A novel Fc-engineered cathepsin D-targeting antibody enhances ADCC, triggers tumor-infiltrating NK cell recruitment, and improves treatment with paclitaxel and enzalutamide in triple-negative breast cancer.

INTRODUCTION: Triple-negative breast cancer (TNBC) prognosis is poor. Immunotherapies to enhance the antibody-induced natural killer (NK) cell antitumor activity are emerging for TNBC that is frequently immunogenic. The aspartic protease cathepsin D (cath-D), a tumor cell-associated extracellular protein with protumor activity and a poor prognosis marker in TNBC, is a prime target for antibody-based therapy to induce NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC). This study investigated whether Fc-engineered anti-cath-D antibodies trigger ADCC, their impact on antitumor efficacy and tumor-infiltrating NK cells, and their relevance for combinatory therapy in TNBC. METHODS: Cath-D expression and localization in TNBC samples were evaluated by western blotting, immunofluorescence, and immunohistochemistry. The binding of human anti-cath-D F1M1 and Fc-engineered antibody variants, which enhance (F1M1-Fc+) or prevent (F1M1-Fc-) affinity for CD16a, to secreted human and murine cath-D was analyzed by ELISA, and to CD16a by surface plasmon resonance and flow cytometry. NK cell activation was investigated by flow cytometry, and ADCC by lactate dehydrogenase release. The antitumor efficacy of F1M1 Fc-variants was investigated using TNBC cell xenografts in nude mice. NK cell recruitment, activation, and cytotoxic activity were analyzed in MDA-MB-231 cell xenografts by immunophenotyping and RT-qPCR. NK cells were depleted using an anti-asialo GM1 antibody. F1M1-Fc+ antitumor effect was assessed in TNBC patient-derived xenografts (PDXs) and TNBC SUM159 cell xenografts, and in combination with paclitaxel or enzalutamide. RESULTS: Cath-D expression on the TNBC cell surface could be exploited to induce ADCC. F1M1 Fc-variants recognized human and mouse cath-D. F1M1-Fc+ activated NK cells in vitro and induced ADCC against TNBC cells and cancer-associated fibroblasts more efficiently than F1M1. F1M1-Fc- was ineffective. In the MDA-MB-231 cell xenograft model, F1M1-Fc+ displayed higher antitumor activity than F1M1, whereas F1M1-Fc- was less effective, reflecting the importance of Fc-dependent mechanisms in vivo. F1M1-Fc+ triggered tumor-infiltrating NK cell recruitment, activation and cytotoxic activity in MDA-MB-231 cell xenografts. NK cell depletion impaired F1M1-Fc+ antitumor activity, demonstrating their key role. F1M1-Fc+ inhibited growth of SUM159 cell xenografts and two TNBC PDXs. In combination therapy, F1M1-Fc+ improved paclitaxel and enzalutamide therapeutic efficacy without toxicity. CONCLUSIONS: F1M1-Fc+ is a promising immunotherapy for TNBC that could be combined with conventional regimens, including chemotherapy or antiandrogens.

Design and selection of optimal ErbB-targeting bispecific antibodies in pancreatic cancer.

The ErbB family of receptor tyrosine kinases is a primary target for small molecules and antibodies for pancreatic cancer treatment. Nonetheless, the current treatments for this tumor are not optimal due to lack of efficacy, resistance, or toxicity. Here, using the novel BiXAb™ tetravalent format platform, we generated bispecific antibodies against EGFR, HER2, or HER3 by considering rational epitope combinations. We then screened these bispecific antibodies and compared them with the parental single antibodies and antibody pair combinations. The screen readouts included measuring binding to the cognate receptors (mono and bispecificity), intracellular phosphorylation signaling, cell proliferation, apoptosis and receptor expression, and also immune system engagement assays (antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity). Among the 30 BiXAbs™ tested, we selected 3Patri-1Cetu-Fc, 3Patri-1Matu-Fc and 3Patri-2Trastu-Fc as lead candidates. The in vivo testing of these three highly efficient bispecific antibodies against EGFR and HER2 or HER3 in pre-clinical mouse models of pancreatic cancer showed deep antibody penetration in these dense tumors and robust tumor growth reduction. Application of such semi-rational/semi-empirical approach, which includes various immunological assays to compare pre-selected antibodies and their combinations with bispecific antibodies, represents the first attempt to identify potent bispecific antibodies against ErbB family members in pancreatic cancer.

Intrinsic features of the cancer cell as drivers of immune checkpoint blockade response and refractoriness.

Immune checkpoint blockade represents the latest revolution in cancer treatment by substantially increasing patients' lifetime and quality of life in multiple neoplastic pathologies. However, this new avenue of cancer management appeared extremely beneficial in a minority of cancer types and the sub-population of patients that would benefit from such therapies remain difficult to predict. In this review of the literature, we have summarized important knowledge linking cancer cell characteristics with the response to immunotherapy. Mostly focused on lung cancer, our objective was to illustrate how cancer cell diversity inside a well-defined pathology might explain sensitivity and refractoriness to immunotherapies. We first discuss how genomic instability, epigenetics and innate immune signaling could explain differences in the response to immune checkpoint blockers. Then, in a second part we detailed important notions suggesting that altered cancer cell metabolism, specific oncogenic signaling, tumor suppressor loss as well as tight control of the cGAS/STING pathway in the cancer cells can be associated with resistance to immune checkpoint blockade. At the end, we discussed recent evidences that could suggest that immune checkpoint blockade as first line therapy might shape the cancer cell clones diversity and give rise to the appearance of novel resistance mechanisms.

Rapid communication: insights into the role of extracellular vesicles during Auger radioimmunotherapy.

PURPOSE: Non-targeted effects, including bystander and systemic effects, play a crucial role during Auger targeted radionuclide therapy. Here, we investigated whether small extracellular vesicles (sEVs) produced by irradiated cells could contribute to the bystander cytotoxic effects in vitro and also to therapeutic efficacy in vivo, after their injection in tumor xenografts. MATERIALS AND METHODS: B16F10 melanoma donor cells were exposed to radiolabeled antibodies (Auger radioimmunotherapy, RIT) for 48 h or to X-rays (donor cells). Then, donor cells were incubated with fresh medium for 2 h to prepare conditioned medium (CM) that was transferred onto recipient cells for bystander effect assessment, or used for sEVs enrichment. Resulting sEVs were incubated in vitro with recipient cells for determining bystander cytotoxicity, or injected in B16F10 melanoma tumors harbored by athymic and C57BL/6 mice. RESULTS: In vitro analysis of bystander cytotoxic effects showed that CM killed about 30-40% of melanoma cells. SEVs isolated from CM contributed to this effect. Moreover, the double-stranded DNA (dsDNA) content was increased in sEVs isolated from CM of exposed cells compared to control (not exposed), but the difference was significant only for the X-ray condition. These results were supported by immunodetection of cytosolic dsDNA in donor cells, a phenomenon that should precede dsDNA enrichment in sEVs. However, sEVs cytotoxicity could not be detected in vivo. Indeed, in athymic and in immunocompetent mice that received four intratumoral injections of sEVs (1/day), tumor growth was not delayed compared with untreated controls. Tumor growth was slightly (not significantly) delayed in immunocompetent mice treated with sEVs from X-ray-exposed cells, and significantly with sEVs purified from CM collected after 48 h of incubation. These results highlight the need to determine the optimal conditions, including radiation absorbed dose and sEVs collection time, to obtain the strongest cytotoxic effects. CONCLUSIONS: This study demonstrates that sEVs could play a role during Auger RIT through bystander effects in vitro. No systemic effects were observed in vivo, under our experimental conditions. However, X-rays experiments showed that sEVs collection time might be influencing the nature of sEVs, a parameter that should also be investigated during Auger RIT.

Improving Biologics' Effectiveness in Clinical Oncology: From the Combination of Two Monoclonal Antibodies to Oligoclonal Antibody Mixtures.

Monoclonal antibodies have revolutionized the treatment of many diseases, but their clinical efficacy remains limited in some other cases. Pre-clinical and clinical trials have shown that combinations of antibodies that bind to the same target (homo-combinations) or to different targets (hetero-combinations) to mimic the polyclonal humoral immune response improve their therapeutic effects in cancer. The approval of the trastuzumab/pertuzumab combination for breast cancer and then of the ipilimumab/nivolumab combination for melanoma opened the way to novel antibody combinations or oligoclonal antibody mixtures as more effective biologics for cancer management. We found more than 300 phase II/III clinical trials on antibody combinations, with/without chemotherapy, radiotherapy, small molecules or vaccines, in the ClinicalTrials.gov database. Such combinations enhance the biological responses and bypass the resistance mechanisms observed with antibody monotherapy. Usually, such antibody combinations are administered sequentially as separate formulations. Combined formulations have also been developed in which separately produced antibodies are mixed before administration or are produced simultaneously in a single cell line or a single batch of different cell lines as a polyclonal master cell bank. The regulation, toxicity and injection sequence of these oligoclonal antibody mixtures still need to be addressed in order to optimize their delivery and their therapeutic effects.

Anti-Müllerian hormone concentration regulates activin receptor-like kinase-2/3 expression levels with opposing effects on ovarian cancer cell survival.

Anti­Müllerian hormone (AMH) type II receptor (AMHRII) and the AMH/AMHRII signaling pathway are potential therapeutic targets in ovarian carcinoma. Conversely, the role of the three AMH type I receptors (AMHRIs), namely activin receptor­like kinase (ALK)2, ALK3 and ALK6, in ovarian cancer remains to be clarified. To determine the respective roles of these three AMHRIs, the present study used four ovarian cancer cell lines (COV434­AMHRII, SKOV3­AMHRII, OVCAR8, KGN) and primary cells isolated from tumor ascites from patients with ovarian cancer. The results demonstrated that ALK2 and ALK3 may be the two main AMHRIs involved in AMH signaling at physiological endogenous and supraphysiological exogenous AMH concentrations, respectively. Supraphysiological AMH concentrations (25 nM recombinant AMH) were associated with apoptosis in all four cell lines and decreased clonogenic survival in COV434­AMHRII and SKOV3­AMHRII cells. These biological effects were induced via ALK3 recruitment by AMHRII, as ALK3­AMHRII dimerization was favored at increasing AMH concentrations. By contrast, ALK2 was associated with AMHRII at physiological endogenous concentrations of AMH (10 pM). Based on these results, tetravalent IgG1­like bispecific antibodies (BsAbs) against AMHRII and ALK2, and against AMHRII and ALK3 were designed and evaluated. In vivo, COV434­AMHRII tumor cell xenograft growth was significantly reduced in all BsAb­treated groups compared with that in the vehicle group (P=0.018 for BsAb 12G4­3D7; P=0.001 for all other BsAbs). However, the growth of COV434­AMHRII tumor cell xenografts was slower in mice treated with the anti­AMRII­ALK2 BsAb 12G4­2F9 compared with that in animals that received a control BsAb that targeted AMHRII and CD5 (P=0.048). These results provide new insights into type I receptor specificity in AMH signaling pathways and may lead to an innovative therapeutic approach to modulate AMH signaling using anti­AMHRII/anti­AMHRI BsAbs.

Anti-Müllerian hormone (AMH) autocrine signaling promotes survival and proliferation of ovarian cancer cells.

In ovarian carcinoma, anti-Müllerian hormone (AMH) type II receptor (AMHRII) and the AMH/AMHRII signaling pathway are potential therapeutic targets. Here, AMH dose-dependent effect on signaling and proliferation was analyzed in four ovarian cancer cell lines, including sex cord stromal/granulosa cell tumors and high grade serous adenocarcinomas (COV434-AMHRII, SKOV3-AMHRII, OVCAR8 and KGN). As previously shown, incubation with exogenous AMH at concentrations above the physiological range (12.5-25 nM) decreased cell viability. Conversely, physiological concentrations of endogenous AMH improved cancer cell viability. Partial AMH depletion by siRNAs was sufficient to reduce cell viability in all four cell lines, by 20% (OVCAR8 cells) to 40% (COV434-AMHRII cells). In the presence of AMH concentrations within the physiological range (5 to 15 pM), the newly developed anti-AMH B10 antibody decreased by 25% (OVCAR8) to 50% (KGN) cell viability at concentrations ranging between 3 and 333 nM. At 70 nM, B10 reduced clonogenic survival by 57.5%, 57.1%, 64.7% and 37.5% in COV434-AMHRII, SKOV3-AMHRII, OVCAR8 and KGN cells, respectively. In the four cell lines, B10 reduced AKT phosphorylation, and increased PARP and caspase 3 cleavage. These results were confirmed in ovarian cancer cells isolated from patients' ascites, demonstrating the translational potential of these results. Furthermore, B10 reduced COV434-MISRII tumor growth in vivo and significantly enhanced the median survival time compared with vehicle (69 vs 60 days; p = 0.0173). Our data provide evidence for a novel pro-survival autocrine role of AMH in the context of ovarian cancer, which was targeted therapeutically using an anti-AMH antibody to successfully repress tumor growth.

Blocking Antibodies Targeting the CD39/CD73 Immunosuppressive Pathway Unleash Immune Responses in Combination Cancer Therapies.

Immune checkpoint inhibitors have revolutionized cancer treatment. However, many cancers are resistant to ICIs, and the targeting of additional inhibitory signals is crucial for limiting tumor evasion. The production of adenosine via the sequential activity of CD39 and CD73 ectoenzymes participates to the generation of an immunosuppressive tumor microenvironment. In order to disrupt the adenosine pathway, we generated two antibodies, IPH5201 and IPH5301, targeting human membrane-associated and soluble forms of CD39 and CD73, respectively, and efficiently blocking the hydrolysis of immunogenic ATP into immunosuppressive adenosine. These antibodies promoted antitumor immunity by stimulating dendritic cells and macrophages and by restoring the activation of T cells isolated from cancer patients. In a human CD39 knockin mouse preclinical model, IPH5201 increased the anti-tumor activity of the ATP-inducing chemotherapeutic drug oxaliplatin. These results support the use of anti-CD39 and anti-CD73 monoclonal antibodies and their combination with immune checkpoint inhibitors and chemotherapies in cancer.

[Anti-tumor monoclonal antibodies: new insights to elicit a long-term immune response]. / Les anticorps monoclonaux anti-tumoraux - Nouvelles perspectives pour générer une réponse immunitaire protectrice et durable.

Tumor-targeting monoclonal antibodies (mAbs) are now widely used for the treatment of cancer patients and their numbers are constantly increasing. Over the past ten years, numerous studies have demonstrated that the anti-tumor role of these antibodies far exceeds that of passive therapies as it was initially described, with the possibility of recruiting innate immune cells to promote activation of the early stages of immune response and to generate a long-term protective anti-tumor memory immune response. Understanding these mechanisms has recently led to the clinical development of a new generation of anti-tumor antibodies modified to increase their ability to interact with immune cells. Finally, the first preclinical and clinical studies have recently demonstrated the interest of developing therapeutic combinations combining anti-tumor mAbs with immune-, chemo- or radiotherapy, to reinforce their immunomodulatory potential and ensure effective and durable anti-tumor protection.

TITLE: Les anticorps monoclonaux anti-tumoraux - Nouvelles perspectives pour générer une réponse immunitaire protectrice et durable. ABSTRACT: Les anticorps monoclonaux (AcM) ciblant les tumeurs sont aujourd'hui largement utilisés pour le traitement de patients atteints de cancer et leur nombre est en constante augmentation. Au cours de ces dix dernières années, de nombreuses études ont montré que l'action anti-tumorale de ces anticorps dépasse largement celle de simples thérapies passives comme cela avait été décrit initialement, avec non seulement le recrutement de cellules immunitaires innées pour favoriser l'activation des étapes précoces de la réponse immunitaire mais aussi avec la génération d'une réponse mémoire anti-tumorale protectrice sur le long-terme. La compréhension de ces mécanismes a récemment conduit au développement clinique d'une nouvelle génération d'AcM anti-tumoraux, modifiés afin d'augmenter leurs capacités à interagir avec les cellules immunitaires. Enfin, les premières études précliniques et cliniques ont démontré l'intérêt de développer des combinaisons thérapeutiques associant ces AcM anti-tumoraux à des immuno-, chimio- ou radiothérapies, afin de renforcer leur potentiel immunomodulateur et d'assurer une protection anti-tumorale efficace et durable.

PD-1 blockade at the time of tumor escape potentiates the immune-mediated antitumor effects of a melanoma-targeting monoclonal antibody.

Tumor antigen-targeting monoclonal antibodies (TA-targeting mAbs) are used as therapeutics in many malignancies and their capacity to mobilize the host immunity puts them at the forefront of anti-cancer immunotherapies. Both innate and adaptive immune cells have been associated with the therapeutic activity of such antibodies, but tumor escape from mAb-induced tumor immune surveillance remains one of the main clinical issues. In this preclinical study, we grafted immunocompetent and immunocompromised mice with the B16F10 mouse melanoma cell line and treated them with the TA99 TA-targeting mAb to analyze the immune mechanisms associated with the tumor response and resistance to TA99 monotherapy. In immunocompetent mice TA99 treatment strongly increased the fraction of CD8 and CD4 effector T cells in the tumor compared with isotype control, highlighting the specific immune modulation of the tumor microenvironment by TA99. However, in most mice, TA99 immunotherapy could not prevent immune effector exhaustion and the recruitment of regulatory CD4 T cells and consequently tumor escape from immune surveillance. Remarkably, anti-PD-1 treatment at the time of tumor emergence restored the Th1 effector functions of CD4 and CD8 T cells as well as of natural killer and γδT cells, which translated into a significant slow-down of tumor progression and extended survival. Our findings provide the first evidence that PD-1 blockade at the time of tumor emergence can efficiently boost the host anti-tumor immune response initiated several weeks before by the TA-targeting mAb. These results are promising for the design of combined therapies to sensitize non-responder or resistant patients.

Dual protein kinase and nucleoside kinase modulators for rationally designed polypharmacology.

Masitinib, a highly selective protein kinase inhibitor, can sensitise gemcitabine-refractory cancer cell lines when used in combination with gemcitabine. Here we report a reverse proteomic approach that identifies the target responsible for this sensitisation: the deoxycytidine kinase (dCK). Masitinib, as well as other protein kinase inhibitors, such as imatinib, interact with dCK and provoke an unforeseen conformational-dependent activation of this nucleoside kinase, modulating phosphorylation of nucleoside analogue drugs. This phenomenon leads to an increase of prodrug phosphorylation of most of the chemotherapeutic drugs activated by this nucleoside kinase. The unforeseen dual activity of protein kinase inhibition/nucleoside kinase activation could be of great therapeutic benefit, through either reducing toxicity of therapeutic agents by maintaining effectiveness at lower doses or by counteracting drug resistance initiated via down modulation of dCK target.

IL-21 promotes the development of a CD73-positive Vγ9Vδ2 T cell regulatory population.

Vγ9Vδ2 T cells contribute to the immune response against many tumor types through their direct cytotoxic activity and capacity to regulate the biological functions of other immune cells, such as dendritic cells and IFN-γ-producing CD8+ T cells. However, their presence in the tumor microenvironment has also been associated with poor prognosis in breast, colon and pancreatic cancers. Additionally, recent studies demonstrated that cytokines can confer some plasticity to Vγ9Vδ2 T cells and promote their differentiation into cells with regulatory functions. Here, we demonstrated that activation of Vγ9Vδ2 T cells isolated from healthy donors and cultured in the presence of IL-21 favors the emergence of a subpopulation of Vγ9Vδ2 T cells that express the ectonucleotidase CD73 and inhibits T cell proliferation in a CD73/adenosine-dependent manner. This subpopulation produces IL-10 and IL-8 and displays lower effector functions and cytotoxic activity than CD73-negative Vγ9Vδ2 T cells. We also showed, in a syngeneic mouse tumor model, the existence of a tumor-infiltrating γδ T cell subpopulation that produces IL-10 and strongly expresses CD73. Moreover, maturation, IL-12 production and induction of antigen-specific T cell proliferation are impaired in DC co-cultured with IL-21-amplified Vγ9Vδ2 T cells. Altogether, these data indicate that IL-21 promotes Vγ9Vδ2 T cell regulatory functions by favoring the development of an immunosuppressive CD73+ subpopulation. Thus, when present in the tumor microenvironment, IL-21 might negatively impact γδ T cell anti-tumor functions.

Post-paralysis tyrosine kinase inhibition with masitinib abrogates neuroinflammation and slows disease progression in inherited amyotrophic lateral sclerosis.

BACKGROUND: In the SOD1(G93A) mutant rat model of amyotrophic lateral sclerosis (ALS), neuronal death and rapid paralysis progression are associated with the emergence of activated aberrant glial cells that proliferate in the degenerating spinal cord. Whether pharmacological downregulation of such aberrant glial cells will decrease motor neuron death and prolong survival is unknown. We hypothesized that proliferation of aberrant glial cells is dependent on kinase receptor activation, and therefore, the tyrosine kinase inhibitor masitinib (AB1010) could potentially control neuroinflammation in the rat model of ALS. METHODS: The cellular effects of pharmacological inhibition of tyrosine kinases with masitinib were analyzed in cell cultures of microglia isolated from aged symptomatic SOD1(G93A) rats. To determine whether masitinib prevented the appearance of aberrant glial cells or modified post-paralysis survival, the drug was orally administered at 30 mg/kg/day starting after paralysis onset. RESULTS: We found that masitinib selectively inhibited the tyrosine kinase receptor colony-stimulating factor 1R (CSF-1R) at nanomolar concentrations. In microglia cultures from symptomatic SOD1(G93A) spinal cords, masitinib prevented CSF-induced proliferation, cell migration, and the expression of inflammatory mediators. Oral administration of masitinib to SOD1(G93A) rats starting after paralysis onset decreased the number of aberrant glial cells, microgliosis, and motor neuron pathology in the degenerating spinal cord, relative to vehicle-treated rats. Masitinib treatment initiated 7 days after paralysis onset prolonged post-paralysis survival by 40 %. CONCLUSIONS: These data show that masitinib is capable of controlling microgliosis and the emergence/expansion of aberrant glial cells, thus providing a strong biological rationale for its use to control neuroinflammation in ALS. Remarkably, masitinib significantly prolonged survival when delivered after paralysis onset, an unprecedented effect in preclinical models of ALS, and therefore appears well-suited for treating ALS.

Inhibition of CD39 enzymatic function at the surface of tumor cells alleviates their immunosuppressive activity.

The ectonucleotidases CD39 and CD73 hydrolyze extracellular adenosine triphosphate (ATP) and adenosine diphosphate (ADP) to generate adenosine, which binds to adenosine receptors and inhibits T-cell and natural killer (NK)-cell responses, thereby suppressing the immune system. The generation of adenosine via the CD39/CD73 pathway is recognized as a major mechanism of regulatory T cell (Treg) immunosuppressive function. The number of CD39⁺ Tregs is increased in some human cancers, and the importance of CD39⁺ Tregs in promoting tumor growth and metastasis has been demonstrated using several in vivo models. Here, we addressed whether CD39 is expressed by tumor cells and whether CD39⁺ tumor cells mediate immunosuppression via the adenosine pathway. Immunohistochemical staining of normal and tumor tissues revealed that CD39 expression is significantly higher in several types of human cancer than in normal tissues. In cancer specimens, CD39 is expressed by infiltrating lymphocytes, the tumor stroma, and tumor cells. Furthermore, the expression of CD39 at the cell surface of tumor cells was directly demonstrated via flow cytometry of human cancer cell lines. CD39 in cancer cells displays ATPase activity and, together with CD73, generates adenosine. CD39⁺CD73⁺ cancer cells inhibited the proliferation of CD4 and CD8 T cells and the generation of cytotoxic effector CD8 T cells (CTL) in a CD39- and adenosine-dependent manner. Treatment with a CD39 inhibitor or blocking antibody alleviated the tumor-induced inhibition of CD4 and CD8 T-cell proliferation and increased CTL- and NK cell-mediated cytotoxicity. In conclusion, interfering with the CD39-adenosine pathway may represent a novel immunotherapeutic strategy for inhibiting tumor cell-mediated immunosuppression.

Plasticity of γδ T Cells: Impact on the Anti-Tumor Response.

The tumor immune microenvironment contributes to tumor initiation, progression, and response to therapy. Among the immune cell subsets that play a role in the tumor microenvironment, innate-like T cells that express T cell receptors composed of γ and δ chains (γδ T cells) are of particular interest. γδ T cells can contribute to the immune response against many tumor types (lymphoma, myeloma, melanoma, breast, colon, lung, ovary, and prostate cancer) directly through their cytotoxic activity and indirectly by stimulating or regulating the biological functions of other cell types required for the initiation and establishment of the anti-tumor immune response, such as dendritic cells and cytotoxic CD8+ T cells. However, the notion that tumor-infiltrating γδ T cells are a good prognostic marker in cancer was recently challenged by studies showing that the presence of these cells in the tumor microenvironment was associated with poor prognosis in both breast and colon cancer. These findings suggest that γδ T cells may also display pro-tumor activities. Indeed, breast tumor-infiltrating γδ T cells could exert an immunosuppressive activity by negatively regulating dendritic cell maturation. Furthermore, recent studies demonstrated that signals from the microenvironment, particularly cytokines, can confer some plasticity to γδ T cells and promote their differentiation into γδ T cells with regulatory functions. This review focuses on the current knowledge on the functional plasticity of γδ T cells and its effect on their anti-tumor activities. It also discusses the putative mechanisms underlying γδ T cell expansion, differentiation, and recruitment in the tumor microenvironment.

Aberrant repair initiated by mismatch-specific thymine-DNA glycosylases provides a mechanism for the mutational bias observed in CpG islands.

The human thymine-DNA glycosylase (TDG) initiates the base excision repair (BER) pathway to remove spontaneous and induced DNA base damage. It was first biochemically characterized for its ability to remove T mispaired with G in CpG context. TDG is involved in the epigenetic regulation of gene expressions by protecting CpG-rich promoters from de novo DNA methylation. Here we demonstrate that TDG initiates aberrant repair by excising T when it is paired with a damaged adenine residue in DNA duplex. TDG targets the non-damaged DNA strand and efficiently excises T opposite of hypoxanthine (Hx), 1,N(6)-ethenoadenine, 7,8-dihydro-8-oxoadenine and abasic site in TpG/CpX context, where X is a modified residue. In vitro reconstitution of BER with duplex DNA containing Hx•T pair and TDG results in incorporation of cytosine across Hx. Furthermore, analysis of the mutation spectra inferred from single nucleotide polymorphisms in human population revealed a highly biased mutation pattern within CpG islands (CGIs), with enhanced mutation rate at CpA and TpG sites. These findings demonstrate that under experimental conditions used TDG catalyzes sequence context-dependent aberrant removal of thymine, which results in TpG, CpA→CpG mutations, thus providing a plausible mechanism for the putative evolutionary origin of the CGIs in mammalian genomes.

Tumor antigen-targeting monoclonal antibody-based immunotherapy: Orchestrating combined strategies for the development of long-term antitumor immunity.

Tumor antigen (TA)-targeting monoclonal antibody (mAb)-based treatments are considered to be one of the most successful strategies in cancer therapy. Besides targeting TAs and inducing tumor cell death, such antibodies interact with immune cells through Fc-dependent mechanisms to induce adaptive memory immune responses. However, multiple inhibitory/immunosuppressive pathways can be induced by tumor cells to limit the establishment of an efficient antitumor response and consequently a sustained clinical response to TA-targeting mAbs. Here, we provide an overview on how TA-targeting mAbs in combination with conventional cancer therapies and/or inhibitors of key immunosuppressive pathways might represent promising approaches to achieve long-term tumor control.

Control of regulatory T cells is necessary for vaccine-like effects of antiviral immunotherapy by monoclonal antibodies.

Regulatory T cells (Tregs) down-regulate immunity and are associated with chronic viral infections, suggesting that their inhibition might be used to treat life-threatening diseases. Using the FrCasE mouse retroviral model, we have recently shown that short mAb-based immunotherapies can induce life-long protective immunity. This finding has a potentially important therapeutical impact because mAbs are increasingly used to treat severe viral infections. We now report that poor anti-FrCasE immunity in infected mice is due to Treg expansion in secondary lymphoid organs because depletion of Tregs restored humoral and cytotoxic T lymphocyte (CTL) antiviral responses. Kinetic analyses show that Treg expansion is not a consequence of chronicity, but rather is associated with viral spread. Moreover, Treg adoptive transfers indicate that production of the immunosuppressive cytokine IL-10 is essential for preventing a protective immune response. Finally, treatment of infected mice with a virus-neutralizing IgG2a shortly after infection prevents Treg expansion and limits immunosuppressive activity. This effect is rapid, necessary for the development of protective immunity, and depends on mAb effector functions. Therefore, manipulating Tregs may be necessary to confer robust antiviral immunity in the context of mAb-based therapy. This concept likely applies to cancer treatment because vaccine-like effects of mAbs have also been observed in certain cancer immunotherapies.