Synthesis and biological evaluation of biaryl alkyl ethers as inhibitors of IDO1.

Starting from the dialkylaniline indoleamine 2,3-dioxygenase 1 (IDO1) inhibitor lead 3 (IDO1 HeLa IC50 = 7.0 nM), an iterative process of synthesis and screening led to cyclized analog 21 (IDO1 HeLa IC50 = 3.6 nM) which maintained the high potency of 3 while addressing issues of lipophilicity, cytochrome P450 (CYP) inhibition, hERG (human potassium ion channel Kv11.1) inhibition, Pregnane X Receptor (PXR) transactivation, and oxidative metabolic stability. An x-ray crystal structure of a biaryl alkyl ether 11 bound to IDO1 was obtained. Consistent with our earlier results, compound 11 was shown to bind to the apo form of the enzyme.

Development of a series of novel o-phenylenediamine-based indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors.

A novel series of o-phenylenediamine-based inhibitors of indoleamine 2,3-dioxygenase (IDO) has been identified. IDO is a heme-containing enzyme, overexpressed in the tumor microenvironment of many cancers, which can contribute to the suppression of the host immune system. Synthetic modifications to a previously described diarylether series resulted in an additional degree of molecular diversity which was exploited to afford compounds that demonstrated significant potency in the HeLa human cervical cancer IDO1 assay. .

Identification and optimization of a novel series of indoleamine 2,3-dioxygenase inhibitors.

The discovery of a series of structurally-novel biaryl urea IDO inhibitors is described. Optimization of a micromolar hit through iterative cycles of synthesis and screening in an assay measuring IDO-mediated intracellular conversion of tryptophan to kynurenine led to potent inhibitors with favorable selectivity and metabolic stability profiles.

CTLA-4 blockade in tumor models: an overview of preclinical and translational research.

Cytotoxic T lymphocyte antigen-4 (CTLA-4) is a key negative regulator of T cell activation. A complex integration of positive and negative co-stimulatory signals in the well-defined B7:CD28/CTLA-4 pathway modulates the generation and maintenance of immune responses. Inhibiting negative regulation through binding of CTLA-4 has been shown to promote stimulation of adaptive immunity and potentiation of T cell activation. CTLA-4-blocking antibodies have demonstrated efficacy in various murine malignancy models when administered as monotherapy; additionally, they have shown synergistic anti-tumor activity when utilized with other agents, such as vaccines, chemotherapy, and radiation. Preclinical studies have supported the rationale for current clinical development of anti-CTLA-4 antibodies, including ipilimumab and tremelimumab, as novel therapeutic strategies to augment anti-tumor immunity in cancer. Both ipilimumab and tremelimumab have been evaluated extensively in melanoma; notably, ipilimumab was recently approved as monotherapy for the treatment of advanced melanoma. Tremelimumab is currently undergoing evaluation in phase II trials as monotherapy in melanoma and malignant mesothelioma, while ipilimumab is under clinical investigation in phase II and III trials in various tumor types, including in melanoma, prostate, and lung cancers as monotherapy and with other therapeutic modalities, such as chemotherapy and radiation. In this review, we will provide a detailed overview of preclinical advances that have delineated many features of CTLA-4 and have helped define its role in T cell response. We will also highlight clinical application of anti-CTLA-4 therapy in cancer and describe knowledge gaps that future studies may address.

Immune modulation through 4-1BB enhances SIV vaccine protection in non-human primates against SIVmac251 challenge.

Costimulatory molecules play a central role in the development of cellular immunity. Understanding how costimulatory pathways can be directed to positively influence the immune response may be critical for the generation of an effective HIV vaccine. Here, we evaluated the ability of intravenous administration of a blocking monoclonal antibody (mAb) directed against the negative costimulatory molecule CTLA-4, and an agonist mAb directed against the positive costimulatory molecule 4-1BB, either alone or in combination, to augment intramuscular SIV DNA immunizations. We then tested the ability these of these responses to impact a high-dose SIVmac251 challenge. Following immunization, the groups infused with the anti-4-1BB mAb exhibited enhanced IFN-γ responses compared to the DNA vaccine only group. Interestingly, although CTLA-4 blockade alone did not enhance IFN-γ responses it did increase the proliferative capacity of the CD4(+) and CD8(+) T cells. The combination of both mAbs enhanced the magnitude of the polyfunctional CD8(+) T cell response. Following challenge, the group that received both mAbs exhibited a significant, ∼2.0 log, decrease in plasma viral load compared to the naïve group the included complete suppression of viral load in some animals. Furthermore, the use of the CTLA-4 blocking antibody resulted in significantly higher viral loads during chronic infection compared to animals that received the 4-1BB mAb, likely due to the higher CD4(+) T cell proliferative responses which were driven by this adjuvant following immunization. These novel studies show that these adjuvants induce differential modulation of immune responses, which have dramatically different consequences for control of SIV replication, suggesting important implications for HIV vaccine development.

Synergistic activity of ixabepilone plus other anticancer agents: preclinical and clinical evidence.

Ixabepilone demonstrates marked synergistic activity in combination with capecitabine, which served as the rationale for the evaluation of this combination in the clinic. Ixabepilone plus capecitabine is currently approved for patients with locally advanced or metastatic breast cancer (MBC) progressing after treatment with an anthracycline and a taxane; approval was based on the results of two phase III trials comparing the combination with capecitabine monotherapy. An array of preclinical studies in multiple solid tumor types show that ixabepilone demonstrates therapeutic synergy with targeted therapies including trastuzumab, bevacizumab, brivanib, and cetuximab; with immune-modulating agents such as anti-CTLA-4 antibody; and with other chemotherapy drugs such as irinotecan and epirubicin. Notably, experiments in several xenograft models show that ixabepilone provides greater antitumor synergism when combined with bevacizumab than either paclitaxel or nab-paclitaxel combined with bevacizumab. These preclinical findings provide a foundation for ongoing phase II clinical trials using ixabepilone in combination with trastuzumab or lapatinib in HER2-positive breast cancer; with bevacizumab in breast cancer, endometrial cancer, renal cancer, and non-small cell lung cancer (NSCLC); with cetuximab in breast cancer, NSCLC, and pancreatic cancer; and with brivanib, dasatinib, sorafinib, sunitinib, or vorinostat in MBC. Preliminary results from several of these trials suggest that ixabepilone-based combinations have promising anticancer activity.

Cloning, expression and characterization of monkey (Macaca fascicularis) CD137.

CD137 plays an important role as a co-stimulatory molecule in activated T cells. Agonistic CD137 specific antibodies have been investigated as therapeutic agents to promote tumor-specific immune responses by direct activation of T cells. As part of the pre-clinical pharmacological evaluation of cynomolgus monkeys, monkey CD137 was cloned and characterized. The deduced amino acid sequence encoded a full-length gene of 254 amino acids 95% identical to human CD137. Sequence variants identified in monkey CD137 include four splice variants lacking the transmembrane domain. These variants were detectable in human including two previously unreported variants. Two missense single nucleotide polymorphisms were detected present in 42 and 50% of 36 monkeys tested. In both monkey and human, mRNA expression of full-length CD137 and splice variants were significantly increased in peripheral blood mononuclear cells (PBMCs) upon stimulation by anti-CD3 antibodies. Recombinant monkey CD137 protein was bound with high affinity by an agonistic anti-human CD137 antibody but not by an anti-mouse CD137 antibody. In summary, compared to human, monkey CD137 showed distinct extracellular domain amino acid sequence and sequence polymorphisms. Thus, antibodies directed against epitopes in this extracellular domain could have differences in pharmacologic activity between cynomolgus monkeys and human or across individual cynomolgus monkeys.

Augmentation of SIV DNA vaccine-induced cellular immunity by targeting the 4-1BB costimulatory molecule.

DNA vaccines are effective at inducing antigen-specific cellular immune responses. Approaches to improve these responses, however, are needed. We examined the effect of stimulating 4-1BB, an activation-inducible T-cell costimulatory receptor, by intravenously co-administering anti-human 4-1BB monoclonal antibody (mAb) in DNA-immunized cynomolgus macaques. Three groups of six cynomolgus macaques were immunized intramuscularly with a DNA vaccine encoding SIV Gag antigen (pSIVgag) at weeks 0, 4 and 8. At days 12, 15, and 19, six macaques received anti-4-1BB 4E9 mAb and six macaques received anti-4-1BB 10C7 mAb. Treatment with 10C7 mAb led to a significant augmentation of SIV Gag-specific IFN-gamma, granzyme B and perforin responses. Treatment with humanized 4E9 mAb also resulted in an enhancement of SIV Gag-specific cellular responses but the magnitude was lower compared to animals receiving 10C7 mAb. These responses persisted up to week 40 and were mostly mediated by CD8(+) T cells. Treatment with anti-4-1BB mAb was more effective in driving the CD8(+) T cells toward a more differentiated CCR7(-)/CD45RA(+) effector state. This study demonstrates that targeting the 4-1BB molecule in vivo results in an enhanced and long-lasting cellular immune response. 4-1BB stimulation may be a promising approach to enhance the effectiveness of DNA vaccines.

CLTA-4 blockade in vivo promotes the generation of short-lived effector CD8 T cells and a more persistent central memory CD4 T cell response.

BACKGROUND: Previously, we examined the effects of in vivo CTLA-4 blockade using a fully human monoclonal antibody as a part of a DNA vaccination regimen in cynomolgus macaques (Macaca fascicularis). We observed that while the antibody had little effect on the IFN-gamma ELISpot response, CTLA-4 blockade enhanced antigen-specific cellular proliferation in both CD4(+) and CD8(+)T-cell compartments. METHODS: We examine the specific effects of CTLA-4 blockade on memory T-cell compartments following the third immunization and 10 months following a fourth immunization, during the memory phase of the immune response. RESULTS: CLTA-4 blockade enhanced CD4(+) and CD8(+) central memory (CD28(hi), CD95(hi)) T-cell responses as well as a short-lived CD8(+) effector (CD28(lo), CD95(hi)) T-cell response. CONCLUSIONS: These data suggest differing effects of CTLA-4 blockade on CD4(+) and CD8(+) T cells with implications on the clinical use of anti-CTLA-4 antibodies for enhancement of vaccine strategies or treatment of human disease.

Adjuvant effect of anti-4-1BB mAb administration in adoptive T cell therapy of cancer.

Administration of anti-4-1BB mAb has been found to be a potent adjuvant when combined with other therapeutic approaches, e.g. chemotherapy, cytokine therapies, anti-OX40 therapy, and peptide or DC vaccines. However, the adjuvant effect of anti-4-1BB mAb administration in adoptive T cell therapy of cancer has not been fully evaluated. In this report, effector T cells were generated in vitro by anti-CD3/anti-CD28 activation of tumor-draining lymph node (TDLN) cells and used in an adoptive immunotherapy model. While T cells or anti-4-1BB alone showed no therapeutic efficacy in mice bearing macroscopic 10-day pulmonary metastases, T cells plus anti-4-1BB mediated significant tumor regression in an anti-4-1BB dose dependent manner. Mice bearing microscopic 3-day lung metastases treated with T cells alone demonstrated tumor regression which was significantly enhanced by anti-4-1BB administration. NK cell depletion abrogated the augmented therapeutic efficacy rendered by anti-4-1BB. Cell transfer between congenic hosts demonstrated that anti-4-1BB administration increased the survival of adoptively transferred TDLN cells. Using STAT4(-/-) mice, we found that modulated IFN gamma secretion in wt TDLN cells after anti-CD3/CD28/4-1BB activation in vitro was lost in similarly stimulated STAT4(-/-) TDLN cells. Additionally, anti-4-1BB administration failed to augment the therapeutic efficacy of T cell therapy in STAT4(-/-) mice. Together, these results indicate that administered anti-4-1BB mAb can serve as an effective adjuvant to augment the antitumor reactivity of adoptively transferred T cells by recruiting the host NK cells; increasing the persistence of infused effector T cells, and modulating the STAT4 molecular signaling pathway.

Anti-CD137 monoclonal antibody administration augments the antitumor efficacy of dendritic cell-based vaccines.

In weakly and poorly immunogenic tumor models, we examined the effects of stimulating CD137 (4-1BB) in vivo by administering anti-CD137 monoclonal antibody after tumor lysate-pulsed dendritic cell (TP-DC) vaccination. TP-DC subcutaneous vaccination induced a transient up-regulation of CD137 on T cells and natural killer (NK) cells within vaccine-primed lymph nodes (VPLNs). In established pulmonary and subcutaneous tumor models, anti-CD137 synergistically enhanced tumor regression after TP-DC vaccination. In the subcutaneous tumor model, the combined therapy resulted in improved survival. Combined therapy also resulted in improved local control of subcutaneous tumor after surgical resection. Anti-CD137 polarized the cytokine release of VPLNs and spleen cells in response to tumor antigen toward a type 1 (interferon-gamma) versus a type 2 (interleukin-4) profile. Cell depletion and the use of knockout animals identified that CD8(+), CD4(+), and NK cells were involved in the tumor rejection response and that CD8(+) cells had the major effector role. Anti-CD137 administration resulted in increased proliferation of adoptively transferred OT-1 CD8(+) T cells in the VPLNs of mice inoculated with B16-OVA TP-DCs. Polarization toward type 1 (interferon-gamma) versus type 2 (interleukin-4) was also observed with the OT-1 cells from VPLNs and spleen cells after anti-CD137 injections. This polarization effect was abrogated by the in vivo depletion of NK cells. These findings indicate that the adjuvant effect of anti-CD137 given in conjunction with TP-DC vaccination is associated with the polarization of T effector cells toward a type 1 response to tumor antigen and is mediated via NK cells.