Antibody-fusion proteins are new, promising derivatives of monoclonal antibodies (mAbs), and some are being used for cancer therapy. Ongoing research efforts are increasing the repertoire and efficacy of mAbs and mAb-based molecules for the treatment of cancer. Antibody-fusion proteins use the antigen recognition capabilities of the mAb to target a tumor antigen, bringing the fusion protein into the tumor microenvironment. Depending upon what other molecule is fused to the mAb component of the molecule (such as other mAbs, cytokines, chemokines, and toxins), a variety of molecular and cellular activities can thereby be localized to the sites of tumor cells. In this review, we discuss different types of antibody-fusion proteins either in clinical trials or in development for multiple malignancies. We also discuss patient-intrinsic factors that affect therapeutic efficacy, including the inhibitory KIR repertoire of a patient's NK cells and the affinity of a patient's Fc receptors for the Fc portion of the mAb molecule. The level of sophistication of antibody-fusion proteins continues to increase with our understanding of patient-intrinsic factors that affect individualized responses to therapy. New and promising fusion proteins that overcome patient-intrinsic limitations are an exciting application of this technology.

Targeted monoclonal antibodies (mAb) can be used therapeutically for tumors with identifiable antigens such as disialoganglioside GD2, expressed on neuroblastoma and melanoma tumors. Anti-GD2 mAbs (αGD2) can provide clinical benefit in patients with neuroblastoma. An important mechanism of mAb therapy is antibody-dependent cellular cytotoxicity (ADCC). Combinatorial therapeutic strategies can dramatically increase the anti-tumor response elicited by mAbs. We combined a novel αGD2 mAb, hu14.18K322A, with an immunostimulatory regimen of agonist CD40 mAb and class B CpG-ODN 1826 (CpG). Combination immunotherapy was more effective than the single therapeutic components in a syngeneic model of GD2-expressing B16 melanoma with minimal tumor burden. NK cell depletion in B6 mice showed that NK cells were required for the anti-tumor effect; however, anti-tumor responses were also observed in tumor-bearing SCID/beige mice. Thus, NK cell cytotoxicity did not appear to be essential. Peritoneal macrophages from anti-CD40 + CpG-treated mice inhibited tumor cells in vitro in an hu14.18K322A antibody-dependent manner. These data highlight the importance of myeloid cells as potential effectors in immunotherapy regimens utilizing tumor-specific mAb and suggest that further studies are needed to investigate the therapeutic potential of activated myeloid cells and their interaction with NK cells.

Hu14.18-IL2 is an immunocytokine (IC) consisting of human IL-2 linked to hu14.18 mAb, which recognizes GD2 disialoganglioside. Phase II clinical trials of intravenous-hu14.18-IL2 (IV-IC) in neuroblastoma and melanoma are underway, and have already demonstrated activity in neuroblastoma. In our Phase II trial, lower neuroblastoma burden at the time of treatment was associated with a greater likelihood of clinical response to IV-IC. We have previously shown that intratumoral-hu14.18-IL2 (IT-IC) compared to IV-IC results in enhanced local and systemic antitumor activity in tumor-bearing mice. We utilized a mouse model to investigate the impact of tumor burden on hu14.18-IL2 treatment efficacy in IV- versus IT-treated animals. Studies presented here describe the analyses of tumor burden at the initiation of treatment and its effects on treatment efficacy, survival, and tumor-infiltrating leukocytes in A/J mice bearing subcutaneous NXS2 neuroblastoma. We show that smaller tumor burden at treatment initiation is associated with increased infiltration of NK and CD8+ T cells and increased overall survival. NXS2 tumor shrinkage shortly after completion of the 3 days of hu14.18-IL2 treatment is necessary for long-term survival. This model demonstrates that tumor size is a strong predictor of hu14.18-IL2-induced lymphocyte infiltration and treatment outcome.

Cancer commonly occurs in the elderly and immunotherapy (IT) is being increasingly applied to this population. However, the majority of preclinical mouse tumor models assessing potential efficacy and toxicities of therapeutics use young mice. We assessed the impact of age on responses to systemic immune stimulation. In contrast to young mice, systemic cancer IT regimens or LPS given to aged mice resulted in rapid and lethal toxicities affecting multiple organs correlating with heightened proinflammatory cytokines systemically and within the parenchymal tissues. This inflammatory response and increased morbidity with age was independent of T cells or NK cells. However, prior in vivo depletion of macrophages in aged mice resulted in lesser cytokine levels, increased survival, and decreased liver histopathology. Furthermore, macrophages from aged mice and normal human elderly volunteers displayed heightened TNF and IL-6 production upon in vitro stimulation. Treatment of both TNF knockout mice and in vivo TNF blockade in aged mice resulted in significant increases in survival and lessened pathology. Importantly, TNF blockade in tumor-bearing, aged mice receiving IT displayed significant anti-tumor effects. These data demonstrate the critical role of macrophages in the age-associated hyper-inflammatory cytokine responses to systemic immunostimulation and underscore the importance of performing preclinical assessments in aged mice.

CD40 ligation has been shown to induce antitumor effects in mice and cancer patients. Most of the studies have focused on the ability of an agonistic anti-CD40 mAb to either directly kill CD40-positive tumor cells or activate T-cell immune responses. In this review the authors focus on the ability of CD40 ligation to activate antitumor effector mechanisms of the cells of innate immunity such as macrophages and NK cells.

Memory T cells exhibit tremendous antigen specificity within the immune system and accumulate with age. Our studies reveal an antigen-independent expansion of memory, but not naive, CD8 (cid:1) T cells after several immunotherapeutic regimens for cancer resulting in a distinctive phenotype. Signaling through T-cell receptors (TCRs) or CD3 in both mouse and human memory CD8 (cid:1) T cells markedly up-regulated programmed death-1 (PD-1) and CD25 (IL-2 receptor (cid:2) chain), and led to antigen-specific tumor cell killing. In contrast, exposure to cytokine alone in vitro or with immunotherapy in vivo did not up-regulate these markers but resulted in expanded memory CD8 (cid:1) T cells expressing NKG2D, granzyme B, and pos-sessing broadly lytic capabilities. Blockade of NKG2D in mice also resulted in significantly diminished antitumor effects after immunotherapy. Treatment of TCR-transgenic mice bearing nonantigen expressing tumors with immunotherapy still resulted in significant antitumor effects. Human melanoma tissue biopsies obtained from patients after topically applied immunodulatory treatment resulted in increased numbers of these CD8 (cid:1) CD25 (cid:3) cells within the tumor site. These findings demonstrate that memory CD8 (cid:1) T cells can express differential phenotypes indicative of adaptive or innate effectors based on the nature of the stimuli in a process conserved across species. up-regulation also occurred after cells were cultured in IL-2 (Figure 2D). These results indicate that cytokine-based stimulation results in a markedly different phenotype on the memory CD8 (cid:1) T-cell population.

Disease recurrence is frequent in high-risk neuroblastoma (NBL) patients even after multi-modality aggressive treatment [a combination of chemotherapy, surgical resection, local radiation therapy, autologous stem cell transplantation, and cis-retinoic acid (CRA)]. Recent clinical studies have explored the use of monoclonal antibodies (mAbs) that bind to disialoganglioside (GD(2)), highly expressed in NBL, as a means to enable immune effector cells to destroy NBL cells via antibody-dependent cell-mediated cytotoxicity (ADCC). Preclinical data indicate that ADCC can be more effective when appropriate effector cells are activated by cytokines. Clinical studies have pursued this by administering anti-GD(2) mAb in combination with ADCC-enhancing cytokines (IL2 and GM-CSF), a regimen that has demonstrated improved cancer-free survival. More recently, early clinical studies have used a fusion protein that consists of the anti-GD(2) mAb directly linked to IL2, and anti-tumor responses were seen in the Phase II setting. Analyses of genes that code for receptors that influence ADCC activity and natural killer (NK) cell function [Fc receptor (FcR), killer immunoglublin-like receptor (KIR), and KIR-ligand (KIR-L)] suggest patients with anti-tumor activity are more likely to have certain genotype profiles. Further analyses will need to be conducted to determine whether these genotypes can be used as predictive markers for favorable therapeutic outcome. In this review, we discuss factors that affect response to mAb-based tumor therapies such as hu14.18-IL2. Many of our observations have been made in the context of NBL; however, we will also include some observations made with mAbs targeting other tumor types that are consistent with results in NBL. Therefore, we hypothesize that the NBL observations discussed here may also be relevant to mAb therapy for other cancers, in which ADCC is known to play a role.

Targeted monoclonal antibodies (mAb) can be used therapeutically for tumors with identifiable antigens such as disialoganglioside GD2, expressed on neuroblastoma and melanoma. αGD2 mAbs have been clinically successful in patients with neuroblastoma. An important mechanism of mAb therapy is Antibody Dependent Cellular Cytotoxicity (ADCC). Combinatorial therapeutic strategies can dramatically increase the anti-tumor response elicited by mAbs. We combined a novel αGD2 mAb, hu14.18K322A, with an immunostimulatory regimen of agonist CD40 mAb and class B CpG-ODN 1826. This combination immunotherapy produced a synergistic anti-tumor response in a syngeneic model of melanoma with minimal tumor burden, resulting in long-term cure of mice. NK depletion in B6 mice showed NK cells were required for the anti-tumor effect. Anti-tumor responses were also observed in tumor-bearing SCID/beige mice; thus NK cell cytotoxicity was not essential. Interrogation of the myeloid population in the peritoneal cavity showed an increase in neutrophils and monocytes with high Fc receptor expression after treatment with αCD40 +CpG. Furthermore, plastic adherent peritoneal cells inhibited tumor cells in vitro in an antibody-dependent manner. These data highlight the importance of myeloid cells as potential effectors in immunotherapy regimens utilizing tumor-specific mAb and suggest further studies are needed to investigate the therapeutic potential of activated myeloid cells.

Abstract Introduction: Hu14.18-IL2 (APN301, Apeiron Biologics) is an immunocytokine (IC) consisting of human IL2 linked to each IgG heavy chain of the hu14.18 mAb, which recognizes the GD2 disialoganglioside. Phase 2 clinical trials of IV hu14.18-IL2 IC in neuroblastoma and melanoma are underway, with activity already demonstrated in neuroblastoma. We have previously shown that intratumoral IC treatment (IT-IC) results in enhanced anti-tumor activity in mouse models. These studies were designed to determine the mechanisms involved in the enhanced activity and to provide justification for future clinical testing of localized IT administration of this and other immunocytokines. Methods: We characterize tumor growth, survival outcomes, histology and phenotype of tumor infiltrating lymphocytes (TILs) by flow cytometry of IT-hu.14.18-IL2 treatment of A/J mice bearing subcutaneous NXS2 neuroblastomas. Looking at these parameters, IT-IC treated mice are compared to IV-IC treated, IT PBS treated and untreated mice. Data: Mice receiving IT-IC show significant increases [by immunohistochemistry (IHC) and by flow cytometry] of NKp46+ Natural Killer (NK) cells and CD8a+ cytotoxic T cells in TIL populations compared to control tumor-bearing mice. Improved survival and inhibition of tumor growth are observed in mice receiving IT-IC vs. untreated mice or mice receiving IV-IC. Comparisons within treatment groups, or independent of treatment groups, show that the number of NK or CD8 T cells in the tumor correlates inversely with change in tumor size. Analyses by IHC and flow-cytometry show greater IC detection in tumor after IT-IC vs. IV-IC. Moreover, IT-IC results in improved IC retention in tumors and increased NKG2D effector receptors on intratumoral NKG2A/C/E cells and on CD8 T cells when compared to control mice or mice receiving IV-IC. The augmented NKG2D seen in TILs was not seen in spleen cells, supporting the localized nature of the intratumoral changes induced by this treatment. Conclusions: In this murine neuroblastoma model, enhanced antitumor effects of IT hu14.18-IL2 compared to IV hu14.18-IL2 are distinguished by increased activated TILs and IC retention, improved survival and inhibition of tumor growth. These observations suggest that localized administration of immunocytokines in human patients may show analogous advantages over IV administration. In accordance, we have designed a phase I/II clinical trial protocol investigating the maximum tolerated dose (MTD) and efficacy endpoints of intratumorally administered hu14.18-IL2 in stage III/IV melanoma patients with recurrent or refractory disease, and are working towards approval for its activation. Supported by R01-CA-32685-27. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1538. doi:1538-7445.AM2012-1538

Memory T cells exhibit tremendous antigen specificity within the immune system and accumulate with age. Our studies reveal an antigen-independent expansion of memory, but not naive, CD8(+) T cells after several immunotherapeutic regimens for cancer resulting in a distinctive phenotype. Signaling through T-cell receptors (TCRs) or CD3 in both mouse and human memory CD8(+) T cells markedly up-regulated programmed death-1 (PD-1) and CD25 (IL-2 receptor α chain), and led to antigen-specific tumor cell killing. In contrast, exposure to cytokine alone in vitro or with immunotherapy in vivo did not up-regulate these markers but resulted in expanded memory CD8(+) T cells expressing NKG2D, granzyme B, and possessing broadly lytic capabilities. Blockade of NKG2D in mice also resulted in significantly diminished antitumor effects after immunotherapy. Treatment of TCR-transgenic mice bearing nonantigen expressing tumors with immunotherapy still resulted in significant antitumor effects. Human melanoma tissue biopsies obtained from patients after topically applied immunodulatory treatment resulted in increased numbers of these CD8(+) CD25(-) cells within the tumor site. These findings demonstrate that memory CD8(+) T cells can express differential phenotypes indicative of adaptive or innate effectors based on the nature of the stimuli in a process conserved across species.

hu14.18–IL-2 (IC) is an immunocytokine consisting of human IL-2 linked to hu14.18 mAb, which recognizes the GD2 disialoganglioside. Phase 2 clinical trials of i.v. hu14.18–IL-2 (i.v.-IC) in neuroblastoma and melanoma are underway and have already demonstrated activity in neuroblastoma. We showed previously that intratumoral hu14.18–IL-2 (IT-IC) results in enhanced antitumor activity in mouse models compared with i.v.-IC. The studies presented in this article were designed to determine the mechanisms involved in this enhanced activity and to support the future clinical testing of intratumoral administration of immunocytokines. Improved survival and inhibition of growth of both local and distant tumors were observed in A/J mice bearing s.c. NXS2 neuroblastomas treated with IT-IC compared with those treated with i.v.-IC or control mice. The local and systemic antitumor effects of IT-IC were inhibited by depletion of NK cells or T cells. IT-IC resulted in increased NKG2D receptors on intratumoral NKG2A/C/E+ NKp46+ NK cells and NKG2A/C/E+ CD8+ T cells compared with control mice or mice treated with i.v.-IC. NKG2D levels were augmented more in tumor-infiltrating lymphocytes compared with splenocytes, supporting the localized nature of the intratumoral changes induced by IT-IC treatment. Prolonged retention of IC at the tumor site was seen with IT-IC compared with i.v.-IC. Overall, IT-IC resulted in increased numbers of activated T and NK cells within tumors, better IC retention in the tumor, enhanced inhibition of tumor growth, and improved survival compared with i.v.-IC.

Natural killer (NK) cells are powerful effector cells that can be directed to eliminate tumor cells through tumor-targeted monoclonal antibodies (mAbs). Some tumor-targeted mAbs have been successfully applied in the clinic and are included in the standard of care for certain malignancies. Strategies to augment the antitumor response by NK cells have led to an increased understanding of how to improve their effector responses. Next-generation reagents, such as molecularly modified mAbs and mAb-cytokine fusion proteins (immunocytokines, ICs) designed to augment NK-mediated killing, are showing promise in preclinical and some clinical settings. Continued research into the antitumor effects induced by NK cells and tumor-targeted mAbs suggests that additional intrinsic and extrinsic factors may influence the antitumor response. Therefore more research is needed that focuses on evaluating which NK cell and tumor criteria are best predictive of a clinical response and which combination immunotherapy regimens to pursue for distinct clinical settings.

Abstract We and others have previously demonstrated that agonistic αCD40 and IL2 immunotherapy (IT) treatment in young mice results in synergistic anti-tumor effects in multiple tumor models. However, efficacy of this and other ITs can be limited by their induction of systemic toxicity. The majority of mouse tumor studies use younger mice (8-12 weeks) which do not accurately represent the age of a typical cancer patient (>50 yrs or 12 months in a mouse). Therefore, we sought to determine the potential toxicity of IT in aged versus young mice. Within 2-3 days after IT treatment, marked toxicity was observed in aged (17-22 months) and middle aged mice (9-12 months) resulting in 100% mortality as opposed to no mortality in young mice (2-4 months). Aged mice had significantly higher serum levels of TNFα, IFNγ, IL6 and IL17a after IT compared to young treated mice, with a proportionate increase with age. Histological evaluation of aged treated mice revealed disseminated lymphocytic infiltrates to the gut, lung and liver; multiple organ damage with hepatic piecemeal necrosis; extensive GI mucosal damage and peribronchiolitis in the lung compared to young treated mice. Finally, IT-treated aged IFNγ-receptor and TNFα-KO mice showed significant decreases in mortality compared to treated WT-aged mice. This data suggests that IT with αCD40/IL2 induces a cytokine storm culminating in multiple organ damage and rapid lethality that correlates with increased serum cytokines and worsens with age.

Abstract Discussion Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-195.

Memory CD8 T cells (CD44hi+) have a wide distribution in peripheral blood, secondary lymphoid and non-lymphoid tissues and are hallmarked by their ability to respond rapidly to antigenic re-challenge. We observed that systemic cytokine immunotherapy (IT) results in the marked expansion of memory CD8 T cells due to the preferential expansion of pre-existing memory T cells and not the conversion of naïve (CD44lo+) T cells to an activated phenotype. These CD8 T cells express NKG2D and have cytolytic activity; however, a lack of increased CD25 and PD-1 expression suggests that the expansion and activation are independent of TCR engagement. Studies of CD8 T cells from OT-1 TCR transgenic mice after IT demonstrated the increased lysis of ova-negative tumor targets; moreover, the OT-1 CD8 T cells possessed a memory phenotype and lacked CD25 expression in the absence of ova vaccination. To determine if memory CD8 T cells play a role in pathogenic situations, mice were infected with influenza and tissues were examined for the presence of CD25-NKG2D+ memory CD8 T cells. Interestingly, these cells expanded rapidly in the lungs of infected mice but not peripheral tissues, indicating that memory T cells may play a role in the clearance of pathogen, regardless of antigen specificity. These data suggest that resident tissue memory CD8 T cells may act in reserve as innate effectors and thus bridge the gap between adaptive and innate immunity in cancer immunotherapy and viral infections.

Memory T-cell responses to cancer antigens may be an effective way to sustain long-term tumor-free survival. However, finding an effective vaccination strategy to induce memory T-cell responses toward tumor associated antigens in patients with existing disease has proven to be extremely difficult. Immune stimulation regimens have been combined with tumor vaccination in an attempt to boost the immune response resulting in better vaccine efficacy. In these instances immune stimulation alone has shown some promise as a primary tumor therapy, but has been less effective at eliciting long-term tumor immunity. Likewise, combining systemic adjuvant therapy with tumor antigen vaccination also demonstrated a lack of sustained anti-tumor immunity in cancer patients. In this review, we discuss whether the immune response generated during immune stimulation is appropriate for supporting memory T-cell generation or whether initial tumor regression and generation of sustained anti-tumor immunity have different immunological signaling requirements.