Focusing and sustaining the antitumor CTL effector killer response by agonist anti-CD137 mAb.

Cancer immunotherapy is undergoing significant progress due to recent clinical successes by refined adoptive T-cell transfer and immunostimulatory monoclonal Ab (mAbs). B16F10-derived OVA-expressing mouse melanomas resist curative immunotherapy with either adoptive transfer of activated anti-OVA OT1 CTLs or agonist anti-CD137 (4-1BB) mAb. However, when acting in synergistic combination, these treatments consistently achieve tumor eradication. Tumor-infiltrating lymphocytes that accomplish tumor rejection exhibit enhanced effector functions in both transferred OT-1 and endogenous cytotoxic T lymphocytes (CTLs). This is consistent with higher levels of expression of eomesodermin in transferred and endogenous CTLs and with intravital live-cell two-photon microscopy evidence for more efficacious CTL-mediated tumor cell killing. Anti-CD137 mAb treatment resulted in prolonged intratumor persistence of the OT1 CTL-effector cells and improved function with focused and confined interaction kinetics of OT-1 CTL with target cells and increased apoptosis induction lasting up to six days postadoptive transfer. The synergy of adoptive T-cell therapy and agonist anti-CD137 mAb thus results from in vivo enhancement and sustainment of effector functions.

Anti-CD137 monoclonal antibodies and adoptive T cell therapy: a perfect marriage?

CD137(4-1BB) costimulation and adoptive T cell therapy strongly synergize in terms of achieving maximal efficacy against experimental cancers. These costimulatory biological functions of CD137 have been exploited by means of introducing the CD137 signaling domain in clinically successful chimeric antigen receptors and to more efficiently expand T cells in culture. In addition, immunomagnetic sorting of CD137-positive T cells among tumor-infiltrating lymphocytes selects for the fittest antitumor T lymphocytes for subsequent cultures. In mouse models, co-infusion of both agonist antibodies and T cells attains marked synergistic effects that result from more focused and intense cytolytic activity visualized under in vivo microscopy and from more efficient entrance of T cells into the tumor through the vasculature. These several levels of dynamic interaction between adoptive T cell therapy and CD137 offer much opportunity to raise the efficacy of current cancer immunotherapies.

T cell costimulation with anti-CD137 monoclonal antibodies is mediated by K63-polyubiquitin-dependent signals from endosomes.

Agonist anti-CD137 (4-1BB) mAbs enhance CD8-mediated antitumor immunity. Agonist anti-human CD137 mAbs binding to four distinct epitopes on the CD137 glycoprotein costimulated T cell activation irrespective of the engaged epitope or its interference with CD137L binding. CD137 perturbation with all these agonist mAbs resulted in Ag and Ab internalization toward an endosomal vesicular compartment. Internalization was observed in activated T lymphocytes from humans and mice, not only in culture but also in Ab-injected living animals. These in vivo experiments were carried out upon systemic i.v. injections with anti-CD137 mAbs and showed CD137 internalization in tumor-infiltrating lymphocytes and in activated human T cells transferred to immunodeficient mice. Efficient CD137 internalization required K63 polyubiquitination and endocytosed CD137-containing vesicles recruited TNFR-associated factor (TRAF) 2 and were decorated with K63 polyubiquitins. CD137 stimulation activates NF-κB through a K63-linked polyubiquitination-dependent route, and CD137-associated TRAF2 becomes K63 polyubiquitinated. Consistent with a role for TRAF2 in CD137 signaling, transgenic mice functionally deficient in TRAF2 showed delayed immunotherapeutic activity of anti-CD137 mAbs. As a whole, these findings advance our knowledge of the mechanisms of action of anti-CD137 immunostimulatory mAbs such as those currently undergoing clinical trials in cancer patients.

CD137 on inflamed lymphatic endothelial cells enhances CCL21-guided migration of dendritic cells.

CD137/TNFR9/41BB was originally described as a surface molecule present on activated T and NK cells. However, its expression is broader among leukocytes, and it is also detected on hypoxic endothelial cells and inflamed blood vessels, as well as in atherosclerotic lesions. Here, we demonstrate that lymphatic endothelial cells (LECs) up-regulate CD137 expression from undetectable baseline levels on stimulation with TNF-α, LPS, and IL-1ß. CD137 cross-linking with an agonistic mAb results in NF-κB nuclear translocation, followed by up-regulation of VCAM and a 3-fold increase in the production of the chemokine CCL21. Accordingly, there is a 50% increase in CCR7-dependent migration toward conditioned medium from activated LECs on CD137 cross-linking with the agonistic mAb or the natural ligand (CD137L). Such an enhancement of cell migration is also observed with monocyte-derived dendritic cells transmigrating across CD137-activated LEC monolayers. Using explanted human dermal tissue, we found that inflamed skin contains abundant CD137(+) lymphatic vessels and that ex vivo incubation of explanted human dermis with TNF-α induces CD137 expression in lymphatic capillaries. More interestingly, treatment with CD137 agonistic antibody induces CCL21 expression and DC accumulation close to lymphatic vessels. Collectively, our results demonstrate that the inflammatory function of lymphatic vessels can be regulated by CD137.

Pilot clinical trial of type 1 dendritic cells loaded with autologous tumor lysates combined with GM-CSF, pegylated IFN, and cyclophosphamide for metastatic cancer patients.

Twenty-four patients with metastatic cancer received two cycles of four daily immunizations with monocyte-derived dendritic cells (DC). DC were incubated with preheated autologous tumor lysate and subsequently with IFN-α, TNF-α, and polyinosinic:polycytidylic acid to attain type 1 maturation. One DC dose was delivered intranodally, under ultrasound control, and the rest intradermally in the opposite thigh. Cyclophosphamide (day -7), GM-CSF (days 1-4), and pegIFN alpha-2a (days 1 and 8) completed each treatment cycle. Pretreatment with cyclophosphamide decreased regulatory T cells to levels observed in healthy subjects both in terms of percentage and in absolute counts in peripheral blood. Treatment induced sustained elevations of IL-12 in serum that correlated with the output of IL-12p70 from cultured DC from each individual. NK activity in peripheral blood was increased and also correlated with the serum concentration of IL-12p70 in each patient. Circulating endothelial cells decreased in 17 of 18 patients, and circulating tumor cells markedly dropped in 6 of 19 cases. IFN-γ-ELISPOT responses to DC plus tumor lysate were observed in 4 of 11 evaluated cases. Tracing DC migration with [(111)In] scintigraphy showed that intranodal injections reached deeper lymphatic chains in 61% of patients, whereas with intradermal injections a small fraction of injected DC was almost constantly shown to reach draining inguinal lymph nodes. Five patients experienced disease stabilization, but no objective responses were documented. This combinatorial immunotherapy strategy is safe and feasible, and its immunobiological effects suggest potential activity in patients with minimal residual disease. A randomized trial exploring this hypothesis is currently ongoing.

Pembrolizumab as Second-Line Therapy for Advanced Hepatocellular Carcinoma: Longer Term Follow-Up from the Phase 3 KEYNOTE-240 Trial.

Introduction: KEYNOTE-240 showed a favorable benefit/risk profile for pembrolizumab versus placebo in patients with sorafenib-treated advanced hepatocellular carcinoma (HCC); however, prespecified statistical significance criteria for overall survival (OS) and progression-free survival (PFS) superiority were not met at the final analysis. Outcomes based on an additional 18 months of follow-up are reported. Methods: Adults with sorafenib-treated advanced HCC were randomized 2:1 to pembrolizumab 200 mg intravenously every 3 weeks or placebo. Dual primary endpoints were OS and PFS assessed per RECIST v1.1 by blinded independent central review (BICR). Secondary endpoints included objective response rate (ORR), assessed per RECIST v1.1 by BICR, and safety. Results: 413 patients were randomized (pembrolizumab, n = 278; placebo, n = 135). As of July 13, 2020, median (range) time from randomization to data cutoff was 39.6 (31.7-48.8) months for pembrolizumab and 39.8 (31.7-47.8) months for placebo. Estimated OS rates (95% CI) were 17.7% (13.4-22.5%) for pembrolizumab and 11.7% (6.8-17.9%) for placebo at 36 months. The estimated PFS rate (95% CI) for pembrolizumab was 8.9% (5.3-13.6%) and 0% for placebo at 36 months. ORR (95% CI) was 18.3% (14.0-23.4%) for pembrolizumab and 4.4% (1.6-9.4%) for placebo. Immune-mediated hepatitis events did not increase with follow-up. No viral hepatitis flare events were reported. Conclusion: With extended follow-up, pembrolizumab continued to maintain improvement in OS and PFS and was associated with a consistent adverse event profile compared with placebo in patients with sorafenib-treated advanced HCC. Although KEYNOTE-240 did not meet prespecified statistical significance criteria at the final analysis, these results together with the antitumor activity of second-line pembrolizumab observed in KEYNOTE-224 and the statistically significant and clinically meaningful OS and PFS benefits of second-line pembrolizumab in patients from Asia observed in KEYNOTE-394 reinforce the clinical activity of pembrolizumab in previously treated patients with advanced HCC.

Workshop on immunotherapy combinations. Society for Immunotherapy of Cancer annual meeting Bethesda, November 3, 2011.

Although recent FDA approvals on ipilimumab and sipuleucel-T represent major milestones, the ultimate success of immunotherapy approaches will likely benefit from appropriate combinations with other immunotherapeutic and/or non-immunotherapeutic approaches. However, implementation of ideal combinations in the clinic may still face formidable challenges in regulatory, drug-availability and intellectual property aspects. The 2011 SITC annual meeting hosted a workshop on combination immunotherapy to discuss: 1) the most promising combinations found in the laboratory; 2) early success of combination immunotherapy in clinical trials; 3) industry perspectives on combination approaches, and 4) relevant regulatory issues. The integrated theme was how to accelerate the implementation of efficacious combined immunotherapies for cancer patients. Rodent animal models are providing many examples of synergistic combinations that typically include more than two agents. However, mouse and human immunology differ in a significant number of mechanisms and hence we might be missing opportunities peculiar to humans. Nonetheless, incisive animal experimentation with deep mechanistic insight remains the best compass that we can use to guide our paths in combinatorial immunotherapy. Combination immunotherapy clinical trials are already in progress and preliminary results are extremely promising. As a key to translate promising combinations into clinic, real and "perceived" business and regulatory hurdles were debated. A formidable step forward would be to be able to test combinations of investigational agents prior to individual approval. Taking together the FDA and the industrial perspective on combinatorial immunotherapy, the audience was left with the clear message that this is by no means an impossible task. The general perception is that the road ahead of us is full of combination clinical trials which hopefully will bring clinical benefit to our cancer patients at a fast pace.

Intratumoral injection of interferon-α and systemic delivery of agonist anti-CD137 monoclonal antibodies synergize for immunotherapy.

CD137 artificial costimulation results in complete tumor rejection in several mouse models. Type I interferons (IFN) exert antitumor effects through an array of molecular functions on malignant cells, tumor stroma and immune system cells. The fact that agonist anti-CD137 mAb induce tumor regressions in mice deficient in the unique receptor for Type I IFNs (IFNAR(-/-) ) indicated potential for treatment combinations. Indeed, combination of intratumor injections of mouse IFN-α and intraperitoneal injections of anti-CD137 mAb synergized as seen on subcutaneous lesions derived from the MC38 colon carcinoma, which is resistant to each treatment if given separately. Therapeutic activity was achieved both against lesions directly injected with IFN-α and against distant concomitant tumors. Experiments in bone marrow chimeras prepared with IFNAR(-/-) and WT mice concluded that expression of the receptor for Type I interferons is mainly required on cells of the hematopoietic compartment. Synergistic effects correlated with a remarkable cellular hyperplasia of the tumor draining lymph nodes (TDLNs). Enlarged TDLNs contained more plasmacytoid and conventional dendritic cells (DC) that more readily cross-presented. Importantly, numbers of both DC subtypes inversely correlated with the tumor size. Numbers of CD8 T cells specific for a dominant tumor antigen were increased at TDLNs by each separate treatment but only with slight augments due to the combination. Combined antitumor effects of the therapeutic strategy were also seen on subcutaneous TC-1 tumors established for 24 days before treatment onset. The described strategy is realistic because (i) agents of each kind are clinically available and (ii) equivalent procedures in humans are feasible.

Synergistic effects of CTLA-4 blockade with tremelimumab and elimination of regulatory T lymphocytes in vitro and in vivo.

Anti-CTLA-4 monoclonal antibodies (mAb) that block the interaction of CTLA-4 with CD80 and CD86 such as tremelimumab and ipilimumab are currently being tested in the clinic for cancer treatment exploiting their properties to de-repress tumor-specific cellular immunity. Addition of the fully human anti-CTLA-4 (tremelimumab) to cultures of human T cells with allogenic dendritic cells (DCs) did not increase proliferation. Magnetic bead-mediated elimination of CD4(+) CD25(+) regulatory T cells (T(reg)) before setting up those alloreactive cultures also largely failed to increase primary proliferation. In contrast, predepletion of CD4(+) CD25(+) T(reg) and culture in the presence of tremelimumab synergistically resulted in increased proliferation and DC:T-cell aggregation. These effects were much more prominent in CD4 than in CD8 T cells. The synergy mechanism can be traced to enhanced CTLA-4 expression in effector cells as a result of T(reg) elimination, thereby offering more targets to the blocking antibody. Human T cells and allogenic DCs (derived both from healthy donors and advanced cancer patients) were coinjected in the peritoneum of Rag2(-/-) IL-2Rγ(-/-) mice. In these conditions, tremelimumab injected intravenously did not significantly enhance alloreactive proliferation unless T(reg) cells had been predepleted. Synergistic effects in vivo were again largely restricted to the CD4 T-cell compartment. In addition, T(reg) depletion and CTLA-4 blockade synergistically enhanced specific cytotoxicity raised in culture against autologous EBV-transformed cell lines. Taken together, these experiments indicate that tremelimumab therapy may benefit from previous or concomitant T(reg) depletion.

Dendritic cells adhere to and transmigrate across lymphatic endothelium in response to IFN-α.

Migration of DC into lymphatic vessels ferries antigenic cargo and pro-inflammatory stimuli into the draining LN. Given that tissues under the influence of viral infections produce type I IFN, it is conceivable that these cytokines enhance DC migration in order to facilitate an antiviral immune response. Cultured lymphatic endothelium monolayers pretreated with TNF-α were used to model this phenomenon under inflammatory conditions. DC differentiated in the presence of either IFN-α2b or IFN-α5 showed enhanced adhesion to cultured lymphatic endothelial cells. These pro-adhesive effects were mediated by DC, not the lymphatic endothelium, and correlated with increased DC transmigration across lymphatic endothelial cell monolayers. Transmigration was guided by chemokines acting on DC, and blocking experiments with mAb indicated a role for LFA-1. Furthermore, incubation of DC with IFN-α led to the appearance of active conformation epitopes on the CD11a integrin chains expressed by DC. Differentiation of mouse DC in the presence of IFN-α also increased DC migration from inflammed footpads toward popliteal LN. Collectively, these results indicate a role for type I IFN in directing DC toward LN under inflammatory conditions.

Paradigms on Immunotherapy Combinations with Chemotherapy.

Checkpoint inhibitors are being added to standard-of-care chemotherapy in multiple clinical trials. Success has been reported in non-small and small cell lung carcinomas and urothelial, head and neck, gastric, and esophageal cancers, and promising results are already available in triple-negative breast and pancreatic malignancies. The potential mechanisms of synergy include immunogenic tumor cell death, antiangiogenesis, selective depletion of myeloid immunosuppressive cells, and lymphopenia, which reduces regulatory T cells and makes room for proliferation of effector T cells. However, chemotherapy regimens have not been optimized for such combinations, perhaps explaining some recent clinical trial disappointments. Approaches to make the most of chemoimmunotherapy include neoadjuvant and adjuvant schemes.Significance: Immunotherapy of cancer based on PD-1/PD-L1 blockade has prompted a revolution in cancer clinical management. Evidence in phase III clinical trials already supports combinations of immunotherapy with standard-of-care chemotherapy for a number of malignant diseases. This review focuses on such evidence and provides an overview of the potential synergistic mechanisms of action and the opportunities to optimize chemoimmunotherapy regimens.

Treatment with anti-CD137 mAbs causes intense accumulations of liver T cells without selective antitumor immunotherapeutic effects in this organ.

BACKGROUND/AIMS: Cancer therapy with agonist anti-CD137 mAbs has been shown to induce immune-mediated tumor rejections in mice, and equivalent agents of this kind are currently being tested in cancer patients. Previous reports indicated that CD137 stimulation induced polyclonal infiltrates of T lymphocytes in the liver. This study characterizes the liver infiltrates and the target dependency of the phenomena and addresses the question of whether tumors nested in the liver are a more favorable target for CD137-based immunotherapy. METHODS: Liver infiltrates were studied with conventional histology and multiple color flow cytometry of total liver leukocytes. CD137(-/-) mice, mice with a single rearrangement of the TCR (OT-1 mice) and Rag(-/-) mice were used to clarify molecular requirements. Mice implanted with MC38 colon carcinomas either subcutaneously or inside the liver were used for comparative studies under treatment with agonist anti-CD137 mAbs. RESULTS: CD137 treatment caused mononuclear inflammation in the portal spaces of the liver, which gave rise to moderate increases in transaminases without signs of cholestasis. Marked increases in the numbers of CD8+ T cells were observed, including CD8+ T lymphocytes co-expressing CD11c. Infiltrates were absent in CD137(-/-) mice and mitigated in mice harboring a single transgenic TCR on their CD8 T cells. Despite the tumor-independent accumulation of T cells in the liver, immunotherapeutic effects were not more prominent against tumors located in this organ. CONCLUSIONS: Target-dependent effects of CD137 stimulation lead to liver infiltration with T cells, but lymphocyte enrichment in this organ does not privilege this site for immunotherapeutic effects against transplanted tumors.

A semi-physiological-based pharmacokinetic/pharmacodynamic model to describe the effects of topotecan on b-lymphocyte lineage cells.

PURPOSE: To develop a semi-physiological-based model describing simultaneously the time course of immature and mature B-lymphocytes after topotecan (TPT) administration to tumor-bearing rats. METHODS: Twenty-four tumor-bearing BDIX male rats received a single 6 mg/kg intra-peritoneal dose of TPT or saline. Mature and immature B-cell levels were measured every two days during three weeks and showed a very different temporal pattern. Both B-cell populations declined rapidly, reaching the nadir at 3-4 days after TPT administration; however, mature cells returned to baseline at day 8, while immature B-cells stayed at nadir until day 9 instead. Data were modeled using the population approach with NONMEM VI. RESULTS: The model developed maintains the proliferation, maturation and degradation elements of previous published models for myelosuppresion. In order to describe the rapid recovery of mature cells, it includes a peripheral compartment providing a constant supply of mature cells to the bloodstream. CONCLUSIONS: The major contribution of the model is its new structure and the dynamical consequences, demonstrating an independent behavior between mature and immature B-cells during recovery. The final model could represent a good basis for the optimization of cytotoxic drugs oriented to attain a maximum antitumor efficacy while minimizing hematological toxicity.

Palettes of vaccines and immunostimulatory monoclonal antibodies for combination.

Various monoclonal antibodies (mAb) target immune system molecules to enhance immunity by costimulating T cells (i.e., CD137, OX40, CD40, GITR) or interfering in coinhibitory signals (i.e., CTLA-4, PD-1). These powerful agents can be guided by cancer vaccines to enhance immunity against tumor but not self tissues. Clinically powerful therapeutic synergies are at hand.

CD137 (4-1BB) Signalosome: Complexity Is a Matter of TRAFs.

CD137 (4-1BB, Tnsfr9) is a member of the TNF-receptor (TNFR) superfamily without known intrinsic enzymatic activity in its cytoplasmic domain. Hence, akin to other members of the TNFR family, it relies on the TNFR-Associated-Factor (TRAF) family of adaptor proteins to build the CD137 signalosome for transducing signals into the cell. Thus, upon CD137 activation by binding of CD137L trimers or by crosslinking with agonist monoclonal antibodies, TRAF1, TRAF2, and TRAF3 are readily recruited to the cytoplasmic domain of CD137, likely as homo- and/or heterotrimers with different configurations, initiating the construction of the CD137 signalosome. The formation of TRAF2-RING dimers between TRAF2 molecules from contiguous trimers would help to establish a multimeric structure of TRAF-trimers that is probably essential for CD137 signaling. In addition, available studies have identified a large number of proteins that are recruited to CD137:TRAF complexes including ubiquitin ligases and proteases, kinases, and modulatory proteins. Working in a coordinated fashion, these CD137-signalosomes will ultimately promote CD137-mediated T cell proliferation and survival and will endow T cells with stronger effector functions. Current evidence allows to envision the molecular events that might take place in the early stages of CD137-signalosome formation, underscoring the key roles of TRAFs and of K63 and K48-ubiquitination of target proteins in the signaling process. Understanding the composition and fine regulation of CD137-signalosomes assembly and disassembly will be key to improve the therapeutic activities of chimeric antigen receptors (CARs) encompassing the CD137 cytoplasmic domain and a new generation of CD137 agonists for the treatment of cancer.

Dynamic cross-regulation of antigen-specific effector and regulatory T cell subpopulations and microglia in brain autoimmunity.

BACKGROUND: Multiple Sclerosis (MS) is considered a T-cell-mediated autoimmune disease with a prototypical oscillatory behavior, as evidenced by the presence of clinical relapses. Understanding the dynamics of immune cells governing the course of MS, therefore, has many implications for immunotherapy. Here, we used flow cytometry to analyze the time-dependent behavior of antigen-specific effector (T(eff)) and regulatory (T(reg)) T cells and microglia in mice model of MS, Experimental Autoimmune Encephalomyelitis (EAE), and compared the observations with a mathematical cross-regulation model of T-cell dynamics in autoimmune disease. RESULTS: We found that T(eff) and T(reg) cells specific to myelin olygodendrocyte glycoprotein (MOG) developed coupled oscillatory dynamics with a 4- to 5-day period and decreasing amplitude that was always higher for the T(eff) populations, in agreement with the mathematical model. Microglia activation followed the oscillations of MOG-specific T(eff) cells in the secondary lymphoid organs, but they were activated before MOG-specific T-cell peaks in the CNS. Finally, we assessed the role of B-cell depletion induced by anti-CD20 therapy in the dynamics of T cells in an EAE model with more severe disease after therapy. We observed that B-cell depletion decreases T(eff) expansion, although its oscillatory behavior persists. However, the effect of B cell depletion was more significant in the T(reg) population within the CNS, which matched with activation of microglia and worsening of the disease. Mathematical modeling of T-cell cross-regulation after anti-CD20 therapy suggests that B-cell depletion may influence the dynamics of T cells by fine-tuning their activation. CONCLUSIONS: The oscillatory dynamics of T-cells have an intrinsic origin in the physiological regulation of the adaptive immune response, which influences both disease phenotype and response to immunotherapy.

Combined immunostimulatory monoclonal antibodies extend survival in an aggressive transgenic hepatocellular carcinoma mouse model.

PURPOSE: Immunostimulatory monoclonal antibodies (ISmAb) that unleash antitumor immune responses are showing efficacy in cancer clinical trials. Anti-B7-H1 (PD-L1) monoclonal antibodies (mAb) block a critical inhibitory pathway in T cells, whereas anti-CD137 and OX40 mAbs provide T-cell costimulation. A combination of these ISmAbs (anti-CD137 + anti-OX40 + anti-B7-H1) was tested using a transgenic mouse model of multifocal and rapidly progressing hepatocellular carcinoma, in which c-myc drives transformation and cytosolic ovalbumin (OVA) is expressed in tumor cells as a model antigen. EXPERIMENTAL DESIGN: Flow-cytometry and immunohistochemistry were used to quantify tumor-infiltrating lymphocytes (TIL) elicited by treatment and assess their activation status and cytolytic potential. Tolerance induction and its prevention/reversal by treatment with the combination of ISmAbs were revealed by in vivo killing assays. RESULTS: The triple combination of ISmAbs extended survival of mice bearing hepatocellular carcinomas in a CD8-dependent fashion and synergized with adoptive T-cell therapy using activated OVA-specific TCR-transgenic OT-1 and OT-2 lymphocytes. Mice undergoing therapy showed clear increases in tumor infiltration by activated and blastic CD8(+) and CD4(+) T lymphocytes containing perforin/granzyme B and expressing the ISmAb-targeted receptors on their surface. The triple combination of ISmAbs did not result in enhanced OVA-specific cytotoxic T lymphocyte (CTL) activity but other antigens expressed by cell lines derived from such hepatocellular carcinomas were recognized by endogenous TILs. Adoptively transferred OVA-specific OT-1 lymphocytes into tumor-bearing mice were rendered tolerant, unless given the triple mAb therapy. CONCLUSION: Extension of survival and dense T-cell infiltrates emphasize the translational potential of combinational immunotherapy strategies for hepatocellular carcinoma. Clin Cancer Res; 19(22); 6151-62. ©2013 AACR.

The HIF-1α hypoxia response in tumor-infiltrating T lymphocytes induces functional CD137 (4-1BB) for immunotherapy.

UNLABELLED: The tumor microenvironment of transplanted and spontaneous mouse tumors is profoundly deprived of oxygenation as confirmed by positron emission tomographic (PET) imaging. CD8 and CD4 tumor-infiltrating T lymphocytes (TIL) of transplanted colon carcinomas, melanomas, and spontaneous breast adenocarcinomas are CD137 (4-1BB)-positive, as opposed to their counterparts in tumor-draining lymph nodes and spleen. Expression of CD137 on activated T lymphocytes is markedly enhanced by hypoxia and the prolyl-hydroxylase inhibitor dimethyloxalylglycine (DMOG). Importantly, hypoxia does not upregulate CD137 in hypoxia-inducible factor (HIF)-1α-knockout T cells, and such HIF-1α-deficient T cells remain CD137-negative even when becoming TILs, in clear contrast to co-infiltrating and co-transferred HIF-1α-sufficient T lymphocytes. The fact that CD137 is selectively expressed on TILs was exploited to confine the effects of immunotherapy with agonist anti-CD137 monoclonal antibodies to the tumor tissue. As a result, low-dose intratumoral injections avoid liver inflammation, achieve antitumor systemic effects, and permit synergistic therapeutic effects with PD-L1/B7-H1 blockade. SIGNIFICANCE: CD137 (4-1BB) is an important molecular target to augment antitumor immunity. Hypoxia in the tumor microenvironment as sensed by the HIF-1α system increases expression of CD137 on tumor-infiltrating lymphocytes that thereby become selectively responsive to the immunotherapeutic effects of anti-CD137 agonist monoclonal antibodies as those used in ongoing clinical trials.

Carcinoma-derived interleukin-8 disorients dendritic cell migration without impairing T-cell stimulation.

BACKGROUND: Interleukin-8 (IL-8, CXCL8) is readily produced by human malignant cells. Dendritic cells (DC) both produce IL-8 and express the IL-8 functional receptors CXCR1 and CXCR2. Most human colon carcinomas produce IL-8. IL-8 importance in malignancies has been ascribed to angiogenesis promotion. PRINCIPAL FINDINGS: IL-8 effects on human monocyte-derived DC biology were explored upon DC exposure to recombinant IL-8 and with the help of an IL-8 neutralizing mAb. In vivo experiments were performed in immunodeficient mice xenografted with IL-8-producing human colon carcinomas and comparatively with cell lines that do not produce IL-8. Allogenic T lymphocyte stimulation by DC was explored under the influence of IL-8. DC and neutrophil chemotaxis were measured by transwell-migration assays. Sera from tumor-xenografted mice contained increasing concentrations of IL-8 as the tumors progress. IL-8 production by carcinoma cells can be modulated by low doses of cyclophosphamide at the transcription level. If human DC are injected into HT29 or CaCo2 xenografted tumors, DC are retained intratumorally in an IL-8-dependent fashion. However, IL-8 did not modify the ability of DC to stimulate T cells. Interestingly, pre-exposure of DC to IL-8 desensitizes such cells for IL-8-mediated in vitro or in vivo chemoattraction. Thereby DC become disoriented to subsequently follow IL-8 chemotactic gradients towards malignant or inflamed tissue. CONCLUSIONS: IL-8 as produced by carcinoma cells changes DC migration cues, without directly interfering with DC-mediated T-cell stimulation.