Newer generations of multi-target CAR and STAb-T immunotherapeutics: NEXT CART Consortium as a cooperative effort to overcome current limitations.

Adoptive T cellular immunotherapies have emerged as relevant approaches for treating cancer patients who have relapsed or become refractory (R/R) to traditional cancer treatments. Chimeric antigen receptor (CAR) T-cell therapy has improved survival in various hematological malignancies. However, significant limitations still impede the widespread adoption of these therapies in most cancers. To advance in this field, six research groups have created the "NEXT Generation CART MAD Consortium" (NEXT CART) in Madrid's Community, which aims to develop novel cell-based immunotherapies for R/R and poor prognosis cancers. At NEXT CART, various basic and translational research groups and hospitals in Madrid concur to share and synergize their basic expertise in immunotherapy, gene therapy, and immunological synapse, and clinical expertise in pediatric and adult oncology. NEXT CART goal is to develop new cell engineering approaches and treatments for R/R adult and pediatric neoplasms to evaluate in multicenter clinical trials. Here, we discuss the current limitations of T cell-based therapies and introduce our perspective on future developments. Advancement opportunities include developing allogeneic products, optimizing CAR signaling domains, combining cellular immunotherapies, multi-targeting strategies, and improving tumor-infiltrating lymphocytes (TILs)/T cell receptor (TCR) therapy. Furthermore, basic studies aim to identify novel tumor targets, tumor molecules in the tumor microenvironment that impact CAR efficacy, and strategies to enhance the efficiency of the immunological synapse between immune and tumor cells. Our perspective of current cellular immunotherapy underscores the potential of these treatments while acknowledging the existing hurdles that demand innovative solutions to develop their potential for cancer treatment fully.

RANK is a poor prognosis marker and a therapeutic target in ER-negative postmenopausal breast cancer.

Despite strong preclinical data, the therapeutic benefit of the RANKL inhibitor, denosumab, in breast cancer patients, beyond the bone, is unclear. Aiming to select patients who may benefit from denosumab, we hereby analyzed RANK and RANKL protein expression in more than 2,000 breast tumors (777 estrogen receptor-negative, ER- ) from four independent cohorts. RANK protein expression was more frequent in ER- tumors, where it associated with poor outcome and poor response to chemotherapy. In ER- breast cancer patient-derived orthoxenografts (PDXs), RANKL inhibition reduced tumor cell proliferation and stemness, regulated tumor immunity and metabolism, and improved response to chemotherapy. Intriguingly, tumor RANK protein expression associated with poor prognosis in postmenopausal breast cancer patients, activation of NFKB signaling, and modulation of immune and metabolic pathways, suggesting that RANK signaling increases after menopause. Our results demonstrate that RANK protein expression is an independent biomarker of poor prognosis in postmenopausal and ER- breast cancer patients and support the therapeutic benefit of RANK pathway inhibitors, such as denosumab, in breast cancer patients with RANK+ ER- tumors after menopause.

The Traf2DNxBCL2-tg Mouse Model of Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma Recapitulates the Biased IGHV Gene Usage, Stereotypy, and Antigen-Specific HCDR3 Selection of Its Human Counterpart.

Chronic lymphocytic leukemia (CLL)/Small lymphocytic lymphoma (SLL) is a heterogeneous disease consisting of at least two separate subtypes, based on the mutation status of the immunoglobulin heavy chain variable gene (IGHV) sequence. Exposure to antigens seems to play a role in malignant transformation and in the selection and expansion of more aggressive CLL clones. Furthermore, a biased usage of particular IGHV gene subgroups and the existence of stereotyped B-cell receptors (BCRs) are distinctive characteristics of human CLL. We have previously described that Traf2DN/BCL2 double-transgenic (tg, +/+) mice develop CLL/SLL with high incidence with aging. In this model, TNF-Receptor Associated Factor (TRAF)-2 deficiency cooperates with B cell lymphoma (BCL)-2 in promoting CLL/SLL in mice by specifically enforcing marginal zone (MZ) B cell differentiation and rendering B cells independent of BAFF for survival. In this report, we have performed the sequencing of the IGHV-D-J rearrangements of B cell clones from the Traf2DN/BCL2-tg+/+ mice with CLL/SLL. The results indicate that these mice develop oligoclonal and monoclonal B cell expansions. Allotransplantation of the oligoclonal populations into immunodeficient mice resulted in the preferential expansion of one of the parental clones. The analysis of the IGHV sequences indicated that 15% were mutated (M) and 85% unmutated (UM). Furthermore, while the Traf2DN/BCL2-tg-/- (wild-type), -/+ (BCL2 single-tg) and +/- (Traf2DNDN single-tg) littermates showed the expression of various IGHV gene subgroups, the CLL/SLL expanded clones from the Traf2DN/BCL2-tg+/+ (double-transgenic) mice showed a more restricted IGHV gene subgroup usage and an overrepresentation of particular IGHV genes. In addition, the HCDR3-encoded protein sequence indicates the existence of stereotyped immunoglobulin (Ig) in the BCRs and strong similarities with BCR recognizing autoantigens and pathogen-associated antigens. Altogether, these results highlight the remarkable similarities between the CLL/SLL developed by the Traf2DN/BCL2-tg+/+ mice and its human counterpart.

From Ugi Multicomponent Reaction to Linkers for Bioconjugation.

Bioconjugation is a key approach for the development of novel molecular entities with clinical applications. The biocompatibility and specificity of biomolecules such as peptides, proteins, and antibodies make these macromolecules ideal carriers for selective targeted therapies. In this context, there is a need to develop new molecular units that cover the requirements of the next generation of targeted pharmaceuticals. Here, we present the design and development of a versatile and stable linker based on a N-alkylated α,α-dialkyl dipeptide for bioconjugation, with a particular focus on antibody-drug conjugates (ADCs). Starting with the well-known Ugi multicomponent reaction, the convenient chemical modification of the prepared adducts allowed us the obtention of versatile bifunctional linkers for bioconjugation. A conjugation strategy was tested to demonstrate the efficiency of the linker. In addition, a novel cytotoxic anti-HER2 ADC was prepared using the Ugi-linker approach.

CD13 as a new tumor target for antibody-drug conjugates: validation with the conjugate MI130110.

BACKGROUND: In the search for novel antibody-drug conjugates (ADCs) with therapeutic potential, it is imperative to identify novel targets to direct the antibody moiety. CD13 seems an attractive ADC target as it shows a differential pattern of expression in a variety of tumors and cell lines and it is internalized upon engagement with a suitable monoclonal antibody. PM050489 is a marine cytotoxic compound tightly binding tubulin and impairing microtubule dynamics which is currently undergoing clinical trials for solid tumors. METHODS: Anti-CD13 monoclonal antibody (mAb) TEA1/8 has been used to prepare a novel ADC, MI130110, by conjugation to the marine compound PM050489. In vitro and in vivo experiments have been carried out to demonstrate the activity and specificity of MI130110. RESULTS: CD13 is readily internalized upon TEA1/8 mAb binding, and the conjugation with PM050489 did not have any effect on the binding or the internalization of the antibody. MI130110 showed remarkable activity and selectivity in vitro on CD13-expressing tumor cells causing the same effects than those described for PM050489, including cell cycle arrest at G2, mitosis with disarrayed and often multipolar spindles consistent with an arrest at metaphase, and induction of cell death. In contrast, none of these toxic effects were observed in CD13-null cell lines incubated with MI130110. Furthermore, in vivo studies showed that MI130110 exhibited excellent antitumor activity in a CD13-positive fibrosarcoma xenograft murine model, with total remissions in a significant number of the treated animals. Mitotic catastrophes, typical of the payload mechanism of action, were also observed in the tumor cells isolated from mice treated with MI130110. In contrast, MI130110 failed to show any activity in a xenograft mouse model of myeloma cells not expressing CD13, thereby corroborating the selectivity of the ADC to its target and its stability in circulation. CONCLUSION: Our results show that MI130110 ADC combines the antitumor potential of the PM050489 payload with the selectivity of the TEA1/8 monoclonal anti-CD13 antibody and confirm the correct intracellular processing of the ADC. These results demonstrate the suitability of CD13 as a novel ADC target and the effectiveness of MI130110 as a promising antitumor therapeutic agent.

Case Report: An EGFR-Targeted 4-1BB-agonistic Trimerbody Does Not Induce Hepatotoxicity in Transgenic Mice With Liver Expression of Human EGFR.

Agonistic monoclonal antibodies (mAbs) targeting the co-stimulatory receptor 4-1BB are among the most effective immunotherapeutic agents across pre-clinical cancer models. However, clinical development of full-length 4-1BB agonistic mAbs, has been hampered by dose-limiting liver toxicity. We have previously developed an EGFR-targeted 4-1BB-agonistic trimerbody (1D8N/CEGa1) that induces potent anti-tumor immunity without systemic toxicity, in immunocompetent mice bearing murine colorectal carcinoma cells expressing human EGFR. Here, we study the impact of human EGFR expression on mouse liver in the toxicity profile of 1D8N/CEGa1. Systemic administration of IgG-based anti-4-1BB agonist resulted in nonspecific immune stimulation and hepatotoxicity in a liver-specific human EGFR-transgenic immunocompetent mouse, whereas in 1D8N/CEGa1-treated mice no such immune-related adverse effects were observed. Collectively, these data support the role of FcγR interactions in the major off-tumor toxicities associated with IgG-based 4-1BB agonists and further validate the safety profile of EGFR-targeted Fc-less 4-1BB-agonistic trimerbodies in systemic cancer immunotherapy protocols.

Intratumoral expression using a NFkB-based promoter enhances IL12 antitumor efficacy.

Interleukin 12 is a promising anti-cancer agent; however, IL12 systemic administration is hampered by side-effects. Although intratumoral administration of IL12 is giving promising results in clinical trials, only a small percentage of patients show a complete therapeutic response. This outcome could be improved by controlling the IL12 expression window. In this work we have tested the efficacy of a self-processing P2A and codon optimized murine IL12 (mIL12Pop) using inflammation-regulated lentivectors in a syngeneic tumor model. Our results show that implantation of cells expressing mIL12Pop employing either the strong constitutive SFFV promoter or a NFkB-based promoter reduced tumor growth, caused CD8+ T cell activation and increased IFNγ production. Importantly, the use of NFkBp-mIL12Pop increased the number of CD8+ TILs and improved the remission rate without increasing IL12-serum concentration. Further experiments suggest that there is a threshold intratumoral IL12 concentration that must be reached to trigger an efficient antitumor response and a limit that once surpassed causes detrimental systemic side effects. Altogether, these results demonstrate that using NFKBp-mIL12Pop significantly increases the overall survival of the mice. In summary, this new inflammation-regulated expression system might be useful for the development of new IL12 delivery systems with improved anti-tumor activity and limited toxicity.

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.

A tumor-targeted trimeric 4-1BB-agonistic antibody induces potent anti-tumor immunity without systemic toxicity.

The costimulation of immune cells using first-generation anti-4-1BB monoclonal antibodies (mAbs) has demonstrated anti-tumor activity in human trials. Further clinical development, however, is restricted by significant off-tumor toxicities associated with FcγR interactions. Here, we have designed an Fc-free tumor-targeted 4-1BB-agonistic trimerbody, 1D8N/CEGa1, consisting of three anti-4-1BB single-chain variable fragments and three anti-EGFR single-domain antibodies positioned in an extended hexagonal conformation around the collagen XVIII homotrimerization domain. The1D8N/CEGa1 trimerbody demonstrated high-avidity binding to 4-1BB and EGFR and a potent in vitro costimulatory capacity in the presence of EGFR. The trimerbody rapidly accumulates in EGFR-positive tumors and exhibits anti-tumor activity similar to IgG-based 4-1BB-agonistic mAbs. Importantly, treatment with 1D8N/CEGa1 does not induce systemic inflammatory cytokine production or hepatotoxicity associated with IgG-based 4-1BB agonists. These results implicate FcγR interactions in the 4-1BB-agonist-associated immune abnormalities, and promote the use of the non-canonical antibody presented in this work for safe and effective costimulatory strategies in cancer immunotherapy.

Bioconjugation through Mesitylene Thiol Alkylation.

The design and generation of complex multifunctional macromolecular structures by bioconjugation is a hot topic due to increasing interest in conjugates with therapeutic applications. In this regard, the development of efficient, selective, and safe conjugation methods is a major objective. In this report, we describe the use of the bis(bromomethyl)benzene scaffold as a linker for bioconjugation with special emphasis on antibody conjugation. We first performed the monothioalkylation of 1,3,5-tris(bromomethyl)benzene, which rendered the reactive dibromotrimethylbenzyl derivatives to be used in thiol bis-alkylation. Next, we introduced into the linker either a bis(Cys)-containing peptide or anti-CD4 and -CD13 monoclonal antibodies, previously subjected to partial reduction of disulfide bonds. Mass spectrometry, UV-vis spectra, and SDS-PAGE experiments revealed that this bis-alkylating agent for bioconjugation preserved both antibody integrity and antibody-antigen binding affinity, as assessed by flow cytometry. Taken together, our results show that the mesitylene scaffold is a suitable linker for thiol-based bioconjugation reactions. This linker could be applicable in the near future for the preparation of antibody drug conjugates.

Dysregulated TRAF3 and BCL2 Expression Promotes Multiple Classes of Mature Non-hodgkin B Cell Lymphoma in Mice.

TNF-Receptor Associated Factor (TRAF)-3 is a master regulator of B cell homeostasis and function. TRAF3 has been shown to bind and regulate various proteins involved in the control of innate and adaptive immune responses. Previous studies showed that TRAF3 overexpression renders B cells hyper-reactive to antigens and Toll-like receptor (TLR) agonists, while TRAF3 deficiency has been implicated in the development of a variety of B cell neoplasms. In this report, we show that transgenic mice overexpressing TRAF3 and BCL2 in B cells develop with high incidence severe lymphadenopathy, splenomegaly and lymphoid infiltrations into tissues and organs, which is the result of the growth of monoclonal and oligoclonal B cell neoplasms, as demonstrated by analysis of VHDJH gene rearrangement. FACS and immunohistochemical analyses show that different types of mature B cell neoplasms arise in TRAF3/BCL2 double-transgenic (tg) mice, all of which are characterized by the loss of surface IgM and IgD expression. However, two types of lymphomas are predominant: (1) mature B cell neoplasms consistent with diffuse large B cell lymphoma and (2) plasma cell neoplasms. The Ig isotypes expressed by the expanded B-cell clones included IgA, IgG, and IgM, with most having undergone somatic hypermutation. In contrast, mouse littermates representing all the other genotypes (TRAF3-/BCL2-; TRAF3+/BCL2-, and TRAF3-/BCL2+) did not develop significant lymphadenopathy or clonal B cell expansions within the observation period of 20 months. Interestingly, a large representation of the HCDR3 sequences expressed in the TRAF3-tg and TRAF3/BCL2-double-tg B cells are highly similar to those recognizing pathogen-associated molecular patterns and damage-associated molecular patterns, strongly suggesting a role for TRAF3 in promoting B cell differentiation in response to these antigens. Finally, allotransplantation of either splenocytes or cell-containing ascites from lymphoma-bearing TRAF3/BCL2 mice into SCID/NOD immunodeficient mice showed efficient transfer of the parental expanded B-cell clones. Altogether, these results indicate that TRAF3, perhaps by promoting exacerbated B cell responses to certain antigens, and BCL2, presumably by supporting survival of these clones, cooperate to induce mature B cell neoplasms in transgenic mice.

Fludarabine Inhibits KV1.3 Currents in Human B Lymphocytes.

Fludarabine (F-ara-A) is a purine analog commonly used in the treatment of indolent B cell malignancies that interferes with different aspects of DNA and RNA synthesis. KV1.3 K+ channels are membrane proteins involved in the maintenance of K+ homeostasis and the resting potential of the cell, thus controlling signaling events, proliferation and apoptosis in lymphocytes. Here we show that F-ara-A inhibits KV currents in human B lymphocytes. Our data indicate that KV1.3 is expressed in both BL2 and Dana B cell lines, although total KV1.3 levels were higher in BL2 than in Dana cells. However, KV currents in the plasma membrane were similar in both cell lines and were abrogated by the specific KV1.3 channel inhibitor PAP-1, indicating that KV1.3 accounts for most of the KV currents in these cell lines. F-ara-A, at a concentration (3.5 µM) similar to that achieved in the plasma of fludarabine phosphate-treated patients (3 µM), inhibited KV1.3 currents by 61 ± 6.3% and 52.3 ± 6.3% in BL2 and Dana B cells, respectively. The inhibitory effect of F-ara-A was concentration-dependent and showed an IC50 value of 0.36 ± 0.04 µM and a nH value of 1.07 ± 0.15 in BL2 cells and 0.34 ± 0.13 µM (IC50 ) and 0.77 ± 0.11 (nH ) in Dana cells. F-ara-A inhibition of plasma membrane KV1.3 was observed irrespective of its cytotoxic effect on the cells, BL2 cells being sensitive and Dana cells resistant to F-ara-A cytotoxicity. Interestingly, PAP-1, at concentrations as high as 10 µM, did not affect the viability of BL2 and Dana cells, indicating that blockage of KV1.3 in these cells is not toxic. Finally, F-ara-A had no effect on ectopically expressed KV1.3 channels, suggesting an indirect mechanism of current inhibition. In summary, our results describe the inhibitory effect of F-ara-A on the activity of KV1.3 channel. Although KV1.3 inhibition is not sufficient to induce cell death, further research is needed to determine whether it might still contribute to F-ara-A cytotoxicity in sensitive cells or be accountable for some of the clinical side effects of the drug.

Synergistic Activity of Deguelin and Fludarabine in Cells from Chronic Lymphocytic Leukemia Patients and in the New Zealand Black Murine Model.

B-cell chronic lymphocytic leukemia (CLL) remains an incurable disease, and despite the improvement achieved by therapeutic regimes developed over the last years still a subset of patients face a rather poor prognosis and will eventually relapse and become refractory to therapy. The natural rotenoid deguelin has been shown to induce apoptosis in several cancer cells and cell lines, including primary human CLL cells, and to act as a chemopreventive agent in animal models of induced carcinogenesis. In this work, we show that deguelin induces apoptosis in vitro in primary human CLL cells and in CLL-like cells from the New Zealand Black (NZB) mouse strain. In both of them, deguelin dowregulates AKT, NFκB and several downstream antiapoptotic proteins (XIAP, cIAP, BCL2, BCL-XL and survivin), activating the mitochondrial pathway of apoptosis. Moreover, deguelin inhibits stromal cell-mediated c-Myc upregulation and resistance to fludarabine, increasing fludarabine induced DNA damage. We further show that deguelin has activity in vivo against NZB CLL-like cells in an experimental model of CLL in young NZB mice transplanted with spleen cells from aged NZB mice with lymphoproliferation. Moreover, the combination of deguelin and fludarabine in this model prolonged the survival of transplanted mice at doses of both compounds that were ineffective when administered individually. These results suggest deguelin could have potential for the treatment of human CLL.

Indole-3-Carbinol Synergizes with and Restores Fludarabine Sensitivity in Chronic Lymphocytic Leukemia Cells Irrespective of p53 Activity and Treatment Resistances.

PURPOSE: Chronic lymphocytic leukemia (CLL) still is lacking a cure. Relapse and development of refractoriness to current treatments are common. New therapies are needed to improve patient prognosis and survival. EXPERIMENTAL DESIGN: Indole-3-carbinol (I3C) is a natural product with antitumor properties already clinically tested. The effect of I3C, F-ara-A, and combinations of both drugs on CLL cells from patients representing different Rai stages, IGHV mutation status, cytogenetic alterations, p53 functionality, and treatment resistances was tested, as well as the toxicity of these treatments in mice. RESULTS: I3C induces cytotoxicity in CLL cells but not in normal lymphocytes. I3C strongly synergized with F-ara-A in all CLL cells tested, including those with p53 deficiency and/or F-ara-A resistance. The mechanism of cell death involved p53-dependent and -independent apoptosis. The combination of I3C + F-ara-A was equally effective in CLL cells irrespective of IGHV mutation stage and patient refractoriness. Moreover, CLL survival and treatment resistance induced by co-culturing CLL cells on stroma cells were overcome by the combinatory I3C + F-ara-A treatment. No toxicity was associated with the combined I3C + fludarabine treatment in mice. CONCLUSIONS: I3C in combination with F-ara-A is highly cytotoxic in CLL cells from refractory patients and those with p53 deficiency. The striking dose reduction index for F-ara-A in combination with I3C would reduce fludarabine toxicity while having a similar or better anti-CLL effectiveness. Moreover, the low toxicity of I3C, already clinically tested, supports its use as adjuvant and combinatory therapy in CLL, particularly for patients with relapsed or refractory disease.

Indole-3-carbinol induces cMYC and IAP-family downmodulation and promotes apoptosis of Epstein-Barr virus (EBV)-positive but not of EBV-negative Burkitt's lymphoma cell lines.

Indole-3-carbinol (I3C) is a natural product found in broadly consumed plants of the Brassica genus, such as broccoli, cabbage, and cauliflower, which exhibits anti-tumor effects through poorly defined mechanisms. I3C can be orally administered and clinical trials have demonstrated that I3C and derivatives are safe in humans. In this study we show that I3C efficiently induces apoptosis in cell lines derived from EBV-positive Burkitt's lymphomas (virus latency I/II), while it does not have any cytotoxic activity against EBV-negative Burkitt's lymphomas and immortalized EBV-infected lymphoblastoid cell lines (virus latency III). The effect of I3C in EBV-positive Burkitt's lymphoma is very specific, since only I3C and its C6-methylated derivative, but not other 3-substituted indoles, have an effect on cell viability. I3C treatment caused apoptosis characterized by loss of mitochondria membrane potential and caspase activation. I3C alters the expression of proteins involved in the control of apoptosis and transcription regulation in EBV-positive Burkitt's lymphoma cell lines. Among those, cMYC, cIAP1/2 and XIAP downmodulation at mRNA and protein level precede apoptosis induction, thus suggesting a role in I3C cytotoxicity. We also showed that I3C and, more particularly, its condensation dimer 3,3'-diindolylmethane (DIM) prolonged survival and reduced tumor burden of mice xenotransplanted with EBV-positive Burkitt's lymphoma Daudi cells. In summary these results, together with previous reports from clinical trials indicating the lack of toxicity in humans of I3C and derivatives, support the use of these compounds as a new therapeutic approach for treating patients with endemic (EBV-positive) Burkitt's lymphoma.

Anti-CCR7 therapy exerts a potent anti-tumor activity in a xenograft model of human mantle cell lymphoma.

BACKGROUND: The chemokine receptor CCR7 mediates lymphoid dissemination of many cancers, including lymphomas and epithelial carcinomas, thus representing an attractive therapeutic target. Previous results have highlighted the potential of the anti-CCR7 monoclonal antibodies to inhibit migration in transwell assays. The present study aimed to evaluate the in vivo therapeutic efficacy of an anti-CCR7 antibody in a xenografted human mantle cell lymphoma model. METHODS: NOD/SCID mice were either subcutaneously or intravenously inoculated with Granta-519 cells, a human cell line derived from a leukemic mantle cell lymphoma. The anti-CCR7 mAb treatment (3 × 200 µg) was started on day 2 or 7 to target lymphoma cells in either a peri-implantation or a post-implantation stage, respectively. RESULTS: The anti-CCR7 therapy significantly delayed the tumor appearance and also reduced the volumes of tumors in the subcutaneous model. Moreover, an increased number of apoptotic tumor cells was detected in mice treated with the anti-CCR7 mAb compared to the untreated animals. In addition, significantly reduced number of Granta-519 cells migrated from subcutaneous tumors to distant lymphoid organs, such as bone marrow and spleen in the anti-CCR7 treated mice. In the intravenous models, the anti-CCR7 mAb drastically increased survival of the mice. Accordingly, dissemination and infiltration of tumor cells in lymphoid and non-lymphoid organs, including lungs and central nervous system, was almost abrogated. CONCLUSIONS: The anti-CCR7 mAb exerts a potent anti-tumor activity and might represent an interesting therapeutic alternative to conventional therapies.

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.

Simultaneous activation of p38 and JNK by arachidonic acid stimulates the cytosolic phospholipase A2-dependent synthesis of lipid droplets in human monocytes.

Exposure of human peripheral blood monocytes to free arachidonic acid (AA) results in the rapid induction of lipid droplet (LD) formation by these cells. This effect appears specific for AA in that it is not mimicked by other fatty acids, whether saturated or unsaturated. LDs are formed by two different routes: (i) the direct entry of AA into triacylglycerol and (ii) activation of intracellular signaling, leading to increased triacylglycerol and cholesteryl ester formation utilizing fatty acids coming from the de novo biosynthetic route. Both routes can be dissociated by the arachidonyl-CoA synthetase inhibitor triacsin C, which prevents the former but not the latter. LD formation by AA-induced signaling predominates, accounting for 60-70% of total LD formation, and can be completely inhibited by selective inhibition of the group IVA cytosolic phospholipase A(2)α (cPLA(2)α), pointing out this enzyme as a key regulator of AA-induced signaling. LD formation in AA-treated monocytes can also be blocked by the combined inhibition of the mitogen-activated protein kinase family members p38 and JNK, which correlates with inhibition of cPLA(2)α activation by phosphorylation. Collectively, these results suggest that concomitant activation of p38 and JNK by AA cooperate to activate cPLA(2)α, which is in turn required for LD formation possibly by facilitating biogenesis of this organelle, not by regulating neutral lipid synthesis.

TNFR-associated factor 2 deficiency in B lymphocytes predisposes to chronic lymphocytic leukemia/small lymphocytic lymphoma in mice.

We have previously shown that transgenic (tg) mice expressing in B lymphocytes both BCL-2 and a TNFR-associated factor 2 (TRAF2) mutant lacking the really interesting new gene and zinc finger domains (TRAF2DN) develop small lymphocytic lymphoma and chronic lymphocytic leukemia with high incidence (Zapata et al. 2004. Proc. Nat. Acad. Sci. USA 101: 16600-16605). Further analysis of the expression of TRAF2 and TRAF2DN in purified B cells demonstrated that expression of both endogenous TRAF2 and tg TRAF2DN was negligible in Traf2DN-tg B cells compared with wild-type mice. This was the result of proteasome-dependent degradation, and rendered TRAF2DN B cells as bona fide TRAF2-deficient B cells. Similar to B cells with targeted Traf2 deletion, Traf2DN-tg mice show expanded marginal zone B cell population and have constitutive p100 NF-κB2 processing. Also, TRAF3, X-linked inhibitor of apoptosis, and Bcl-X(L) expression levels were increased, whereas cellular inhibitors of apoptosis 1 and 2 levels were drastically reduced compared with those found in wild-type B cells. Moreover, consistent with previous results, we also show that TRAF2 was required for efficient JNK and ERK activation in response to CD40 engagement. However, TRAF2 was deleterious for BCR-mediated activation of these kinases. In contrast, TRAF2 deficiency had no effect on CD40-mediated p38 MAPK activation but significantly reduced BCR-mediated p38 activation. Finally, we further confirm that TRAF2 was required for CD40-mediated proliferation, but its absence relieved B cells of the need for B cell activating factor for survival. Altogether, our results suggest that TRAF2 deficiency cooperates with BCL-2 in promoting chronic lymphocytic leukemia/small lymphocytic lymphoma in mice, possibly by specifically enforcing marginal zone B cell accumulation, increasing X-linked inhibitor of apoptosis expression, and rendering B cells independent of B cell activating factor for survival.