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Langerhans cell histiocytosis (LCH) is a disorder with highly diverse clinical manifestations. We explored if age, sex, race, organ system involved, and therapy approaches determine patient survival in the era of modern treatments. LCH patient data reported to the Surveillance, Epidemiology, and End Results (SEER) program in 2010-2016 (n=1282; age: 0 to 100 y) was analyzed. Age-specific LCH incidence flattening to a low level suggests an age cutoff for pediatric patients of 20 years. The overall survival probability is lower for patients 21 to 100 years old ( P0.0001), irrespective of sex and race. The commonest sites involved in the 0- to 20-year age group were bone, skin, and bone marrow; this shifted to lung, bone, and skin as the commonest disease sites in patients 21 to 100 years of age. The treatments applied differed between age groups, as younger versus older patients were more likely to receive chemotherapy-based treatment (48.4% vs. 17%; P0.0001). There also was a trend toward nonwhite versus white patients being less likely to receive chemotherapy-based treatment (31.7% vs. 38.2%; P =0.067). Whereas there are treatment disparities related to LCH patient age and perhaps race, patient age is the strongest predictor of survival, with patients 21 to 100 years of age with lung, lymph node, skin, and bone marrow disease having the worst outcomes ( P0.0001).

Outcomes for children and adolescents with relapsed and refractory Hodgkin lymphoma (HL) are poor, with approximately 50% of patients experiencing a subsequent relapse. The anti-CD30 antibody-drug conjugate brentuximab vedotin improved progression-free survival (PFS) when used as consolidation after autologous stem cell transplant (ASCT) in adults with high-risk relapsed/refractory HL. Data on brentuximab vedotin as consolidative therapy after ASCT in pediatric patients with HL are extremely limited, with only 11 patients reported in the literature. We performed a retrospective analysis of 67 pediatric patients who received brentuximab vedotin as consolidation therapy following ASCT for the treatment of relapsed/refractory HL to describe the experience of this regimen in the pediatric population. This is the largest cohort reported to date. We found that brentuximab vedotin was well tolerated with a safety profile similar to adult patients. With a median follow up of 37 months, the 3-year PFS was 85%. These data suggest a potential role for the use of brentuximab vedotin as consolidation therapy after ASCT for children with relapsed/refractory Hodgkin lymphoma.

Establishing the diagnosis of classical Hodgkin lymphoma (cHL) in a timely fashion can be a challenge, as there are no pathognomonic signs or symptoms. The percutaneous needle biopsy (CNB) of the tumor gained increased popularity due to its minimal invasiveness and expeditious recovery from procedure. The diagnostic accuracy of CNB depends upon patient size, mass accessibility, and feasibility to obtain diagnostic tissue assuming that the tumor is histologically homogenous. We report on a patient with cHL who underwent multiple CNB from different sites before the diagnosis was established. Yet, only the incisional biopsy of the most metabolically-active tumor site resulted in diagnostic success. It is imperative to thoroughly consider both the biopsy method and the biopsy site for establishing correct diagnosis.

To the editor:Progress continues to be made regarding the treatment of children and adolescents with aggressive B-cell Non-Hodgkin Lymphoma (NHL). Recent data demonstrated patients with mature B-ce...

Acute promyelocytic leukemia (APML), characterized by the reciprocal translocation between chromosomes 15 and 17 [t(15;17)], is a result of proliferation of myeloid cells maturation which is interrupted at the promyelocytic stage. The central, and the most important, distinguishing feature of APML is a predisposition to disseminated intravascular coagulation (DIC). The overall prognosis of APML is very good, with 90% of patients achieving complete remission. We find it important to remind pediatric practitioners, both in the ambulatory and urgent care room settings, of presenting signs and symptoms of leukemia, as well as, up-to-date on management of such fulminant scenarios as DIC. Intracranial hemorrhage (ICH) is one of the commonest, and frequently fulminant complication of APML seen after initiation of induction chemotherapy. We report on a young female presenting with non-specific upper respiratory illness symptoms and recurrent headache, who was found to already have ICH and to be in DIC in the setting of APML at the time of initial evaluation. This case illustrates importance of thorough assessment and prompt consideration of wide differential diagnosis, which became somewhat limited and biased towards web-based telemedicine in the COVID-19 pandemics era.

Background: Although the prognosis for children with Hodgkin lymphoma (HL) is excellent, up to 25% of patients experience relapse and require salvage therapy. Event-free survival (EFS) for patients with relapsed or refractory disease (R/R) who require high-dose therapy with autologous stem cell transplantation (ASCT) is between 31-67% (Daw, 2011), and efforts to improve these outcomes are an area of urgent clinical need. Brentuximab vedotin (BV) is an anti-CD30 antibody-drug conjugate combined with monomethyl auristatin E. The phase III AETHERA study (NCT01100502) was a randomized, placebo-controlled trial that demonstrated consolidation with BV following ASCT for adults with R/R HL improved progression-free survival (PFS) in comparison to placebo (5-year PFS 59% vs. 41%) in patients identified as having a high-risk of post-ASCT progression (Moskowitz, 2015, 2018). However, there is limited data on post-ASCT BV consolidation in pediatric patients. Here, we report a retrospective multi-center study of BV as consolidation post-ASCT in pediatric patients with R/R HL. Patients and Methods: We performed a retrospective analysis of pediatric patients aged ≤ 21 years who received BV as consolidative therapy following ASCT for the treatment of relapsed/refractory HL. Data was collected on disease risk factors (refractory disease or relapse ≤ 12 months after frontline therapy, best response of partial remission or stable disease to salvage therapy, presence of extranodal involvement, presence of B symptoms, and number of systemic treatments pre-ASCT ≥ 2), treatment history, BV-related toxicity, and response to therapy. The primary endpoint was 3-year EFS. Events were defined as relapse, progression of disease, or death from any cause. Results: Seventy patients were identified from 14 academic centers. Eighteen received BV >90 days after ASCT and were excluded due to significant delay in initiation of BV. Of the remaining 52 patients, the median age at diagnosis was 15 years (range 4-21), and the median age at diagnosis of R/R disease was 16 years (range 8-22). Twenty-eight patients (54%) were male, 13 (25%) had B symptoms, 19 (37%) had extranodal disease. Six patients (12%) had primary refractory disease and 24 patients (47%) relapsed The median time from ASCT to the start of BV consolidation was 52 days (range 30-90 days). The most frequent adverse events were peripheral neuropathy of any grade (sensory 19/52 [37%]; motor (14/52 [27%]), and cytopenias (grade 3 or 4: (25/52 [48%]), including neutropenia (13/52 [25%]), thrombocytopenia (7/52 [14%]), and anemia (5/52 [10%]). Three patients (6%) experienced infusion-related reactions. The median number of BV cycles completed post-ASCT was 12 (range 1-17), and 16 patients (31%) completed the full 16 cycles of BV. The most common reason for premature termination was adverse effects (22/36 [61%]). Additional reasons included: physician plan for shorter duration (4/36 [11%]), patient/family decision (3/36 [8%]), and progression (1/36 [3%]). Treatment was still ongoing in 3 patients and reason for premature termination was not reported for 17/36 patients (47%). With a median follow of 2.8 years (range 0.1 to 6.25 years) the three-year EFS was 92% [95%CI: 83-100]. Patients with 0-1 risk factors (15/52 [29%]) and ≥ 2 risk factors (37/52 [71%]), had a 3-year EFS of 100% and 90% [95%CI: 79-100], respectively. Fifty patients (96%) are alive with no evidence of disease, while 2 (4%) patients are alive with disease. No deaths have occurred. Conclusion: The use of post-ASCT BV consolidation for pediatric patients with R/R HL is associated with a favorable safety and tolerability profile. In this cohort, the combination of salvage therapy, HD-chemotherapy/ASCT, and BV consolidation resulted in extremely promising outcomes and warrants further investigation in a prospective pediatric trial. Figure 1 Figure 1. Disclosures Leger: Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbott Diagnostics: Research Funding. Roth: Merck: Consultancy; Janssen: Consultancy.

Cytotoxic T-lymphocyte-associated protein 4 (CTLA4) haploinsufficiency (HI) is an immune dysregulation disorder characterized by T lymphocyte hyperactivation, and generalized lymphoproliferative and autoimmune disorders. Patients with CTLA4 haploinsufficiency are also prone to developing various malignancies. Due to the rare nature of this condition, the longitudinal evolution of CTLA4 haploinsufficiency clinical manifestations remains to be described. We report on over a decade-long course of a young adult men patient with known CTLA4 haploinsufficiency, who sequentially developed multiple autoimmune conditions along with two distinct episodes of classical Hodgkin Lymphoma (cHL) of unique histologies. Notably, the patient had serological and molecular evidence of Epstein Barr virus (EBV) infection at the time of diagnosis of both cHL. The allogeneic stem cell transplantation consolidated durable remission of cHL and CTLA-4 haploinsufficiency-related clinical manifestations, but failed to enable the patient to permanently suppress EBV viremia, which raises concerns for the development of other EBV infection-related conditions in the future. This case report adds on the current understanding of the natural clinical history of patients with CTLA4 haploinsufficiency, and their unique susceptibility to developing cHL.

We studied the effectiveness of monoclonal anti-CD40 + cytosine-phosphate-guanosine-containing oligodeoxynucleotide 1826 (CpG-ODN) immunotherapy (IT) in mice treated with multidrug chemotherapy (CT) consisting of vincristine, cyclophosphamide and doxorubicin. Combining CT with IT led to synergistic anti-tumour effects in C57BL/6 mice with established B16 melanoma or 9464D neuroblastoma. CT suppressed the functions of T cells and natural killer (NK) cells, but primed naive peritoneal macrophages (Mφ) to in vitro stimulation with lipopolysaccharide (LPS), resulting in augmented nitric oxide (NO) production. IT, given after CT, did not restore the responsiveness of T cells and NK cells, but further activated Mφ to secrete NO, interferon-γ (IFN-γ) and interleukin (IL)-12p40 and to suppress the proliferation of tumour cells in vitro. These functional changes were accompanied by immunophenotype alterations on Mφ, including the up-regulation of Gr-1. CD11b(+) F4/80(+) Mφ comprised the major population of B16 tumour-infiltrating leucocytes. CT + IT treatment up-regulated molecules associated with the M1 effector Mφ phenotype [CD40, CD80, CD86, major histocompatibility complex (MHC) class II, IFN-γ, tumour necrosis factor-α (TNF-α) and IL-12] and down-regulated molecules associated with the M2 inhibitory Mφ phenotype (IL-4Rα, B7-H1, IL-4 and IL-10) on the tumour-associated Mφ compared with untreated controls. Together, the results show that CT and anti-CD40 + CpG-ODN IT synergize in the induction of anti-tumour effects which are associated with the phenotypic repolarization of tumour-associated Mφ.

Under different circumstances, tumors can inhibit or activate macrophage (Mφ) effector functions. We studied the mechanisms of tumor-Mφ interactions leading to Mφ activation. The results show that L5178Y mouse T cell lymphoma cells can prime naive mouse Mφ to subsequent LPS stimulation, resulting in increased NO production and antilymphoma effects in vitro. L5178Y cells, but not naive splenocytes, primed Mφ to ligation of TLR4 but not TLR9. L5178Y-primed Mφ incubated with LPS showed down-regulation of CD40 and up-regulation of NKG2D expression. Although L5178Y T cell lymphoma cells primed naive mouse Mφ, several other mouse and human cells lines failed to prime mouse Mφ. Neither L5178Y-conditioned supernatants nor coculture of Mφ and L5178Y cells in Transwells resulted in priming, indicating that direct L5178Y cell-Mφ contact was needed. Several receptor-ligand pairs are reciprocally expressed on Mφ and L5178Y cell membranes and can be potentially involved in Mφ priming. Of these, the CD40-CD154 pair played the most important role, as blocking the interaction of these molecules substantially reduced in vitro Mφ priming. Furthermore, simultaneous blocking of interactions between CD40-CD154, NKG2D-H60, and CD18-ICAM-1/2 led to complete abrogation of Mφ-mediated NO secretion and complete inhibition of Mφ-mediated tumor cell cytostasis. The priming of Mφ to LPS with L5178Y cells was also observed in vivo. These results suggest that contact with certain tumor cells via CD40, NKG2D, and CD18 molecules on the Mφ may facilitate Mφ-mediated antitumor immune surveillance.

Antitumor effects of CD40 ligation appear to involve distinct antitumor effector cells in different experimental models. In this study, we tested whether T cells were required for antitumor effects of agonistic anti-CD40 mAb (alphaCD40) against immunogenic versus poorly immunogenic tumors. Treatment of mice bearing poorly immunogenic B16 melanoma and its more immunogenic variant, B16-hsp72.1, with alphaCD40 resulted in a similar level of tumor growth suppression. Depletion of T cells did not reduce the antitumor effects in these 2 tumor models. To generate antitumor T cell responses, C57BL/6 mice were immunized with irradiated B16-hsp72.1. Treatment of these vaccinated mice challenged with a high dose of B16-hsp72.1 tumor cells with alphaCD40 induced tumor growth suppression, which was reduced by T-cell depletion, demonstrating that T cells were involved in the antitumor effect of alphaCD40. However, immunized mice depleted of T cells and treated with alphaCD40 were still able to suppress tumor growth as compared to tumor growth in immunized, T cell-depleted mice not treated with alphaCD40, suggesting that T cells were not required for the antitumor effect of alphaCD40. To confirm a lack of correlation between tumor immunogenicity and T-cell requirement in antitumor effects of CD40 ligation, we found that alphaCD40 induced tumor growth suppression in nude and SCID/beige mice bearing highly immunogenic tumors such as Meth A sarcoma, suggesting that macrophages may play a role. Indeed, both poorly immunogenic and highly immunogenic tumors were sensitive to in vitro growth inhibition by macrophages from alphaCD40-treated mice. Taken together, our results indicate that antitumor effects induced by alphaCD40, even against immunogenic tumors, can be observed in the absence of T cells and may involve macrophages.

We have previously shown that macrophages (Mφ) can be activated by CD40 ligation to become cytotoxic against tumor cells in vitro. Here we show that treatment of mice with agonistic anti-CD40 mAb (anti-CD40) induced up-regulation of intracellular TLR9 in Mφ and primed them to respond to CpG-containing oligodeoxynucleotides (CpG), resulting in synergistic activation. The synergy between anti-CD40 and CpG was evidenced by increased production of IFN-γ, IL-12, TNF-α, and NO by Mφ, as well as by augmented apoptogenic effects of Mφ against tumor cells in vitro. The activation of cytotoxic Mφ after anti-CD40 plus CpG treatment was dependent on IFN-γ but not TNF-α or NO, and did not require T cells and NK cells. Anti-CD40 and CpG also synergized in vivo in retardation of tumor growth in both immunocompetent and immunodeficient mice. Inactivation of Mφ in SCID/beige mice by silica treatment abrogated the antitumor effect. Taken together, our results show that Mφ can be activated via CD40/TLR9 ligation to kill tumor cells in vitro and inhibit tumor growth in vivo even in immunocompromised tumor-bearing hosts, indicating that this Mφ-based immunotherapeutic strategy may be appropriate for clinical testing.

We have shown previously that agonistic anti-CD40 mAb induced T cell-independent antitumor effects in vivo. In this study, we investigated mechanisms of macrophage activation with anti-CD40 mAb treatment, assessed by the antitumor action of macrophages in vitro. Intraperitoneal injection of anti-CD40 mAb into C57BL/6 mice resulted in activation of peritoneal macrophages capable of suppressing B16 melanoma cell proliferation in vitro, an effect that was greatly enhanced by LPS and observed against several murine and human tumor cell lines. Anti-CD40 mAb also primed macrophages in vitro to mediate cytostatic effects in the presence of LPS. The tumoristatic effect of CD40 ligation-activated macrophages was associated with apoptosis and killing of tumor cells. Activation of macrophages by anti-CD40 mAb required endogenous IFN-γ because priming of macrophages by anti-CD40 mAb was abrogated in the presence of anti-IFN-γ mAb, as well as in IFN-γ-knockout mice. Macrophages obtained either from C57BL/6 mice depleted of T and NK cells by Ab treatment, or from scid/beige mice, were still activated by anti-CD40 mAb to mediate cytostatic activity. These results argued against the role of NK and T cells as the sole source of exogenous IFN-γ for macrophage activation and suggested that anti-CD40 mAb-activated macrophages could produce IFN-γ. We confirmed this hypothesis by detecting intracytoplasmic IFN-γ in macrophages activated with anti-CD40 mAb in vivo or in vitro. IFN-γ production by macrophages was dependent on IL-12. Taken together, the results show that murine macrophages are activated directly by anti-CD40 mAb to secrete IFN-γ and mediate tumor cell destruction.

Established s.c. NXS2 murine neuroblastoma tumors exhibited transient resolution after suboptimal therapy using the hu14.18-IL2 immunocytokine (IC). The hu14.18-IL2 IC is a fusion protein that has linked a molecule of interleukin 2 (IL-2) to the COOH terminus of each of the IgG heavy chains on the humanized anti-GD2 monoclonal antibody hu14.18. To induce more potent and longer lasting in vivo antitumor effects, we tested hu14.18-IL2 IC in a regimen combining it with constant infusion IL-2 in NXS2 tumor-bearing mice. The addition of the constant infusion IL-2 augmented the antitumor response induced by treatment with the hu14.18-IL2 IC in animals with experimentally induced hepatic metastases and in animals bearing localized s.c. tumors. The combined treatment induced prolonged tumor eradication in most animals bearing s.c. tumors and involved both natural killer cells and T cells. The enhanced ability of this combined treatment to prevent tumor recurrence was not observed when a larger dose of hu14.18-IL2 IC, similar in IL-2 content to the IC plus systemic IL-2 regimen, was tested as single-agent therapy. Animals showing prolonged tumor eradication of established tumors after the combined hu14.18-IL2 plus IL-2 regimen exhibited a protective T-cell-dependent antitumor memory response against NXS2 rechallenge.

The huKS-IL2 immunocytokine (IC) consists of IL2 fused to a mAb against EpCAM, while the hu14.18-IL2 IC recognizes the GD2 disialoganglioside. They are under evaluation for treatment of EpCAM(+) (ovarian) and GD2(+) (neuroblastoma and melanoma) malignancies because of their proven ability to enhance tumor cell killing by antibody-dependent cell-mediated cytotoxicity (ADCC) and by antitumor cytotoxic T cells. Here, we demonstrate that huKS-IL2 and hu14.18-IL2 bind to tumor cells via their antibody components and increase adhesion and activating immune synapse (AIS) formation with NK cells by engaging the immune cells' IL-2 receptors (IL2R). The NK leukemia cell line, NKL (which expresses high affinity IL2Rs), shows fivefold increase in binding to tumor targets when treated with IC compared to matching controls. This increase in binding is effectively inhibited by blocking antibodies against CD25, the α-chain of the IL2R. NK cells isolated from the peritoneal environment of ovarian cancer patients, known to be impaired in mediating ADCC, bind to huKS-IL2 via CD25. The increased binding between tumor and effector cells via ICs is due to the formation of AIS that are characterized by the simultaneous polarization of LFA-1, CD2 and F-actin at the cellular interface. AIS formation of peritoneal NK and NKL cells is inhibited by anti-CD25 blocking antibody and is 50-200% higher with IC versus the parent antibody. These findings demonstrate that the IL-2 component of the IC allows IL2Rs to function not only as receptors for this cytokine but also as facilitators of peritoneal NK cell binding to IC-coated tumor cells.

The hu14.18-IL2 (EMD 273063) IC, consisting of a GD2-specific mAb genetically engineered to two molecules of IL-2, is in clinical trials for treatment of GD2-expressing tumors. Anti-tumor activity of IC in vivo and in vitro involves NK cells. We studied the kinetics of retention of IC on the surface of human CD25+CD16– NK cell lines (NKL and RL12) and GD2+ M21 melanoma after IC binding to the cells via IL-2R and GD2, respectively. For NK cells, ∼50% of IC was internalized by 3 h and ∼90% by 24 h of cell culture. The decrease of surface IC levels on NK cells correlated with the loss of their ability to bind to tumor cells and mediate antibody-dependent cellular cytotoxicity in vitro. Unlike NK cells, M21 cells retained ∼70% of IC on the surface following 24 h of culture and maintained the ability to become conjugated and lysed by NK cells. When NKL cells were injected into M21-bearing SCID mice, IT delivery of IC augmented NK cell migration into the tumor. These studies demonstrate that once IC binds to the tumor, it is present on the tumor surface for a prolonged time, inducing the recruitment of NK cells to the tumor site, followed by tumor cell killing.

We have earlier demonstrated T-cell-independent antitumor effects of a combination of anti-CD40 monoclonal antibody (mAb) and CpG oligodeoxynucleotides (CpG) which involved macrophages. As some immunotherapeutic treatments can be potentiated by chemotherapy, we tested if cyclophosphamide (CY) would enhance the antitumor effect of anti-CD40 mAb+CpG. Treatment of B16 melanoma-bearing mice with CY and anti-CD40 mAb+CpG resulted in a significant reduction in tumor growth in immunocompetent mice compared with either CY alone or anti-CD40 mAb with CpG. This enhanced antitumor effect was maintained in severe combined immunodeficiency mice, as measured by both tumor growth and overall survival. Natural killer cells were not required for this antitumor effect as it was also observed in severe combined immunodeficiency/beige mice. Moreover, although CY treatment of immunocompetent mice suppressed natural killer cell activity, it did not negatively affect the antitumor activity of their macrophages when assayed in vitro. Depletion of macrophages in vivo reduced the antitumor effect of CY and anti-CD40 mAb+CpG. These results suggest that therapeutic strategies to activate macrophages may have potential for clinical application in cancer patients receiving chemotherapy.

Purpose: Radiofrequency ablation (RFA) is a common treatment modality for surgically unresectable tumors. However, there is a high rate of both local and systemic recurrence. Experimental Design: In this preclinical study, we sought to enhance the antitumor effect of RFA by combining it with huKS-IL2 immunocytokine [tumor-specific monoclonal antibody fused to interleukin-2 (IL2)] in mice bearing CT26-KS colon adenocarcinoma. Mice were treated with RFA, huKS-IL2 via intratumoral injection, or combination therapy. Results: Treatment of mice bearing s.c. tumors with RFA and huKS-IL2 resulted in significantly greater tumor growth suppression and enhanced survival compared with mice treated with RFA or huKS-IL2 alone. When subtherapeutic regimens of RFA or huKS-IL2 were used, tumors progressed in all treated mice. In contrast, the combination of RFA and immunocytokine resulted in complete tumor resolution in 50% of mice. Treatment of a tumor with RFA and intratumoral huKS-IL2 also showed antitumor effects against a distant untreated tumor. Tumor-free mice after treatment with RFA and huKS-IL2 showed immunologic memory based on their ability to reject subsequent challenges of CT26-KS and the more aggressive parental CT26 tumors. Flow cytometry analysis of tumor-reactive T cells from mice with complete tumor resolution showed that treatment with RFA and huKS-IL2 resulted in a greater proportion of cytokine-producing CD4 T cells and CD8 T cells compared with mice treated with RFA or huKS-IL2 alone. Conclusions: These results show that the addition of huKS-IL2 to RFA significantly enhances the antitumor response in this murine model, resulting in complete tumor resolution and induction of immunologic memory.

The Eμ-TCL1 transgenic mouse spontaneously develops a CD5+ B cell lymphoproliferative disorder similar to human chronic lymphocytic leukemia (CLL). Given the ineffectual T cell antitumor responses in this mouse model of CLL, we sought to determine whether combined treatment with anti-CD40 mAb (αCD40) and CpG-containing oligodeoxynucleotides (CpG) could exert immunotherapeutic effects. We have previously shown that macrophages activated by sequential ligation of CD40 and TLR9 could become cytotoxic against solid tumor cell lines both in vitro and in vivo. In the current study, we find that αCD40 plus CpG-activated macrophages induce tumor B cell apoptosis in vitro and that αCD40 plus CpG treatment markedly retards tumor growth in immunodeficient SCID/Beige mice following transplantation of primary tumor B cells. Our results suggest a novel immunotherapeutic strategy for CLL that may be effective even in the face of tumor or chemotherapy-induced T cell immunodeficiency.

Immunocytokines (IC), consisting of tumor-specific monoclonal antibodies fused to the immunostimulatory cytokine interleukin 2 (IL2), exert significant antitumor effects in several murine tumor models. We investigated whether intratumoral (IT) administration of IC provided enhanced antitumor effects against subcutaneous tumors. Three unique ICs (huKS-IL2, hu14.18-IL2, and GcT84.66-IL2) were administered systemically or IT to evaluate their antitumor effects against tumors expressing the appropriate IC-targeted tumor antigens. The effect of IT injection of the primary tumor on a distant tumor was also evaluated. Here, we show that IT injection of IC resulted in enhanced antitumor effects against B16-KSA melanoma, NXS2 neuroblastoma, and human M21 melanoma xenografts when compared to intravenous (IV) IC injection. Resolution of both primary and distant subcutaneous tumors and a tumor-specific memory response were demonstrated following IT treatment in immunocompetent mice bearing NXS2 tumors. The IT effect of huKS-IL2 IC was antigen-specific, enhanced compared to IL2 alone, and dose-dependent. Hu14.18-IL2 also showed greater IT effects than IL2 alone. The antitumor effect of IT IC did not always require T cells since IT IC induced antitumor effects against tumors in both SCID and nude mice. Localization studies using radiolabeled (111)In-GcT84.66-IL2 IC confirmed that IT injection resulted in a higher concentration of IC at the tumor site than IV administration. In conclusion, we suggest that IT IC is more effective than IV administration against palpable tumors. Further testing is required to determine how to potentially incorporate IT administration of IC into an antitumor regimen that optimizes local and systemic anticancer therapy.

We have previously demonstrated T cell-independent antitumor and antimetastatic effects of CD40 ligation that involved natural killer (NK) cells. As CD40 molecules are expressed on the surface of macrophages (Mφ), we hypothesized that Mφ may also serve as antitumor effector cells when activated by CD40 ligation. Progression of subcutaneous NXS2 murine neuroblastomas was delayed significantly by agonistic CD40 monoclonal antibody (anti-CD40 mAb) therapy in immunocompetent A/J mice, as well as in T and B cell-deficient severe combined immunodeficiency (SCID) mice. Although NK cells can be activated by anti-CD40 mAb, anti-CD40 mAb treatment also induced a significant antitumor effect in SCID/beige mice in the absence of T and NK effector cells, even when noncytolytic NK cells and polymorphonuclear cells (PMN) were depleted. Furthermore, in vivo treatment with anti-CD40 mAb resulted in enhanced expression of cytokines and cell surface activation markers, as well as Mφ-mediated tumor inhibition in A/J mice, C57BL/6 mice, and SCID/beige mice, as measured in vitro. A role for Mφ was shown by reduction in the antitumor effect of anti-CD40 mAb when Mφ functions were inhibited in vivo by silica. In addition, activation of peritoneal Mφ by anti-CD40 mAb resulted in survival benefits in mice bearing intraperitoneal tumors. Taken together, our results show that anti-CD40 mAb immunotherapy of mice can inhibit tumor growth in the absence of T cells, NK cells, and PMN through the involvement of activated Mφ.

Graft-versus-host disease (GVHD) remains a major cause of morbidity and mortality in allogeneic stem cell transplantation (alloSCT). Donor T cells that accompany stem cell grafts cause GVHD by attacking recipient tissues; therefore, all patients receive GVHD prophylaxis by depletion of T cells from the allograft or through immunosuppressant drugs. In addition to providing a graft-versus-leukemia effect, donor T cells are critical for reconstituting T cell–mediated immunity. Ideally, immunity to infectious agents would be transferred from donor to host without GVHD. Most donors have been exposed to common pathogens and have an increased precursor frequency of memory T cells against pathogenic antigens. We therefore asked whether memory CD62L–CD44+ CD4+ T cells would induce less GVHD than unfractionated or naive CD4+ T cells. Strikingly, we found that memory CD4 cells induced neither clinical nor histologic GVHD. This effect was not due to the increased number of CD4+CD25+ regulatory T cells found in the CD62L–CD44+ fraction because memory T cells depletion of these cells did not cause GVHD. Memory CD4 cells engrafted and responded to antigen both in vivo and in vitro. If these murine results are applicable to human alloSCT, selective administration of memory T cells could greatly improve post-transplant immune reconstitution.

Introduction: The hu14.18-IL2 immunocytokine (IC) links rhIL2 to humanized anti-GD2 monoclonal antibody hu14.18 and inhibits NXS2 tumor growth in A/J mice. This study combined systemic IL2 with hu14.18-IL2 therapy to increase antitumor effects. Methods: A/J mice with subcutaneous NXS2 tumors received rhIL2 (50,000 IU/d by continuous infusion pump) × 7 d, IC (5 micrograms/d × 5), IC (22 micrograms/d × 5), or combined rhIL2 and IC (5 micrograms/d). Mice were also depleted of NK or T cells during combined therapy. Proliferation assays of human and murine cells with high-affinity IL2Rs (T cells) or intermediate-affinity IL2Rs (NK cells) were performed. Results: The combination of systemic IL2 and IC therapy resulted in durable resolution of NXS2 tumors. This antitumor effect was greater than that achieved by either rhIL2 or IC alone (p = 0.026 and 0.007). The efficacy of the combined treatment was dependent on NK and T cells. Although IL2 in the form of an immunocytokine and rhIL2 induce similar proliferation of human NK and T cells, the immunocytokine is less potent than rhIL2 at stimulating murine NK and T cells (3 to 20-fold less, respectively). Conclusions: The combination of systemic IL2 and hu14.18-IL2 is more effective than either therapy alone in A/J mice bearing subcutaneous NXS2 tumors and involves both NK and T cells. This may be the result of NK cell activation and expansion prior to IC treatment and reflect the stronger IL2-mediated action of rhIL2 than IC on murine IL2R-bearing cells.

Hu14.18-IL2 (EMD 273063) IC, consisting of GD2-specific mAb and IL2, is in trials for treatment of melanoma and neuroblastoma. Preclinical studies showed augmented antitumor activity when IC is injected IT vs. IV. Human IL2R+CD16+NK cells mediate ex vivo ADCC in the presence of IC that binds them to GD2+ tumor cells. We studied the kinetics of IC presence on the surface of human M21 melanoma and CD25+CD16-NK cell lines, NKL and RL12, after IC binding to the cells via GD2 and IL2R, respectively. For NK cells, ~50% of IC was internalized by 6 h, and ~90% by 24 h of cell culture. Excess free IL2 in the culture did not slow internalization kinetics for these NK cells. IL2-deprivation for 24 h prior to IC arming of NK cells decreased IL2Rα expression and overall IC binding but did not affect internalization kinetics of the bound IC. For NK cells that were prelabeled with IC, the decrease of surface IC levels over 24 h correlated with reduction of ADCC in vitro. Unlike NK cells, M21 cells retained ~70% of IC on the surface following 24 h of culture. When CFSE-labeled NKL cells were injected IV into M21-bearing SCID mice, they migrated better into the tumor when IC was delivered IT than when they were pre-armed with IC before implantation. These preliminary studies suggest that once IC gets to tumor sites, it may stay on the tumor surface for a prolonged time, and that IT injections of IC enhance the recruitment of NK cells to the tumor site.

We present that L5178Y murine T-cell lymphoma cells can prime naïve murine macrophages (Mϕ) to subsequent LPS stimulation, resulting in antitumor effects in vitro against L5178Y cells. Priming occurred in both cell-type and TLR specific manner, as L5178Y tumor cells (TC), but not naïve splenocytes, primed Mϕ to ligation of TLR4 but not TLR9. L5178Y-primed Mϕ also showed down-regulation of CD40 and CD86 and up-regulation of B7H1, with no changes of TLR4, B7H4, NKG2D and MHC-II expression. Among different TC lines, the priming effect was limited to murine TC of T cell (L5178Y and YAC-1) but not B cell (A20) or nonmyeloid (B16) origin. Human TC lines including Jurkat (T cell), Daudi (B cell), and M21 (melanoma) failed to substantially prime Mϕ. Neither L5178Y-conditioned supernatants nor co-culture of Mϕ and L5178Y TC in transwells resulted in priming, indicating that direct L5178Y TC-Mϕ contact was needed. Furthermore, TLR4-ligation must not precede contact with L5178Y TC in order to induce tumoritoxic Mϕ. Among several receptor-ligand pairs reciprocally expressed on Mϕ and L5178Y TC and potentially involved in the Mϕ priming, CD40 (expressed on Mϕ) and CD40L (expressed on L5178Y TC) appeared to play the most important role, as blocking their interaction with anti-CD40L mAb substantially reduced in vitro tumoritoxicity. Hence, contact with TC may facilitate Mϕ-mediated immune surveillance against certain cancers.