Anti-4-1BB monoclonal antibody enhances rejection of large tumor burden by promoting survival but not clonal expansion of tumor-specific CD8+ T cells

Anti-4-1BB monoclonal antibody (mAb) has been shown to induce antitumor immunity by a CD4/CD8-dependent mechanism, but its direct effect on tumor-specific CD8+ T cells in tumor rejection is unclear. Here we used transgenic CD8+ T cells against the unmutated tumor rejection antigen P1A to analyze whether this mAb can promote CD8+ T-cell function against large tumors in the absence of CD4+ T-helper cells. RAG-2(-/-) mice were challenged with P1A-expressing plasmacytoma J558. Once tumor size reached a diameter of 0.85-1.75 cm, mice were treated with P1A-specific CD8+ CTL (P1CTL) in conjunction with anti-4-1BB mAb or control IgG. All of the mice showed a partial regression of tumor, but mice treated with anti-4-1BB mAb exhibited markedly enhanced tumor rejection, delayed tumor progression, and prolonged survival. Correspondingly, we observed a substantial increase in the number of P1CTL in anti-4-1BB mAb-treated mice. Surprisingly, anti-4-1BB mAb did not accelerate division of the tumor-specific CD8+ T cells, and the increase in tumor-specific T-cell number was due to reduced activation-induced cell death. These results indicate that anti-4-1BB mAb can promote CD8+ T cell-mediated protection against large tumors in the absence of CD4+ T-cell help by promoting P1CTL survival without increasing initial clonal expansion.     

Combined natural killer T-cell based immunotherapy eradicates established tumors in mice

A rational monoclonal antibody (mAb)-based antitumor therapy approach has previously been shown to eradicate various established experimental and carcinogen-induced tumors in a majority of mice. This therapy comprised an agonistic mAb reactive with tumor necrosis factor-related apoptosis-inducing ligand receptor (DR5), expressed by tumor cells, an agonistic anti-CD40 mAb to mature dendritic cells, and an agonistic anti-4-1BB mAb to costimulate CD8(+) T cells. Because agonists of CD40 have been toxic in patients, we were interested in substituting anti-CD40 mAb with other dendritic cell-maturing agents, such as glycolipid ligands recognized by invariant natural killer T (iNKT) cells. Here, we show that CD1d-restricted glycolipid ligands for iNKT cells effectively substitute for anti-CD40 mAb and reject established experimental mouse breast and renal tumors when used in combination with anti-DR5 and anti-4-1BB mAbs (termed "NKTMab" therapy). NKTMab therapy-induced tumor rejection was dependent on CD4(+) and CD8(+) T cells, NKT cells, and the cytokine IFN-gamma. NKTMab therapy containing either alpha-galactosylceramide (alpha-GC) or alpha-C-galactosylceramide (alpha-c-GC) at high concentrations induced similar rates of tumor rejection in mice; however, toxicity was observed at the highest doses of alpha-GC (>250 ng/injection), limiting the use of this glycolipid. By contrast, even very low doses of alpha-c-GC (25 ng/injection) retained considerable antitumor activity when used in combination with anti-DR5/anti-4-1BB, and thus, alpha-c-GC showed a considerably greater therapeutic index. In summary, sequential tumor cell apoptosis and amplification of dendritic cell function by NKT cell agonists represents an exciting and novel approach for cancer treatment.     

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

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.     

Systemic administration of interleukin-12 can restore the anti-tumor potential of B16 melanoma-draining lymph node cells impaired at a late tumor-bearing state

We investigated the effect of the systemic administration of interleukin (IL)-12 on the anti-tumor potential of tumor-draining lymph nodes (LNs). Tumor-draining LN cells on day 10 after s.c. inoculation of B16 melanoma showed a significant anti-tumor effect against established pulmonary metastases after in vitro expansion, whereas those on either day 20 or 30 exhibited an impaired anti-tumor potential. However, i.p. injections of IL-12 (0.5 μg) on days 18, 20 and 22 significantly increased the total number of tumor-draining LN cells on day 24 and, furthermore, restored their anti-tumor potential after in vitro expansion. In addition, i.p. injection of IL-12 (0.1 μg) on days 20, 22, 24, 26 and 28 significantly suppressed the growth of s.c.-inoculated B16 melanoma and finally cured the tumors in 6 of 12 mice (50%), whereas dissection of the tumor-draining LNs on day 18, prior to the IL-12 treatment, decreased both the IL-12-induced anti-tumor effect and the percentage of cured mice (8.3%). Cured mice acquired a specific protective immunity. Collectively, our results indicate that the systemic administration of IL-12 can restore the immunotherapeutic potential of tumor-draining LNs, which was impaired at the late tumor-bearing state, and that the IL-12-induced systemic anti-tumor activity is preceded by the restoration of an anti-tumor response in tumor-draining LNs. Int. J. Cancer 75:400–405, 1998. © 1998 Wiley-Liss, Inc.     

Mechanisms involved in synergistic anticancer immunity of anti-4-1BB and anti-CD4 therapy

Anti-4-1BB-mediated anticancer effects were potentiated by depletion of CD4+ cells in B16F10 melanoma-bearing C57BL/6 mice. Anti-4-1BB induced the expansion and differentiation of polyclonal tumor-specific CD8+ T cells into IFN-gamma-producing CD11c+CD8+ T cells. The CD4+ cell depletion was responsible for facilitating immune cell infiltration into tumor tissues and removing some regulatory barriers such as T regulatory and indoleamine-2,3-dioxygenase (IDO)+ dendritic cells. Both monoclonal antibodies (mAb) contributed to the efficient induction of MHC class I molecules on the tumor cells in vivo. The effectors that mediated the anti-4-1BB effect were NKG2D+KLRG1+CD11c+CD8+ T cells that accumulated preferentially in the tumor tissues. Blocking NKG2D reduced the therapeutic effect by 20% to 26%, which may indicate that NKG2D contributes partially to tumor killing by the differentiated CD8+ T cells. Our results indicate that the combination of the two mAbs, agonistic anti-4-1BB and depleting anti-CD4, results in enhanced production of efficient tumor-killing CTLs, facilitation of their infiltration, and production of a susceptible tumor microenvironment.     

Local injection of OK432 can augment the TH1-type T-cell response in tumor-draining lymph node cells and increase their immunotherapeutical potential

The effect of local injections with streptococcal preparation OK432 on the therapeutical potential of tumor-draining lymph node (LN) cells was investigated in mice. Peritumoral injections with OK432 on days 2, 4, 6, 8 and 10 showed no effect on the in vivo growth of s.c. inoculated B16F10 melanoma. The B16F10-draining OK432-treated LN cells, however, showed a high level of anti-B16F10 cytolytic activity after an in vitro culture first with both anti-CD3 monoclonal antibody (MAb) and activated B cell blasts, and subsequently with interleukin (IL)-2 without in vitro restimulation. Such in vitro expanded LN cells showed a remarkable antitumor effect against pulmonary metastasis of B16F10 melanoma, even without the concurrent administration of IL-2. In addition, the therapeutical protocol was also found to be moderately effective against poorly immunogenic MCA fibrosarcoma, and the in vivo antitumor effect was specific to the tumor from which the LNs were harvested. Interestingly, 2 kinds of comparative analyses of the cytokines revealed that the B16F10-bearing state induced the draining LN cells to develop a Th2-type response. However, the OK432 treatment was able to effectively augment their Th1-type response. Collectively, our results suggest that peritumoral injections with OK432 significantly increased the therapeutical potential of the tumor-draining LN cells by augmenting their Th1-type response.Int. J. Cancer 70:606–611. © 1997 Wiley-Liss Inc.     

Polarization effects of 4-1BB during CD28 costimulation in generating tumor-reactive T cells for cancer immunotherapy

Using murine tumor-draining lymph node (TDLN) cells, we investigated the polarization effect of 4-1BB (CD137) during CD28 costimulation in generating antitumor T cells. Costimulation of TDLN cells through the newly induced 4-1BB molecules, CD3, and CD28 using monoclonal antibodies significantly enhanced cell proliferation. The greater cell yield with 4-1BB signaling appeared to be related to the inhibition of activation-induced cell death. Activation of TDLN cells through 4-1BB in addition to CD3/CD28 signaling shifted T-cell responses toward a type 1 cytokine pattern because 4-1BB ligation plus CD3/CD28 stimulation significantly augmented type 1 cytokine (e.g., IFN-gamma) and granulocyte macrophage colony-stimulating factor secretion. By contrast, type 2 cytokine (e.g., interleukin 10) secretion by the activated TDLN cells was significantly reduced. The in vivo antitumor reactivity of TDLN cells activated through 4-1BB in conjunction with CD3/CD28 pathways was examined using an adoptive immunotherapy model. The number of pulmonary metastases was significantly reduced and survival was prolonged after the transfer of anti-CD3/anti-CD28/anti-4-1BB-activated TDLN cells compared with an equivalent number of cells activated without anti-4-1BB. The antitumor effect through 4-1BB involvement during CD28 costimulation was dependent on IFN-gamma production and abrogated after IFN-gamma neutralization. By contrast, interleukin 10 neutralization resulted in significantly enhanced tumor regression. These results indicate that costimulation of TDLN cells through newly induced 4-1BB and CD3/CD28 signaling can significantly increase antitumor reactivity by shifting T-cell responses toward a type 1 cytokine pattern while concomitantly decreasing type 2 responses.     

Effective anti-angiogenic therapy of established tumors in mice by naked anti-human endoglin (CD105) antibody: differences in growth rate and therapeutic response between tumors growing at different sites

We investigated anti-angiogenic/vascular targeting therapy of established tumors in immunocompetent mice using an anti-human endoglin (EDG; CD105) monoclonal antibody (mAb) SN6j. SN6j weakly cross-reacted with murine endothelial cells but reacted neither with colon-26 murine colon carcinoma cells nor with 4T1 murine mammary carcinoma cells. Systemic administration of naked (unconjugated) SN6j showed significant growth suppression of established tumors of colon-26 and 4T1 cells in immunocompetent BALB/c mice (P<0.05). Moreover, the overall survival rate of SN6j-treated mice was significantly higher than that of control IgG-treated mice (P<0.01). During these studies, we found that two different types of tumor formed in BALB/c and immunodeficient SCID mice when three different types of tumor cells (colon-26, 4T1 and MCF-7 human breast cancer cells) were inoculated subcutaneously. One type of tumor grew in the skin-side tissue (i.e., epidermis, corium, or subcutis), and mainly invaded into the corium and epidermis. The other type grew in the muscle-side tissue (i.e., fascia, muscle, or peritoneum/pleura). We termed the former SS tumors and the latter MS tumors. MS tumors grew faster than SS tumors. This differential growth of MS and SS tumors was observed in three different animal models, i.e., colon-26 tumors and 4T1 tumors in BALB/c mice, and MCF-7 tumors in SCID mice. In the therapeutic study of colon-26 and 4T1 tumors with SN6j, MS tumors were less responsive to therapy than SS tumors although SN6j showed significant antitumor efficacy against both tumors (P<0.05). The results show that antitumor therapy can yield different therapeutic outcomes depending on the tumor growth sites even for the same tumor. A differential survival between mice with the two types of tumor was also observed when mice were untreated (P<0.01).     

NK and CD8+ T cell-mediated eradication of poorly immunogenic B16-F10 melanoma by the combined action of IL-12 gene therapy and 4-1BB costimulation

In previous reports, systemic administration of a stimulatory monoclonal antibody directed against the 4-1BB receptor had no effect on survival or tumor burden in mice inoculated with the poorly immunogenic B16-F10 melanoma. We combined IL-12 gene transfer with 4-1BB costimulation to explore a previously noted cooperative anti-tumor effect against this model tumor. We hypothesize that the innate immune response mediated by IL-12-activated natural killer (NK) cells initiates the activation of the immune system, leading to the priming of T cells, whereas 4-1BB costimulation enhances the function of primed tumor-specific T cells. The effect of the combination therapy on the growth of subcutaneous (s.c.) tumors and pulmonary metastasis was examined. The combination therapy significantly retarded the growth of subcutaneously-inoculated tumors, and 50% of tumor-bearing mice survived with complete tumor regression. In contrast, neither IL-12 gene transfer nor anti-4-1BB antibody administration alone was as effective. Enhanced CTL activity against both B16-F10 tumor cells and TRP-2-pulsed EL4 syngeneic tumor cells was observed in tumor-bearing animals treated with the combination therapy 2 weeks after treatment and, in long-term survivors from this combination therapy, at >120 days. In a pulmonary metastatic model, only the combination therapy generated significant protection against metastasis. In vivo depletion of NK or CD8(+) but not CD4(+) subsets eliminated the protective immunity. Furthermore, NK cell depletion significantly reduced both tumor-specific CTL activity and the number of tumor-specific IFN-gamma-producing cells, suggesting that this synergistic effect requires the participation of both NK and CD8(+) T cells.     

Combination therapy with cisplatin and anti-4-1BB: synergistic anticancer effects and amelioration of cisplatin-induced nephrotoxicity

Anti-4-1BB and cisplatin showed synergistic anticancer effects in the CT-26 colon carcinoma model, producing complete regression in >60% of mice with either preventive or therapeutic treatment. The tumor-free mice formed long-lasting CD8(+) T cell-dependent tumor-specific memory. Anti-4-1BB induced rapid repopulation of T and B cells from cisplatin-mediated lymphopenia and differentiation and expansion of IFN-gamma(+)CD11c(+)CD8(+) T cells. Cisplatin facilitated expansion of na?ve, effector, and memory CD8(+) T cells; combination therapy produced almost twice as many lymphoid cells as anti-4-1BB alone. Cisplatin increased 4-1BB on antigen-primed T cells and induced 4-1BB de novo on kidney tubular epithelium. Cross-linking of 4-1BB protected the T cells and kidney epithelium from cisplatin-mediated apoptosis by increasing expression of antiapoptotic molecules. Thus, cisplatin-induced 4-1BB provided a mechanism for amelioration of the lymphopenia and nephrotoxicity inherent in cisplatin treatment. We concluded that chemoimmunotherapy with anti-4-1BB and cisplatin is synergistic in tumor killing and prevention of organ-specific toxicity.     

Sequential involvement of two distinct CD4+ regulatory T cells during the course of transplantable tumor growth and protection from 3-methylcholanthrene-induced tumorigenesis by CD25-depletion.

The involvement of two phenotypically different regulatory T cells in different stages of tumor growth was investigated. Treatment of BALB/c mice with anti-CD25 monoclonal antibody (mAb) (PC61), but not anti-CD4 mAb (GK1.5) before RL male 1 or Meth A inoculation caused tumor rejection. On the other hand, treatment of BALB/c mice with anti-CD4 mAb (GK1.5) but not anti-CD25 mAb (PC61) on day 6 after inoculation of the same tumors caused rejection. The findings suggest that CD4+CD25+ T cells downregulated the rejection response in the early stage of tumor growth. On the other hand, putative CD4+CD25- T cells downregulated the tumor rejection response in the late stage. Both CD4+CD25+ and putative CD4+CD25- T cells appeared to inhibit the efficient generation of cytotoxic T lymphocytes (CTL). The present study also demonstrated that the treatment of BALB/c mice with anti-CD25 mAb (PC61) at 4 or 6 weeks after 3-methylcholanthrene (3-MC) inoculation retarded tumor occurrence and prolonged survival.     

Agonist anti-CD137 mAb act on tumor endothelial cells to enhance recruitment of activated T lymphocytes

Agonist monoclonal antibodies (mAb) to the immune costimulatory molecule CD137, also known as 4-1BB, are presently in clinical trials for cancer treatment on the basis of their costimulatory effects on primed T cells and perhaps other cells of the immune system. Here we provide evidence that CD137 is selectively expressed on the surface of tumor endothelial cells. Hypoxia upregulated CD137 on murine endothelial cells. Treatment of tumor-bearing immunocompromised Rag(-/-) mice with agonist CD137 mAb did not elicit any measurable antiangiogenic effects. In contrast, agonist mAb stimulated tumor endothelial cells, increasing cell surface expression of the adhesion molecules intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, and E-selectin. When adoptively transferred into mice, activated T lymphocytes derived from CD137-deficient animals entered more avidly into tumor tissue after treatment with agonist mAb. This effect could be neutralized with anti-ICAM-1 and anti-VCAM-1 blocking antibodies. Thus, stimulation of CD137 not only enhanced T-cell activation but also augmented their trafficking into malignant tissue, through direct actions on the blood vessels that irrigate the tumor. Our findings identify an additional mechanism of action that can explain the immunotherapeutic effects of agonist CD137 antibodies.     

Estrogen receptor positive breast cancer metastasis: altered hormonal sensitivity and tumor aggressiveness in lymphatic vessels and lymph nodes

Breast cancers commonly spread to lymph nodes (LNs). If the primary tumors are estrogen receptor (ER) and/or progesterone receptor (PR) positive, then the likelihood that LN metastases express receptors exceeds 80%. However, due to lack of ER+ models, little is known about the role of hormones in breast cancer spread or the effects of the LN microenvironment on hormone responsiveness. We have developed metastasis models using ZsGreen labeled MCF-7 and T47D human breast cancer cells. Tumors are tracked in living mice by whole-body imaging, and macrometastases or micrometastases are detected by intravital imaging or fluorescence microscopy. Tumor growth is estrogen dependent and required for intratumoral lymphangiogenesis. Seventy-five percent of all tumors and >95% of larger tumors generate LN metastases. Occasionally more distant metastases are also observed. "Triads" of primary tumors, tumor-filled draining lymphatic vessels, and tumor-filled LNs from the same mouse show that (a) proliferation, as measured by 5-bromo-2'-deoxyuridine uptake, is higher in the LN than in the primary tumor. (b) High ER levels are extensively down-regulated by estradiol in primary tumors. However, there is partial failure of ER down-regulation in LNs associated with (c) reduced PR expression. This suggests that ER are dysfunctional in the LN microenvironment and perhaps hormone resistant. (d) CD44 is sparsely expressed in primary tumor cells but homogeneously overexpressed in cells transiting the lymphatics and populating LNs. We hypothesize that CD44 expression targets tumor cells for transport to, and uptake in, LNs. If so, the CD44 pathway could be targeted therapeutically to slow or prevent LN metastases.     

Oncolytic virus and anti-4-1BB combination therapy elicits strong antitumor immunity against established cancer

Oncolytic virotherapy using vaccinia virus (Vv) has shown some encouraging antitumor responses in mouse models and patients, but the breadth of efficacy in clinical trials has been somewhat limited. Given that antitumor effects have correlated with increased host immune responses, we hypothesized that improved therapeutic outcomes may be achieved by using oncolytic virus (OV) in combination with a potent immune agonist reagent. In this study, we carried out a preclinical evaluation of a genetically engineered strain of oncolytic vaccinia virus (Vvdd) for its capacity to induce antitumor responses when combined with an agonist antibody (Ab) specific for the costimulatory molecule 4-1BB (CD137). In immune-competent syngeneic mouse models of cancer, this combination therapy significantly reduced the growth of established subcutaneous tumors relative to either treatment alone. Importantly, the development of pulmonary metastatic lesions was also reduced. Tumor growth inhibition was associated with increased numbers of CD11b(+) and CD11c(+) myeloid cells in the tumor draining lymph nodes, greater infiltration of CD8(+) effector T and natural killer (NK) cells, and a more sustained presence of neutrophils at the tumor site. Depletion of T or NK cells or neutrophils reduced efficacy, confirming their contribution to an effective therapeutic response. We further extended this conclusion through results from IFNγ-deficient mice. In summary, our findings offered a proof-of-concept for a combinatorial approach to enhance the antitumor efficacy of an OV, suggesting a strategy to improve their use as an immunotherapeutic treatment for cancer.     

Suppression of Tregs by anti‐glucocorticoid induced TNF receptor antibody enhances the antitumor immunity of interferon‐α gene therapy for pancreatic cancer

We have reported that interferon (IFN)‐α can attack cancer cells by multiple antitumor mechanisms including the induction of direct cancer cell death and the enhancement of an immune response in several pancreatic cancer models. However, an immunotolerant microenvironment in the tumors is often responsible for the failure of the cancer immunotherapy. Here we examined whether the suppression of regulatory T cells (Tregs) within tumors can enhance an antitumor immunity induced by an intratumoral IFN‐α gene transfer. First we showed that an intraperitoneal administration of an agonistic anti‐glucocorticoid induced TNF receptor (GITR) monoclonal antibody (mAb), which is reported to suppress the function of Tregs, significantly inhibited subcutaneous tumor growth in a murine pancreatic cancer model. The anti‐GITR mAb was then combined with the intratumoral injection of the IFN‐α‐adenovirus vector. The treatment with the antibody synergistically augmented the antitumor effect of IFN‐α gene therapy not only in the vector‐injected tumors but also in the vector‐uninjected tumors. Immunostaining showed that the anti‐GITR mAb decreased Foxp3⁺ cells infiltrating in the tumors, while the intratumoral IFN‐α gene transfer increased CD4⁺ and CD8⁺ T cells in the tumors. Therefore, the combination therapy strongly inclined the immune balance of the tumor microenvironment in an antitumor direction, leading to a marked systemic antitumor effect. The CCR5 expression on Tregs was downregulated in the antibody‐treated mice, which may explain the decrease of tumor‐infiltrating Tregs. The combination of Treg‐suppression by GITR mAb and the tumor immunity induction by IFN‐α gene therapy could be a promising therapeutic strategy for pancreatic cancer.     

Intracellular tumor-associated antigens represent effective targets for passive immunotherapy

Monoclonal antibody (mAb) therapy against tumor antigens expressed on the tumor surface is associated with clinical benefit. However, many tumor antigens are intracellular molecules that generally would not be considered suitable targets for mAb therapy. In this study, we provide evidence challenging this view through an investigation of the efficacy of mAb directed against NY-ESO-1, a widely expressed immunogen in human tumors that is expressed intracellularly rather than on the surface of cells. On their own, NY-ESO-1 mAb could neither augment antigen-specific CD8(+) T-cell induction nor cause tumor eradication. To facilitate mAb access to intracellular target molecules, we combined anti-NY-ESO-1 mAb with anticancer drugs to accentuate the release of intracellular NY-ESO-1 from dying tumor cells. Strikingly, combination therapy induced a strong antitumor effect that was accompanied by the development of NY-ESO-1-specific effector/memory CD8(+) T cells that were not elicited by single treatments alone. The combinatorial effect was also associated with upregulation of maturation markers on dendritic cells, consistent with the organization of an effective antitumor T-cell response. Administration of Fc-depleted F(ab) mAb or combination treatment in Fcγ receptor-deficient host mice abolished the therapeutic effect. Together, our findings show that intracellular tumor antigens can be captured by mAbs and engaged in an efficient induction of CD8(+) T-cell responses, greatly expanding the possible use of mAb for passive cancer immunotherapy.     

Anti-TIM3 antibody promotes T cell IFN-γ-mediated antitumor immunity and suppresses established tumors

Strategies to activate and rescue exhausted tumor-specific T cells, including the use of monoclonal antibodies (mAb) that block the negative costimulatory receptors CTLA-4 and PD-1 are proving very effective, but TIM3 has been relatively neglected as a target. Here we report an extensive characterization of the therapeutic activity and mechanism of action of an anti-mouse TIM3 mAb against experimental and carcinogen-induced tumors. For the first time we specifically define the mechanism of antitumor action of anti-TIM3 requiring IFN-γ producing CD8(+) T cells and CD4(+) T cells, and a higher ratio of tumor infiltrating CD8(+):CD4(+) T cells correlating with therapeutic success. Interestingly, in some models, anti-TIM3 appeared to be effective sometime before the appearance and accumulation of significant TIM3(+)PD-1(+) T cell populations in tumor bearing mice. Anti-TIM3 displayed modest prophylactic and therapeutic activity against a small fraction of carcinogen-induced sarcomas, but comparative and combination studies of anti-TIM3 with anti-CTLA-4 and anti-PD-1 against experimental and carcinogen-induced tumors suggested that these agents might be well-tolerated and very effective in combination.     

Combination of anti‐CD4 antibody treatment and donor lymphocyte infusion ameliorates graft‐versus‐host disease while preserving graft‐versus‐tumor effects in murine allogeneic hematopoietic stem cell transplantation

Allogeneic hematopoietic stem cell transplantation (allo‐HSCT) is not only a well‐established immunotherapy for hematologic malignancies, but is potentially useful for treating solid tumors refractory to available therapies. However, application of allo‐HSCT to solid tumors is limited, despite the beneficial antitumor effects, by the risk of graft‐versus‐host disease (GVHD). CD4⁺ T cells have been implicated in several aspects of GVHD, and suppress antitumor CD8⁺ T‐cell responses. In the present study, we investigated clinically applicable allo‐HSCT protocols designed to maximize antitumor effects while reducing the risk of GVHD. We used a mouse model of allo‐HSCT with s.c. tumors. We found that myeloablative conditioning was associated with better inhibition of tumor growth but with severe acute GVHD. Early treatment with anti‐CD4 mAb substantially ameliorated GVHD while preserving antitumor effects, leading to improved survival in myeloablative allo‐HSCT. Late treatment with anti‐CD4 mAb also ameliorated GVHD to some extent. Donor lymphocyte infusion in GVHD mice treated with anti‐CD4 mAb further suppressed tumor growth without exacerbating GVHD. Collectively, our results suggest that myeloablative allo‐HSCT followed by anti‐CD4 mAb treatment and donor lymphocyte infusion could be a potent and safe immunotherapy for patients with cancers refractory to available therapies.     

Eradication of established renal cell carcinoma by a combination of 5-fluorouracil and anti-4-1BB monoclonal antibody in mice

Renal cell carcinoma (RCC), one of the most incurable malignancies, is highly resistant to chemotherapy and radiotherapy. Cytokine immunotherapy has been the standard approach, but the overall response rate is still very low. Administration of agonistic anti-4-1BB monoclonal antibody (mAb) has been shown to induce regression of several animal tumors but its effect on RCC is unknown. We show here that monotherapy with either anti-4-1BB mAb or the cytotoxic drug, 5-fluorouracil (5-FU), has little effect on established RCC, Renca tumors, but combination therapy with anti-4-1BB mAb and 5-FU eradicates the tumors in more than 70 % of mice. The regressing tumor tissues from mice receiving the combination therapy contained more apoptotic tumor cells and tumor infiltrating lymphocytes than tumor tissues from mice receiving 5-FU or anti-4-1BB mAb monotherapy. The number of lymphocytes in the spleens and tumor- draining lymph nodes (TDLNs) of the combination therapy mice was greatly increased compared to that of control or 5-FU monotherapy mice. Mice that had recovered due to the combination therapy rapidly rejected rechallenge with the tumor, pointing to the establishment of long-lasting tumor-specific memory. Our results indicate that targeting tumors with 5-FU, and immune cells with 4-1BB stimulation, could be a useful strategy for treating incurable RCC.