can be overcome through antibody multimerization.

Immunomodulatory mAbs, led by the anti-CTLA4 mAb ipilimumab, are an exciting new class of drugs capable of promoting anticancer immunity and providing durable control of some tumors. Close analysis of a number of agents has revealed a critical yet variable role for Fcγ receptors in their efficacy. In this article, we reveal that agonistic anti-CD40 mAbs have an absolute requirement for cross-linking by inhibitory FcγRIIB when used systemically to treat established BCL1 syngeneic lymphoma, and therapy is lost when using a mouse IgG2a mAb not cross-linked by FcγRIIB. Furthermore, in FcγRIIB-deficient mice the lymphoma itself can provide FcγRIIB to cross-link anti-CD40 on neighboring cells, and only when this is blocked does therapy fail. The dependence on FcγRIIB for immunostimulatory activity was not absolute, however, because when anti-CD40 mAbs were administered systemically with the TLR3 agonist polyinosinic:polycytidylic acid or were given subcutaneously, activatory FcγR could also provide cross-linking. Using this mechanistic insight, we designed multimeric forms of anti-CD40 mAb with intrinsic FcγR-independent activity that were highly effective in the treatment of lymphoma-bearing mice. In conclusion, FcγR-independent anti-CD40 activation is a viable strategy in vivo. These findings have important translational implications, as humans, unlike mice, do not have IgG that binds strongly to FcγRIIB; therefore FcγR-independent derivatives represent an attractive therapeutic option.

Humanized mice as a model for aberrant responses in human T cell immunotherapy.

Immune-deficient mice, reconstituted with human stem cells, have been used to analyze human immune responses in vivo. Although they have been used to study immune responses to xenografts, allografts, and pathogens, there have not been models of autoimmune disease in which the mechanisms of the pathologic process can be analyzed. We have found that reconstituted "humanized" mice treated with anti-CTLA-4 Ab (ipilimumab) develop autoimmune disease characterized by hepatitis, adrenalitis, sialitis, anti-nuclear Abs, and weight loss. Induction of autoimmunity involved activation of T cells and cytokine production, and increased infiltration of APCs. When anti-CTLA-4 mAb-treated mice were cotreated with anti-CD3 mAb (teplizumab), hepatitis and anti-nuclear Abs were no longer seen and weight loss did not occur. The anti-CD3 blocked proliferation and activation of T cells, release of IFN-γ and TNF, macrophage infiltration, and release of IP-10 that was induced with anti-CTLA-4 mAb. We also found increased levels of T regulatory cells (CD25(+)CD127(-)) in the spleen and mesenteric lymph nodes in the mice treated with both Abs and greater constitutive phosphorylation of STAT5 in T regulatory cells in spleen cells compared with mice treated with anti-CTLA-4 mAb alone. We describe a model of human autoimmune disease in vivo. Humanized mice may be useful for understanding the mechanisms of biologics that are used in patients. Hepatitis, lymphadenopathy, and other inflammatory sequelae are adverse effects of ipilimumab treatment in humans, and this study may provide insights into this pathogenesis and the effects of immunologics on autoimmunity.

CTLA-4 Is Expressed by Activated Mouse NK Cells and Inhibits NK Cell IFN-γ Production in Response to Mature Dendritic Cells

NK cells express an array of activating and inhibitory receptors that determine NK cell responses upon triggering by cognate ligands. Although activating NK cell receptors recognize mainly ligands expressed by stressed, virus-infected, or transformed cells, most inhibitory receptors engage MHC class I, preventing NK cell activation in response to healthy cells. In this study, we provide insight into the regulation and function of additional receptors involved in mouse NK cell responses: CTLA-4 and CD28. CTLA-4 and CD28 engage the same ligands, B7-1 and B7-2, which are primarily expressed by APCs, such as dendritic cells. Our data demonstrate that activation of mouse NK cells with IL-2 induces the expression of CTLA-4 and upregulates CD28. CTLA-4 expression in IL-2-expanded NK cells was further up- or downregulated by IL-12 or TGF-β, respectively. Using gene-deficient NK cells, we show that CD28 induces, and CTLA-4 inhibits, IFN-γ release by NK cells upon engagement by the recombinant ligand, B7-1, or upon coculture with mature dendritic cells. Notably, we show that mouse NK cells infiltrating solid tumors express CD28 and CTLA-4 and respond to stimulation with recombinant B7-1, suggesting that the NK cell responses mediated by the CD28/CTLA-4:B7-1/B7-2 system could be of importance during malignant disease. Accordingly, our study might have implications for immunotherapy of cancer based on blocking anti-CTLA-4 mAbs.

Current status of cancer immunotherapy

To prove clinical benefits of cancer vaccine is currently difficult, except for one phase III trial has documented improved overall survival with the vaccine, Sipuleucel-T, although induction of anti-tumor immune responses through cancer vaccine is theoretically promising and would be straightforward. In contrast, immune checkpoint blockade with anti-CTLA4 mAb and anti-PD-1 mAb has demonstrated clear evidence of objective responses including improved overall survival and tumor shrinkage, driving renewed enthusiasm for cancer immunotherapy in multiple cancer types. In addition, there is a promising novel cancer immunotherapy, CAR therapy-a personalized treatment that involves genetically modifying a patient's T-cells to make them target tumor cells. We are now facing new era of cancer immunotherapy.

2B4 (CD244) induced by selective CD28 blockade functionally regulates allograft-specific CD8+ T cell responses

Mounting evidence in models of both autoimmunity and chronic viral infection suggests that the outcome of T cell activation is critically impacted by the constellation of co-stimulatory and co-inhibitory receptors expressed on the cell surface. Here, we identified a critical role for the co-inhibitory SLAM family member 2B4 (CD244) in attenuating primary antigen-specific CD8(+) T cell responses in the presence of immune modulation with selective CD28 blockade. Our results reveal a specific up-regulation of 2B4 on antigen-specific CD8(+) T cells in animals in which CD28 signaling was blocked. However, 2B4 up-regulation was not observed in animals treated with CTLA-4 Ig (abatacept) or CD28 blockade in the presence of anti-CTLA-4 mAb. 2B4 up-regulation after CD28 blockade was functionally significant, as the inhibitory impact of CD28 blockade was diminished when antigen-specific CD8(+) T cells were deficient in 2B4. In contrast, 2B4 deficiency had no effect on CD8(+) T cell responses during unmodified rejection or in the presence of CTLA-4 Ig. We conclude that blockade of CD28 signals in the presence of preserved CTLA-4 signals results in the unique up-regulation of 2B4 on primary CD8(+) effectors, and that this 2B4 expression plays a critical functional role in controlling antigen-specific CD8(+) T cell responses.

Local CTLA4 blockade effectively restrains experimental pancreatic adenocarcinoma growth in vivo

Antibody-mediated blockade of CTLA4 has been shown to be effective in treating a select group of patients with late-stage melanoma. The precise mechanism underlying the clinical activity of CTLA4 immunotherapy is poorly understood, although recent experimental findings indicate that antibody-mediated depletion of regulatory T cells (Tregs) in the tumor microenvironment plays a key role in efficacious antitumor responses. In the current study, we used an experimental model of pancreatic adenocarcinoma to compare the antitumor efficacy of peritumoral low-dose anti-CTLA4 monoclonal antibody (mAb) administration to that of a commonly utilized systemic high-dose anti-CTLA4 regimen. We selected pancreatic adenocarcinoma as it presents a particular challenge to clinicians due to its aggressive behavior, metastatic spread and limited treatment options. Furthermore, Fc gamma receptor (FcγR)-dense myeloid cells commonly infiltrate pancreatic tumors, such that these tumor types exhibit increased susceptibility to CTLA4 antibody-targeted Treg depletion via antibody-dependent cell-mediated cytotoxicity (ADCC). Locally administered anti-CTLA4 mAb effectively reduced tumor growth at a low dose and no additional anti-tumor effects were apparent when increasing the dose or number of injections. No significant difference in overall survival was seen when comparing locally administered low-dose with standard systemic high-dose CTLA4 blockade therapy, and both delivery routes led to increased tumor-infiltrating effector T cells and reduced Treg cells. As opposed to low-dose peritumoral treatment, high-dose systemic therapy stimulated the accumulation of Tregs in secondary lymphoid organs, an effect that could potentially counteract the antitumor immunotherapeutic benefit of CTLA4 blockade. Our study confirms previous findings that local administration of low-dose anti-CTLA4 antibody generates sustained antitumor effects and provides rationale to devise ultrasound-guided intratumoral anti-CTLA4 antibody injection regimens to treat patients with pancreatic adenocarcinoma and other types of solid tumors. In support, clinical relevancy could include reduced immune-related adverse events by limiting systemic antibody spread to immune cell-dense organs.

B7h (ICOS-L) Maintains Tolerance at the Fetomaternal Interface

In a successful pregnancy, the semiallogeneic fetus is not rejected by the maternal immune system, which implies tolerance mechanisms protecting fetal tissues from maternal immune attack. Here we report that the ICOS-B7h costimulatory pathway plays a critical role in maintaining the equilibrium at the fetomaternal interface. Blockade of this pathway increased fetal resorption and decreased fetal survival in an allogeneic pregnancy model (CBA female × B6 male). Locally in the placenta, levels of regulatory markers such as IDO and TGF-β1 were reduced after anti-B7h monoclonal antibody treatment, whereas levels of effector cytokines (eg, IFN-γ) were significantly increased. In secondary lymphoid organs, enhanced IFN-γ and granzyme B production (predominantly by CD8(+) T cells) was observed in the anti-B7h-treated group. The deleterious effect of B7h blockade in pregnancy was maintained only in CD4 knockout mice, not in CD8 knockout mice, which suggests a role for CD8(+) Tcells in immune regulation by the ICOS-B7h pathway. In accord, regulatory CD8(+) T cells (in particular, CD8(+)CD103(+) cells) were significantly decreased after anti-B7h monoclonal antibody treatment, and adoptive transfer of this subset abrogated the deleterious effect of B7h blockade in fetomaternal tolerance. Taken together, these data support the hypothesis that B7h blockade abrogates tolerance at the fetomaternal interface by enhancing CD8(+) effector response and reducing local immunomodulation mediated by CD8(+) regulatory T cells.

Abstract PR11: Enhanced in vivo persistence and therapeutic activity of combined transferred polyclonal melanoma-reactive CTL generated in presence of IL-21 and anti-CTLA4.

Abstract Monotherapy with a blocking human anti-CTLA4 mAb achieves disease control in 20-28% of patients (pts), but failure to benefit in the remaining patients may reflect inadequate induction and/or expansion of melanoma reactive T cells following CTLA4 blockade. We have been exploring a combined biologic strategy involving the adoptive transfer of melanoma-reactive CD8+ cytotoxic T lymphocytes (CTL) with concurrent administration of anti-CTLA4 as a means to enhance and broaden the anti-tumor effect of the transferred CTL. Autologous melanoma-specific CTL were generated by stimulating CTL lines in the presence of the γc-chain cytokine Interleukin-21 (IL-21), which programs responding antigen-specific cells to retain characteristics of central memory T cells (Tcm). Responding cells were sorted for binding of a peptide-MHC (pMHC) multimer to obtain a purified tumor-reactive polyclonal product. We report on 7 pts with bulky, therapy-resistant melanoma who received 1010/m2 polyclonal IL-21-derived CTL lines targeting MART1. The infusions were preceded by intravenous cyclophosphamide 300 mg/m2 and followed by low-dose subcutaneous IL-2 (250,000 IU/m2 BID) x 14 days to promote in vivo expansion and survival. Anti-CTLA4 (ipilimumab) was administered at 3mg/kg every three weeks for four doses, starting on the day after the CTL infusion. The treatment was well tolerated, with no unexpected or serious (grade ≥3) related toxicities. 5/7 evaluable pts exhibited stable disease (SD) or partial responses (PR) by RECIST 1.1 criteria at 6 weeks, and 4/6 exhibited SD at 12 weeks. One pt who had failed previous ipilimumab monotherapy and prior CTL therapy targeting MART1 without CTLA4 blockade had a noteworthy response to the current protocol. He had SD at 12 weeks, then experienced a delayed objective response with 66% reduction in tumor mass at 28+ weeks. Persistence of the infused CTL at frequencies >2x baseline levels was documented at 6 weeks (6/6 evaluable pts) and 12 weeks (4/4 evaluable pts), whereas such levels of persistence beyond 6 weeks was previously typically observed in only 15-20% of pts in previous studies. The cells infused into each pt expressed moderate/detectable levels of CD28 prior to infusions, but analysis of persisting CTL detected by specific multimer binding in vivo in responding pts revealed progressively higher expression of CD28, consistent with enhanced CD28:CD80/86 interactions in the presence of CTLA4 blockade providing survival advantages. Additionally, infused multimer+ T cells continued to proliferate (multimer+ cells expressed Ki-67), secreted multiple cytokines (IFNγ, TNFα and IL-2) in response to cognate antigen, and acquired phenotypic characteristics of Tcm (including expression of CD27, CD127, CD62L and CCR7) within 12 weeks of observation after infusion. Most significantly, all pts who had clinical benefit (prolonged SD or PR) developed de novo anti-tumor responses (epitope spreading) to non-targeted antigens (NY-ESO-1, Tyrosinase, gp100 and MAGE A3) within 12 weeks, potentially reflecting release of tumor antigens following tumor lysis by the infused CTL in a pro-inflammatory, pro-immunogenic context induced by concurrent anti-CTLA4 blockade. Our preliminary results suggest that treatment with polyclonal, melanoma-targeting CTL generated in the presence of IL-21 and combined with ipilimumab is safe. Blockade of CTLA4 may enhance the activity/persistence of the infused melanoma-specific T cells in vivo. Additionally, CTL-induced tumor cell lysis in the presence of CTLA4 blockade may promote induction of anti-tumor responses to non-targeted antigens, enhancing the biologic activity of the combined regimen. This abstract is also presented as Poster A14. Citation Format: Aude G. Chapuis, John A. Thompson, Kim A. Margolin, David Byrd, Shailender Bhatia, Sylvia M. Lee, Ivy P. Lai, Ilana M. Roberts, Heather L. Sloan, Philip D. Greenberg, Cassian Yee. Enhanced in vivo persistence and therapeutic activity of combined transferred polyclonal melanoma-reactive CTL generated in presence of IL-21 and anti-CTLA4. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr PR11.

Abstract P4-04-01: Combination of intratumoral CpG with systemic anti-OX40 and anti-CTLA4 mAbs eradicates established triple negative breast tumors in mice

Abstract Background: Evidence is mounting across diverse datasets and scientific techniques implicating immune dysregulation in poor outcomes of high grade and/or triple negative breast cancers. A multimodal strategy to stimulate the immune system could be an effective therapeutic approach for breast cancer. CpG oligodeoxynucleotides are single-stranded DNA that stimulate dendritic cells and B cells by binding to Toll-like receptor 9 (TLR9). OX40 (CD134) is predominantly expressed on activated T cells and the binding of OX40 with an agonistic antibody (anti-OX40) leads to the proliferation and survival of T cells. Agonistic anti-OX40 antibodies can also reverse the suppressive effects of Treg cells. A third avenue is to target Cytotoxic T lymphocyte associated antigen (CTLA4), which plays a negative regulatory role in T cell activation and is also constitutively expressed on Treg cells. Blocking CTLA4 with an antagonistic antibody (anti-CTLA4) has been shown to enhance anti-tumor responses. In this study, we tested the combination of intratumoral (IT) CpG with systemic anti-OX40 plus anti-CTLA4 in a mouse model of triple negative breast cancer. Methods: 4T1, an aggressive, triple-negative mammary carcinoma, was implanted orthotopically into syngeneic BALB/C mice. Treatment was initiated when tumors were ∼0.5 cm in the largest dimension. Mice were divided into 8 groups. Various combinations of PBS control, CpG, taxol, and anti-OX40+anti-CTLA4 mAbs were tested (Tables 1 and 2). CpG, 100 µg, was injected IT for 5 consecutive days (days 1–5); Taxol, 16 mg/kg, was administered IP on day 1; anti-OX40 (400 µg) and anti-CTLA4 (100 µg) were given IP on days 1 and 5. Mice were sacrificed once tumors became ulcerated or reached a diameter >2 cm. Results: As shown in Table 1, CpG alone, taxol alone, or the anti-OX40 + anti-CTLA4 combination each delayed the growth of established 4T1 tumors when compared to the untreated control group. However, the majority of the mice (except one in CpG treated group) died from tumor progression. The combination of CpG + anti-OX40 + anti-CTLA4 had the best anti-tumor effects, with 4 out of 5 mice having demonstrated complete regression of their tumors (Table 2). The addition of taxol to the combination of CpG + anti-OX40 + anti-CTLA4 did not improve the anti-tumor effect, and while there was a substantial delay in growth, only one animal achieved a complete regression. Conclusion: Our study demonstrated that the combination of IT CpG with systemic anti-OX40 + anti-CTLA4 mAbs can cure mice with established triple negative breast tumors, supporting the possibilities of applying immune modulation approach in human triple negative tumors. Studies are on-going to further dissect the immunological mechanisms involved. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-04-01.

Hypophysitis Induced by Monoclonal Antibodies to Cytotoxic T Lymphocyte Antigen 4: Challenges from a New Cause of a Rare Disease

Specific human monoclonal antibodies antagonize cytotoxic T-lymphocyte antigen 4 (anti-CTLA-4 mAbs), a negative regulator of the immune system, inducing unrestrained T-cell activation. In patients with advanced or metastatic melanoma, one of these agents, ipilimumab, produced considerable disease control rates and, for the first time, a clear improvement in overall survival outcomes. However, accumulating clinical experience with anti-CTLA-4 mAbs identified a novel syndrome of autoimmune and autoinflammatory side effects, designated as "immune-related adverse events," including mainly rash, colitis, and hepatitis. Autoimmune hypophysitis has emerged as a distinctive side effect induced by anti-CTLA-4 mAbs. This condition may be life threatening because of adrenal insufficiency if not promptly recognized, but it may easily be diagnosed and treated if clinically suspected. Hypopituitarism caused by these agents is rarely reversible and prolonged or life-long substitutive hormonal treatment is often required. The precise mechanism of injury to the pituitary triggered by anti-CTLA-4 mAbs is yet to be fully elucidated.

Immunothérapie des cancers

Immunotherapy of cancer has long been considered as an attractive therapeutic approach but with no impact on clinical practice. Two clinical protocols of immunotherapy, one based on a cancer vaccine in patients with prostate cancer or melanoma and the other using an immunomodulator targeting T cells (anti-CTLA4 mAb) in melanoma patients, have demonstrated clinical efficacy in two phaseIII clinical trials. To improve these encouraging clinical results, biomarkers to better select patients which may benefit from this therapy are actively searched. In addition, immunosuppression associated with cancer has to be overcome to allow a better immunostimulation. In contrast to chemotherapy, clinical variables to monitor the efficacy of immunotherapy has to be revisited and overall survival appears to be a better endpoint than clinical response defined by the RECIST criteria. Combination of immunotherapy with conventional treatments (chemotherapy, anti-angiogenic, etc.) should further improve this approach both in its effectiveness and in its clinical indications.

Blockade of CTLA-4 Decreases the Generation of Multifunctional Memory CD4+ T Cells In Vivo

CTLA-4 is known as a central inhibitor of T cell responses. It terminates T cell activation and proliferation and induces resistance against activation induced cell death. However, its impact on memory formation of adaptive immune responses is still unknown. In this study, we demonstrate that although anti-CTLA-4 mAb treatment during primary immunization of mice initially enhances the number of IFN-γ-producing CD4(+) T cells, it does not affect the size of the memory pool. Interestingly, we find that the CTLA-4 blockade modulates the quality of the memory pool: it decreases the amount of specialized "multifunctional" memory CD4(+) T cells coproducing IFN-γ, TNF-α, and IL-2 in response to Ag. The reduction of these cells causes an immense decrease of IFN-γ-producing T cells after in vivo antigenic rechallenge. Chimeric mice expressing CTLA-4-competent and -deficient cells unmask, which these CTLA-4-driven mechanisms are mediated CD4(+) T cell nonautonomously. In addition, the depletion of CD25(+) T cells prior to the generation of Ag-specific memory cells reveals that the constitutively CTLA-4-expressing natural regulatory T cells determine the quality of memory CD4(+) T cells. Taken together, these results indicate that although the inhibitory molecule CTLA-4 damps the primary immune response, its engagement positively regulates the formation of a high-quality memory pool equipped with multifunctional CD4(+) T cells capable of mounting a robust response to Ag rechallenge.

Abstract SY24-02: Development of recombinant vaccines for the prevention and therapy of human carcinomas

Abstract SY24-02: Development of recombinant vaccines for the prevention and therapy of human carcinomas

Current Experience With CTLA4-blocking Monoclonal Antibodies for the Treatment of Solid Tumors

Monoclonal antibodies (mAbs) specific for cytotoxic T lymphocyte-associated antigen 4 (CTLA4) are a novel form of immunotherapy for treatment of patients with advanced cancers. These anti-CTLA4 mAbs prevent normal downregulation of the immune system, thus prolonging and enhancing T-cell activation and potentially promoting an antitumor immune response. Clinical studies in patients with advanced cancers have indicated that CTLA4 blockade with mAbs is associated with antitumor activity in a small percentage of patients and has a manageable toxicity profile. The key limitations for broader applicability of this mode of therapy are better definition of the mechanism that leads to tumor rejection and the validation of favorable observations in single-arm studies into prospectively randomized clinical trials.

8-Methoxypsoralen Plus Ultraviolet A Therapy Acts via Inhibition of the IL-23/Th17 Axis and Induction of Foxp3+ Regulatory T Cells Involving CTLA4 Signaling in a Psoriasis-Like Skin Disorder

To elucidate the molecular action of 8-methoxypsoralen plus UVA (PUVA), a standard dermatological therapy, we used K5.hTGF-beta1 transgenic mice exhibiting a skin phenotype and cytokine abnormalities with strong similarities to human psoriasis. We observed that impaired function of CD4+CD25+ regulatory T cells (Tregs) and increased cytokine levels of the IL-23/Th17 pathway were responsible for the psoriatic phenotype in this mouse model. Treatment of K5.hTGF-beta1 transgenic mice with PUVA suppressed the IL-23/Th17 pathway, Th1 milieu, as well as transcription factors STAT3 and orphan nuclear receptor RORgammat. PUVA induced the Th2 pathway and IL-10-producing CD4+CD25+Foxp3+Tregs with disease-suppressive activity that was abolished by anti-CTLA4 mAb treatment. These findings were paralleled by macroscopic and microscopic clearance of the diseased murine skin. Anti-IL-17 mAb treatment also diminished the psoriatic phenotype of the mice. This indicated that both induced Tregs involving CTLA4 signaling and inhibition of the IL-23/Th17 axis are central for the therapeutic action of PUVA.

Association of adverse events and tumor response in melanoma patients treated with tremelimumab.

e13104 Background: Several analyses of data from trials of anti-CTLA4 mAbs have suggested a relationship between antitumor activity and certain AEs. Such associations are confounded by the fact that responding patients receive more doses of drug and have longer follow- up. An analysis was done using data from 2 pivotal tremelimumab melanoma studies to determine whether certain AEs in the first treatment cycle (90 days) predict response to tremelimumab. This time period was chosen in order to decrease the difference in follow up time between responders and nonresponders. Methods: Tremelimumab was administered at 15 mg/kg Q90days in studies A3671008 and A3671009. Adverse events were reported for 90 days post last dose or until start of new therapy. RECIST responders were determined by independent radiological review for A3671008 and by sponsor using investigator measurements for A3671009. The following AE categories were analyzed: diarrhea/colitis, all grades and grade ≥3; endocrine events, rash, and pruritus. Results: 37 of 325 treated pts in A3671009 and 16 of 246 treated pts in A3671008 achieved response. All responders and 67% of nonresponders remained on study at least 80 days. Most tumor responders received ≥4 doses and were on study for ≥1 year. Most nonresponders received only a single dose. Rash but not pruritus was more frequent in the first cycle in responders compared to nonresponders. Rates of diarrhea and of severe diarrhea in the first cycle were similar in responders and nonresponders. None of the responders reported endocrine events in the first cycle. Conclusions: Endocrine and GI AEs occurring in the first cycle did not predict response. The higher incidence of endocrine and GI events AEs reported on study in pts with tumor response is due to the greater number of doses received and longer time on study. Responders were more likely than nonresponders to report rash, but not pruritus, in the first cycle; however, about half of the responders did not experience rash in the first cycle, so appearance of rash should not be used to decide whether to continue therapy. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Pfizer Pfizer

Combination Therapy of Established Tumors by Antibodies Targeting Immune Activating and Suppressing Molecules

The blockade of immune suppression against antitumor responses is a particularly attractive strategy when combined with agents that promote tumor-specific CTLs. In this study, we have attempted to further improve the CTL induction and potent antitumor efficacy of a combination mAb-based therapy (termed "trimAb therapy") that comprises tumor cell death-inducing anti-death receptor 5 mAb and immune activating anti-CD40 and anti-CD137 mAbs. Among trimAb-treated tumors, the infiltration of CD4(+) Foxp3(+) cells was greater in progressing tumors compared with stable tumors. Blockade of CTLA-4 (CD152)-mediated signals by an antagonistic mAb substantially increased the tumor rejection rate of trimAb therapy, although the immune responses of draining lymph node cells were not augmented. Interestingly, by comparison, additional treatment with agonistic anti-glucocorticoid-induced TNF receptor mAb, antagonistic anti-programmed death-1 (CD279) mAb, or agonistic anti-OX40 (CD134) mAb significantly augmented immune responses of draining lymph node cells, but did not augment the therapeutic effect of trimAb. CD4 T cell depletion reduced the antitumor effect of anti-CTLA-4 mAb treatment alone, but did not reduce the tumor rejection rate of trimAb in conjunction with anti-CTLA-4 mAb. Thus, the blockade of the CTLA-4-mediated inhibitory signal in tumor infiltrating CTL may be the most effective strategy to augment the effect of immune therapies that generate tumor-specific CTL.

Two Distinct Mechanisms of Augmented Antitumor Activity by Modulation of Immunostimulatory/Inhibitory Signals

Blockade of CTL-associated antigen-4 (CTLA-4), an inhibitory immunomodulatory molecule on T cells, has been shown to enhance T-cell responses and induce tumor rejection, and a number of clinical trials with anti-CTLA-4 blocking monoclonal antibody (mAb) are under way. However, accumulating evidence indicates that anti-CTLA-4 mAb increases the number of CD4+CD25+Foxp3+ regulatory T cells (Treg) and that anti-CTLA4 mAb alone is often insufficient to reject established tumors in mice and humans. Thus, finding maneuvers to control Tregs and other immunosuppressive mechanisms remains a critical challenge. The potential to enhance antitumor immune responses by combining anti-CTLA-4 mAb with anti-glucocorticoid-induced tumor necrosis factor receptor family related gene (GITR) mAb, a costimulatory molecule that abrogates directly/indirectly Treg-mediated immune suppression or anti-CD25 mAb that depletes Tregs was analyzed with two tumor models, CT26 (a murine colon carcinoma cell line) and CMS5a (a murine fibrosarcoma cell line). Anti-CTLA-4/anti-GITR mAb combination treatment exhibited far stronger antitumor effects compared with either antibody alone. This strong antitumor effect was attributed to (a) increased numbers of CD8+ T cells infiltrating tumor sites in anti-CTLA-4 mAb-treated mice and (b) increased cytokine secretion and Treg resistance of tumor-specific CD8+ T cells with strongly upregulated CD25 expression in anti-GITR mAb-treated mice, indicating distinct quantitative/qualitative changes induced by modulating CTLA-4 and GITR signaling. This study shows that combined treatment with different immune modulators can augment antitumor immune responses and provides justification for exploring anti-CTLA-4/anti-GITR mAb combination treatment in the clinic.

Abstract 5329: Blocking multiple coinhibitory pathways for immunotherapy of established melanoma

Abstract Previous studies have shown that coinhibitory molecules like PD-L1 and B7-H3 are upregulated on a variety of tumor cells and high expression correlates with poor prognosis. Recent studies also showed that coinhibitory receptors like CTLA4, PD-1, LAG3 and CD160 were up-regulated in exhausted CD8 T cells during chronic infection, and blocking of coinhibitory pathways could reverse the exhaustion phenotype and restore CD8 T cell effector functions. Our previous studies have shown that HVEM binding to CD160 delivered a coinhibitory signal to T cell activation. Deletion of the CRD1 domain of HVEM (HDD1) converted HVEM from a dominant coinhibitory molecule into an exclusively costimulatory molecule, making HDD1 a good candidate to boost anti-tumor immune responses. Our preliminary studies showed that HVEM was up-regulated in human melanoma cell lines, and mice challenged with B16 melanoma had increased expression of HVEM on a subset of B cells. To determine if targeting the HVEM pathway could enhance anti-melanoma immune responses, we transduced B16F10 tumor cells with the mutant costimulatory HVEM (HDD1). Our data showed that engagement of HDD1 enhanced NF-kB signaling, which correlated with enhanced susceptibility of B16 cells to irradiation-induced apoptosis. Our in vivo data showed that tumor vaccination with irradiated B16-HDD1 combined with dual blockade of CTLA4 and PD1 using UC10 and 29F.1A12 mAbs (triple treatment, on day 0, 3, 6 and 9) significantly enhanced survival from 0 to 75% when the treatment started the same day as tumor challenge (50,000 live B16F10 cells subcutaneously). In contrast, treatment with any component alone or in dual combination resulted in suboptimal efficacies ranging from 30-40% survival. The enhanced survival with triple treatment was associated with enhanced infiltration of neutrophils, CD4 and CD8 T cells into tumor sites. Trp2 tumor antigen-specific CD8 T cell production of IFN-gamma was also increased in triple treated mice compared to control groups. In addition, in an established tumor model, when the triple treatment started on day 10 after the tumor was established, triple treatment gave 30% survival rate. When another anti-CTLA4 mAb, 9H10, was used instead of UC10, survival with triple treatment was increased to 80%. In addition, depletion of CD4 T cells using GK1.5 mAb further enhanced survival to tumor challenge; however, this correlated with enlarged draining lymph nodes associated with severe skin irritation, indicating possible autoimmune responses due to depletion of regulatory CD4 T cells. Depletion of CD8 T cells during triple treatment only slightly reduced the efficacy of triple treatment, indicating that other effector cells such as CD4 and NK cells also contribute to the anti-tumor immune response. In summary, our data demonstrated that triple treatment could be used as an efficient immunotherapy for melanoma through enhancing tumor specific immune responses. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5329.

What Is the Role of Cytotoxic T Lymphocyte–Associated Antigen 4 Blockade in Patients with Metastatic Melanoma?

With increasing knowledge of the molecular basis of the immune system and mechanisms of tumor tolerance, novel approaches to treating malignant diseases refractory to standard therapies are being investigated. Monoclonal antibodies (mAbs) that bind cytotoxic T lymphocyte-associated antigen (CTLA)-4 can block inhibitory signals normally generated through this receptor, thus prolonging and sustaining T-cell activation and proliferation. These antibodies are being developed and tested in patients with metastatic melanoma. This article reviews data published or presented at scientific congresses describing the clinical safety and antitumor activity of two different anti-CTLA-4 mAbs: tremelimumab (CP-675,206) and ipilimumab (MDX-010). Overall, although the response rate has not been consistently higher than the response rates associated with other treatments, the induction of durable responses and the favorable safety profile observed with anti-CTLA-4 mAbs are encouraging. However, the true advantage of these new drugs may depend largely on the characterization of predictive biomarkers of activity and subsequent targeting of responsive patients.