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Purpose: Docosahexaenoyl difluorodeoxycytidine (DHA-dFdC) is an amide with potent, broad-spectrum antitumor activity. In the present study, DHA-dFdC's ability to induce immunogenic cell death (ICD) was tested using CT26 mouse colorectal cancer cells, an established cell line commonly used for identifying ICD inducers, as well as Panc-02 mouse pancreatic cancer cells., Methods: The three primary surrogate markers of ICD (i.e., calreticulin (CRT) surface translocation, ATP release, and high mobility group box 1 protein (HMGB1) release) were measured in vitro. To confirm DHA-dFdC's ability to induce ICD in vivo, the gold standard mouse vaccination studies were conducted using both CT26 and Panc-02 models. Additionally, the effect of DHA-dFdC on tumor response to anti-programmed cell death protein 1 monoclonal antibody (anti-PD-1 mAb) were tested in mice with pre-established Panc-02 tumors. RNA sequencing experiments were conducted on PANC-1 human pancreatic cancer cells treated with DHA-dFdC, dFdC, or vehicle control in vitro., Results: DHA-dFdC elicited CRT surface translocation and ATP and HMGB1 release in both cell lines. Immunization of mice with CT26 or Panc-02 cells pretreated with DHA-dFdC prevented or delayed the development of corresponding secondary live challenge tumor. DHA-dFdC enabled Panc-02 tumors to respond to anti-PD-1 mAb. RNA sequencing experiments revealed that DHA-dFdC and dFdC differentially impacted genes related to the KRAS, TP53, and inflammatory pathways, and DHA-dFdC enriched for the unfolded protein response (UPR) compared to control, providing insight into DHA-dFdC's potential mechanism of inducing ICD., Conclusion: DHA-dFdC is a bona fide ICD inducer and can render pancreatic tumors responsive to anti-PD-1 mAb therapy., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Debate is around the optimal immunization regimen for cancer vaccines since too intense vaccination schedules may exhaust reactive lymphocytes. GX301 is a telomerase-based cancer vaccine whose safety and immunological effects were tested in a phase I trial applying an eight administrations schedule. Main objective of this study was to comparatively analyse safety and immunological response to three GX301 regimens in metastatic castration-resistant prostate cancer patients with response/disease stability after docetaxel chemotherapy. This was a multicentre, randomized, parallel-group, open-label trial registered with EudraCT (2014-000095-26) and ClinicalTrials.gov (NCT02293707, 2014). Ninety-eight patients were randomized to receive either eight (regimen 1), four (regimen 2) or two (regimen 3) vaccine administrations. Sixty-three patients were assessable for the primary immunological end-point. Vaccine-specific immune responses were evaluated by intracellular staining for IFN, elispot and cytotoxic assay at 90 and 180 days from baseline. No major side effects were recorded. A 54% overall immune responder rate was observed with 95% of patients showing at least one vaccine-specific immune response. Rate of immunological responders and number of immunizations were proportionally related, suggesting superiority of regimens 1 and 2 over regimen 3. Overall survival did not differ among regimens in both immunological responders and non-responders and was inversely associated (P = 0.002) with increase in the number of circulating CD8 + T regulatory cells at 180 days. These data indicate that GX301 cancer vaccine is safe and immunogenic in metastatic castration-resistant prostate cancer patients. Schedules with high number of administrations should be preferred in future studies due to their better immunological outcome., (© 2021. The Author(s).)

Background: Selected patients with advanced non-small cell lung cancer (NSCLC) benefit from immunotherapy, especially immune checkpoint inhibitors such as PD-1 (programmed cell death protein 1) inhibitor. Peripheral blood biomarkers would be most convenient to predict treatment outcome and immune-related adverse events (irAEs) in candidate patients. This study explored associations between inflammation-related peripheral blood markers and onset of irAEs and outcome in patients with advanced NSCLC receiving PD-1 inhibitors., Methods: A retrospective analysis was conducted of 102 patients with advanced NSCLC receiving PD-1 inhibitors from January 2017 to May 2019. Cox regression models were employed to assess the prognostic effect of low/high neutrophil/lymphocyte ratio (NLR), lactate dehydrogenase (LDH), and prognostic nutrition index (PNI) on overall survival (OS) and progression-free survival (PFS). Logistic regression models were used to analyze the correlation between peripheral blood markers and the onset of irAEs., Result: NLR < 5, LDH < 240 U/L, or PNI ≥ 45 was favorably associated with significantly better outcomes compared with higher, higher, or lower values, respectively. The multivariate analysis determined that these parameters were independently associated with both better PFS (p = 0.049, 0.046, 0.014, respectively) and longer OS (p = 0.007, 0.031, < 0.001, respectively). Patients with three favorable factors among NLR, LDH, and PNI had better PFS and OS than did those with two, one, or none. PNI and NLR were associated with the onset of irAEs., Conclusion: In patients with advanced NSCLC treated with PD-1 inhibitors, pretreatment NLR, LDH, and PNI may be useful predictive markers of clinical outcome and irAEs.

Purpose: Circularly permuted TRAIL (CPT) has exhibited promising efficacy as a mono-therapy or in combination with thalidomide for patients with multiple myeloma (MM). In this phase 2 study, the safety and efficacy of CPT in combination with thalidomide and dexamethasone (CPT + TD) was evaluated in patients with pretreated relapsed/refractory MM (RRMM)., Methods: Patients who received at least two previous therapies for MM were randomly assigned at a 2:1 ratio to receive treatment with CPT + TD or thalidomide and dexamethasone (TD). The primary endpoint was the overall response rate (ORR), and the secondary endpoints included progression-free survival (PFS), duration of response (DOR) and safety., Results: Overall, 47 patients were assigned to the CPT + TD group, and 24 patients were recruited to the TD group. The ORR in the CPT + TD group was 38.3 vs. 25.0% in the TD group. The median PFS time was 6.7 months for the CPT + TD group and 3.1 months for the TD group. The median DORs for the CPT + TD and TD groups were 7.1 and 3.2 months, respectively. Most of the adverse effects (AEs) were grade 1 or 2. Serious AEs were reported in 19.7% of the patients. No treatment-related deaths were reported., Conclusion: CPT plus TD could serve as a new therapeutic strategy for patients with RRMM. A randomized, double-blind, placebo-controlled confirmatory study is currently under way.

Purpose: Ipilimumab is designed to block cytotoxic T-lymphocyte antigen-4 to augment antitumor T cell responses. In studies of predominantly Caucasian patients with advanced melanoma, ipilimumab was associated with durable response, long-term survival benefit, and a manageable safety profile. This phase II study assessed the safety of ipilimumab in Japanese patients with unresectable stage III or IV melanoma., Methods: Patients received ipilimumab 3 mg/kg every 3 weeks for four doses. The database lock for the original analysis was in August 2014. Overall survival, progression-free survival, and data on deaths were based on an updated, follow-up analysis (database lock April 2015)., Results: Data are reported from 20 patients. Fifteen patients (75 %) received all four doses of ipilimumab during induction. Twelve patients (60 %) had at least one drug-related adverse event (AE), and no patients discontinued due to a drug-related AE. There were no deaths related to study drug. The most common drug-related AEs were rash (n = 7), pyrexia (n = 3), increased aspartate aminotransferase (AST; n = 3), and increased alanine aminotransferase (ALT; n = 3). Twelve patients (60 %) reported immune-related AEs (irAEs); most frequent were skin (n = 9) and liver (n = 3) disorders. Grade 3 irAEs were ALT and AST elevation (n = 2) and diabetes mellitus (n = 1). Two patients had a partial response and two had stable disease, yielding a 20 % disease control rate. Median overall survival and progression-free survival were 8.71 and 2.74 months, respectively., Conclusion: Ipilimumab 3 mg/kg had a manageable AE profile in this Japanese patient population with clinical outcomes similar to that in Caucasian patients. CLINICALTRIALS., Gov Identifier: NCT01990859.

Polysaccharide K (PSK) is a widely used mushroom extract that has shown anti-tumor and immunomodulatory effects in both preclinical and clinical studies. Therefore, it is important to understand the mechanism of actions of PSK. We recently reported that PSK can activate toll-like receptor 2 and enhances the function of NK cells. The current study was undertaken to study the effect of PSK on gamma delta (γδ) T cells, another important arm of the innate immunity. In vitro experiments using mouse splenocytes showed that γδ T cells produce IFN-γ after treatment with PSK and have up-regulated expression of CD25, CD69, and CD107a. To investigate whether the effect of PSK on γδ T cells is direct or indirect, purified γδ T cells were cultured either alone or together with bone marrow-derived DC in a co-culture or trans-well system and then stimulated with PSK. Results showed that direct cell-to-cell contact between γδ T cells and DC is required for optimal activation of γδ T cells. There was also reciprocal activation of DC by PSK-activated γδ T cells, as demonstrated by higher expression of costimulatory molecules and enhanced production of IL-12 by DC in the presence of γδ T cells. PSK can also co-stimulate γδ T cells with anti-TCR and anti-CD3 stimulation, in the absence of DC. Finally, in vivo treatment with PSK activates γδ T cells among the tumor infiltrating lymphocytes, and depleting γδ T cells during PSK treatment attenuated the anti-tumor effect of PSK. All together, these results demonstrated that γδ T cells are activated by PSK and contribute to the anti-tumor effect of PSK.

Background: Cetuximab is an anti-epidermal growth factor receptor (EGFR) monoclonal antibody that prolongs survival in the treatment for head and neck cancer (HNC), but only in 10-20 % of patients. An immunological mechanism of action such as natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC) has been suggested. We investigated the effects of activating toll-like receptor (TLR)-8 to enhance activity of cetuximab-stimulated, FcγR-bearing cells., Objective: To determine the capability of TLR8-stimulation to enhance the activation and function of NK cells and dendritic cells (DC) in the presence of cetuximab-coated HNC cells., Methods: Peripheral blood mononuclear cells (PBMC), NK, DC, and CD8(+) T cells were isolated and analyzed using (51)Cr release ADCC, flow cytometry analysis, cytokine ELISA, and EGFR853-861 tetramer staining., Results: TLR8 stimulation of unfractionated PBMC led to enhanced cetuximab-mediated ADCC in healthy donors (p < 0.01) and HNC patients (p < 0.001), which was dependent on NK cells. Secretion of Th1 cytokines TNFα (p < 0.0001), IFNγ (p < 0.0001), and IL-12p40 (p < 0.005) was increased. TLR8 stimulation of PBMC augmented cetuximab-enhanced NK cell degranulation (p < 0.001). TLR8-stimulated NK cells enhanced DC maturation markers CD80, CD83, and CD86 in co-culture with cetuximab-treated HNC cells. TLR8 stimulation of NK-DC co-cultures significantly increased DC priming of EGFR-specific CD8(+) T cells in the presence of cetuximab., Discussion: VTX-2337 and cetuximab combination therapy can activate innate and adaptive anti-cancer immune responses. Further investigation in human trials will be important for determining the clinical benefit of this combination and for determining biomarkers of response.

Purpose: NPC-1C is a chimeric immunoglobulin IgG1 developed from antigen tested in the Hollinshead tumor vaccine trials that recognizes an immunogenic MUC5AC-related tumor-associated antigen. In this article, we describe the pre-clinical characterization of this antibody that is currently being tested in human clinical trials., Experimental Design: The specificity of NPC-1C for pancreatic and colorectal cancer cell lines was tested by flow cytometry assays and immunohistochemical staining. Antibody-dependent cell cytotoxicity was measured using a tumor cell line lysis assay. Anti-tumor efficacy and biodistribution were assessed in nude mice bearing human pancreatic tumor xenografts., Results: Human tumor cell binding measured by flow cytometry ranged from 52 to 94 % of cells stained positive with NPC-1C in three colorectal and one pancreatic cell lines, while IHC demonstrated staining of 43 % of colon cancers and 48 % of pancreatic cancer tissues, with little or no cross-reactivity of NPC-1C with normal colon or pancreas tissues. In vitro NPC-1C-mediated tumor cell killing occurred in a median of 44.5 % of four colorectal and three pancreatic tumor cell lines. In vivo anti-tumor efficacy in a human pancreatic CFPAC-1 tumor xenograft model was demonstrated with a twofold to threefold reduction in tumor growth in the NPC-1C-treated mice compared to saline and human IgG controls. Pharmacodynamic studies indicate NPC-1C localizes in antigen-positive tumors and has minimal uptake in normal mouse tissues., Conclusions: NPC-1C, a chimeric monoclonal antibody that reacts with a MUC5AC-related antigen expressed by pancreatic and colorectal tumor tissues, has promising preclinical activity in pancreatic and colorectal adenocarcinoma.

Purpose: Transferrin receptor (TfR) is a cell membrane-associated glycoprotein involved in the cellular uptake of iron and the regulation of cell growth. Recent studies have shown elevated expression levels of TfR on cancer cells compared with normal cells. We previously designed a TfR-lytic hybrid peptide, which combines the TfR-binding peptide and a lytic peptide, and reported that it bound specifically to TfR and selectively killed cancer cells. Furthermore, the intravenous administration of TfR-lytic peptide in an athymic mouse model significantly inhibited tumor progression. To evaluate the immunogenicity of this peptide as a novel and potent anticancer agent, we investigated whether TfR-lytic hybrid peptide elicits cellular and humoral immune responses to produce antibodies. We also examined the toxicity of this peptide in syngeneic mice., Methods: We performed hematologic and blood chemistry test and histological analysis and assessed hemolytic activity to check toxicity. To evaluate the immunogenicity, measurement of murine interferon-gamma and detection of TfR-lytic-specific antibody by ELISA were demonstrated., Results: No T cell immune response or antibodies were detected in the group treated with TfR-lytic hybrid peptide. No hematologic toxicity, except for a decrease in leukocytes, was observed, and no remarkable influence on metabolic parameters and organs (liver, kidney, and spleen) was noted., Conclusions: Therefore, TfR-lytic hybrid peptide might provide an alternative therapeutic option for patients with cancer.

Phase I testing of the hu14.18-IL2 immunocytokine in melanoma patients showed immune activation, reversible toxicities, and a maximal tolerated dose of 7.5 mg/m(2)/day. In this phase II study, 14 patients with measurable metastatic melanoma were scheduled to receive hu14.18-IL2 at 6 mg/m(2)/day as 4-h intravenous infusions on Days 1, 2, and 3 of each 28 day cycle. Patients with stable disease (SD) or regression following cycle 2 could receive two additional treatment cycles. The primary objective was to evaluate antitumor activity and response duration. Secondary objectives evaluated adverse events and immunologic activation. All patients received two cycles of treatment. One patient had a partial response (PR) [1 PR of 14 patients = response rate of 7.1 %; confidence interval, 0.2-33.9 %], and 4 patients had SD and received cycles 3 and 4. The PR and SD responses lasted 3-4 months. All toxicities were reversible and those resulting in dose reduction included grade 3 hypotension (2 patients) and grade 2 renal insufficiency with oliguria (1 patient). Patients had a peripheral blood lymphocytosis on Day 8 and increased C-reactive protein. While one PR in 14 patients met protocol criteria to proceed to stage 2 and enter 16 additional patients, we suspended stage 2 due to limited availability of hu14.18-IL2 at that time and the brief duration of PR and SD. We conclude that subsequent testing of hu14.18-IL2 should involve melanoma patients with minimal residual disease based on compelling preclinical data and the confirmed immune activation with some antitumor activity in this study.

Bispecific tetravalent antibodies (TandAb) directed against the B cell surface marker CD19 and activating receptors on T or NK cells (CD19 × CD3 or CD19 × CD16) have shown promising effects in vitro and in preclinical studies. Here, we examine the cytotoxic efficacy of T and NK cells from patients with B cell Non-Hodgkin's Lymphoma (NHL) against B-lymphoma cells following the binding of the matching TandAb. The addition of CD19 × CD16 TandAb led to a threefold increase in NK cell activation in the presence of B-lymphoma cells. Similarly, T cells displayed a sevenfold increase in cytotoxic activity after the addition of CD19 × CD3 TandAb. Comparison of T and NK cell effector function of patients and healthy controls showed comparable levels of cytotoxic activity in response to lymphoma cells and no reduction in functional activity due to age, disease stage or the type and amount of previous therapy. Thus, T and NK cells of patients with B cell NHL are fully capable of being activated by therapeutic crosslinking antibodies. These results provide a rationale for the use of TandAbs for patients with B cell NHL, particularly in cases where remission with minimal residual disease could be achieved by cytotoxic chemotherapy.

The nexus of chemotherapeutic intervention and the immunomodulation of IgE-related phenomena are not well understood. The relationship bears importance in better understanding the causal and/or resultant effects of one on the other and their collective role in the management and sequelae of the cancer patient. This review discusses the relationship of chemotherapy on immunoglobulins with a focus on IgE and other related biological processes including hypersensitivity reactions and proposes models toward effective management of the cancer patient in this regard.

Hypersensitivity reactions (HSRs) to chemotherapy drugs, such as taxanes and platins, and to monoclonal antibodies limit their therapeutic use due to the severity of some reactions and the fear of inducing a potentially lethal reaction in highly sensitized patients. Patients who experience hypersensitivity reactions face the prospect of abandoning first-line treatment and switching to a second-line, less effective therapy. Some of these reactions are mast cell-mediated hypersensitivity reactions, a subset of which occur through an immunoglobulin (IgE)-dependent mechanism, and are thus true allergies. Others involve mast cells without a demonstrable IgE mechanism. Whether basophils can participate in these reactions has not been demonstrated. Rapid drug desensitization (RDD) is a procedure that induces temporary tolerance to a drug, allowing a medication allergic patient to receive the optimal agent for his or her disease. Through RDD, patients with IgE and non-IgE HSRs can safely be administered important medications while minimizing or completely inhibiting adverse reactions. Due to the clinical expansion and success of RDD, the molecular mechanisms inducing the temporary tolerization have been investigated and are partially understood, allowing for safer and more effective protocols. This article reviews the current literature on molecular mechanisms of RDD with an emphasis in our recent contributions to this field as well as the indications, methods and outcomes of RDD for taxanes, platins, and monoclonal antibodies.

Purpose: Ipilimumab, a fully human monoclonal antibody specific to CTLA-4, has been shown to improve overall survival in metastatic melanoma patients. As a consequence of CTLA-4 blockade, ipilimumab treatment is associated with proliferation and activation of peripheral T cells. To better understand various tumor-associated components that may influence the clinical outcome of ipilimumab treatment, gene expression profiles of tumors from patients treated with ipilimumab were characterized., Experimental Design: Gene expression profiling was performed on tumor biopsies collected from 45 melanoma patients before and 3 weeks after the start of treatment in a phase II clinical trial., Results: Analysis of pre-treatment tumors indicated that patients with high baseline expression levels of immune-related genes were more likely to respond favorably to ipilimumab. Furthermore, ipilimumab appeared to induce two major changes in tumors from patients who exhibited clinical activity: genes involved in immune response showed increased expression, whereas expression of genes for melanoma-specific antigens and genes involved in cell proliferation decreased. These changes were associated with the total lymphocyte infiltrate in tumors, and there was a suggestion of association with prolonged overall survival in these patients. Many IFN-γ-inducible genes and Th1-associated markers showed increased expression after ipilimumab treatment, suggesting an accumulation of this particular type of T cell at the tumor sites, which might play an important role in mediating the antitumor activity of ipilimumab., Conclusions: These results support the proposed mechanism of action of ipilimumab, suggesting that cell-mediated immune responses play an important role in the antitumor activity of ipilimumab.

Purpose: MEHD7945A is a novel dual-action monoclonal antibody in which each of the two antigen-binding fragments is capable of binding to EGFR and HER3 with high affinity. It is being evaluated as a potential therapy for human cancer. The purpose of these studies was to characterize the pharmacokinetics (PK) of MEHD7945A in mouse and monkey and predict its human PK and efficacious dose., Methods: PK of MEHD7945A was determined in SCID beige mice and cynomolgus monkeys after administration of single intravenous doses. Human PK profiles were projected from monkey PK profiles using a species-invariant time method, and human population PK parameters were estimated using a nonlinear, two-compartment model comprising specific (target-mediated) and nonspecific clearance pathways. The antitumor efficacy in mice bearing human tumor xenografts was used in conjunction with human PK projections to estimate human efficacious doses., Results: The total clearance of MEHD7945A decreased with increase in dose in both mouse and monkey. The nonspecific clearance in monkey was estimated to be 14 mL/day/kg. The predicted nonspecific clearance range in humans was 6-10 mL/day/kg. Doses of 8-12 mg/kg administered every 2 weeks in humans were predicted to achieve exposure of 300 day μg/mL per week to match the efficacious exposure observed in xenograft models., Conclusions: The PK of MEHD7945A was nonlinear in mouse and monkey in the dose range tested. The nonspecific clearance in monkey was approximately twofold higher than typical humanized IgG1 antibodies. The projected human efficacious dose and dose regimen appear to be achievable in patients.

Disrupting tumor-mediated mechanisms suppressing host immunity represents a novel approach to tumor immunotherapy. Depletion of regulatory T cells (Tregs) increases endogenous anti-tumor immunity and the efficacy of active immunotherapy in experimental tumor models. HLA-A2.1/HLA-DR1 (A2.1/DR1) × BALB- neuT+ (neuT+) triple transgenic mice represent an improvement over neuT+ mice for evaluating vaccination regimens to overcome tolerance against HER-2/neu. We questioned whether depletion of Tregs with Denileukin diftitox (Ontak) enhances the efficacy of a therapeutic vaccine consisting of HER-2(85-94) (p85) CTL and HER-2(776-790) (p776) Th peptides against the growth of TUBO.A2 transplantable tumor in male A2.1/DR1 × neuT+ Tg mice. While the therapeutic vaccine primed the tumor-reactive CD8+ CTLs and CD4+ effector T lymphocytes (Teffs) compartment, inducing activation, tumor infiltration, and tumor rejection or delay in tumor growth, treatment with Ontak 1 day prior to vaccination resulted in enhanced CD4+ and CD8+ T-cell-mediated vaccine-specific immune responses in the periphery. This was closely associated with greater infiltration and a striking change in the intratumor balance of Tregs and vaccine-specific CTLs/Teffs that directly correlated with markedly enhanced antitumor activity. The data suggest that Tregs control both CD4+ and CD8+ T-cell activity within the tumor, emphasize the importance of the intratumor ratio of vaccine-specific lymphocytes to Tregs, and demonstrate significant inversion of this ratio and correlation with tumor rejection during Ontak/vaccine immunotherapy.

Purpose: AGS-1C4D4 is a human monoclonal antibody against prostate stem cell antigen (PSCA), a cell-surface protein expressed by most prostate cancers. AGS-1C4D4 is produced in Chinese hamster ovary (CHO) cells and has an identical sequence to AGS-PSCA, an anti-PSCA antibody produced in mouse hybridoma cells that has completed Phase I testing. Preclinical studies demonstrated comparability of AGS-1C4D4 to AGS-PSCA with respect to pharmacokinetics (PK) and tumor inhibition. However, because of differences in antibody-dependent cellular cytotoxicity between AGS-PSCA and AGS-1C4D4, a limited Phase I trial using AGS-1C4D4 was performed evaluating safety and PK., Patients and Methods: Thirteen patients with metastatic castration-resistant prostate cancer were enrolled. AGS-1C4D4 was administered intravenously every 3 weeks for four planned doses at 6, 12, 24, or 48 mg/kg. Primary endpoints were safety and PK. Secondary endpoints were immunogenicity and clinical activity. Disease assessments were conducted every 12 weeks and included radiographic and PSA evaluations. Patients with stable disease could receive extended treatment beyond four infusions., Results: Adverse events were primarily grade 1-2, without any grade 3-4 drug-related toxicities or infusion reactions. Anti-AGS-1C4D4 antibodies were not detected. Similar to AGS-PSCA, serum AGS-1C4D4 concentrations declined biphasically and elimination was characterized by slow clearance (CL) and a long terminal half-life (t (1/2)). Median CL for the four dose levels ranged from 0.10 to 0.14 ml/h kg, and t (1/2) ranged from 2.2 to 2.9 weeks. No PSA reductions ≥50% were observed. Six patients (46%) had radiographically stable disease, lasting a median of 24 weeks., Conclusion: AGS-1C4D4 was well-tolerated and demonstrated linear PK. Despite preclinical differences in antibody-dependent cellular cytotoxicity, AGS-1C4D4 and AGS-PSCA have similar safety and PK profiles. The recommended Phase II dose is 48 mg/kg.

Some anticancer chemotherapeutics, such as anthracyclines and oxaliplatin, elicit immunogenic apoptosis, meaning that dying cancer cells are engulfed by dendritic cells and tumor antigens are efficiently presented to CD8+ T cells, which control residual tumor cells. Immunogenic apoptosis is characterized by pre-apoptotic cell surface exposure of calreticulin (CRT), which usually resides into the endoplasmic reticulum. We investigated the ability of the n3-polyunsaturated fatty acid docosahexaenoic acid (22:6n3, DHA) to induce pre-apoptotic CRT exposure on the surface of the human PaCa-44 pancreatic and EJ bladder cancer cell lines. Cells were treated with 150 μM DHA for different time periods, and, by immunoblot and immunofluorescence, we showed that DHA induced CRT exposure, before the apoptosis-associated phosphatidylserine exposure. As for the known immunogenic compounds, CRT exposure was inhibited by the antioxidant GSH, the pan-caspase zVAD-FMK, and caspase-8 IETD-FMK inhibitor. We provide the first evidence that DHA induces CRT exposure, representing thus a novel potential anticancer immunogenic chemotherapeutic agent.

Recent studies suggested that the histidine residues at 118 and 122 play an important role for the toxicity of staphylococcal enterotoxin C subtype 2 (SEC2), and the substitutions of both histidines with alanine can severely impair the fever activity of SEC2. We hypothesized that promising SEC2 antitumor agent with low toxicity and enhanced superantigen activity can be constructed by introducing related mutations at protein functional sites of SEC2. We showed that the SEC2 mutants H122A and H118A/H122A exhibited improved superantigen activity after introducing the point mutations at Thr20 and Gly22. A resultant mutant, named as SAM-3, has considerable abilities to inhibit the growth of H22 and Hepa1-6 tumor cells in vitro and colon 26 solid tumor in vivo. Furthermore, SAM-3 also exhibits significantly reduced toxicity compared with native SEC2. The study provides a novel strategy for designing promising superantigen immunotherapeutic agent. The constructed SEC2 mutant SAM-3 can be used as a powerful candidate for cancer immunotherapy and could compensate the deficiency caused by toxicity of native SEC2 in clinic.

Background: The primary objective was to study the antitumor activity of prolonged subcutaneous dosing of systemic 852A, a Toll-like receptor-7 agonist (TLR-7), in recurrent breast, ovarian and cervix cancer. Secondary objectives included assessment of safety and immune system activation., Methods: Adults with recurrent breast, ovarian or cervix cancer failing multiple therapies received 0.6 mg/m(2) of 852A subcutaneously twice weekly for 12 weeks. Doses increased by 0.2 mg/m(2)/week to a maximum of 1.2 mg/m(2). Serum was collected to assess immune activation., Results: Fifteen patients enrolled: 10 ovarian, 2 cervix and 3 breast. Three completed all 24 injections. There were two grade 2 (decreased ejection fractions), nine grade 3 (1 cardiovascular, 1 anorexia, 3 dehydration, 2 infections, 2 renal) and two grade 4 (hepatic and troponin elevation) unanticipated toxicities. Cardiac toxicities included three cardiomyopathies (2 asymptomatic) and one stress-related non-ST elevated myocardial infarction. Five patients discontinued therapy due to possibly associated side effects. One who had stable disease (SD) following 24 doses received 17 additional doses. A cervix patient with SD following 24 doses received chemotherapy after progressing 3 months later, and remains disease free at 18 months. Immune activation, as evidenced by increased IP-10 and IL-1ra, was observed., Conclusions: In this first human experience of a TLR-7 agonist delivered subcutaneously using a prolonged dosing schedule, 852A demonstrated sustained tolerability in some patients. Clinical benefit was modest, but immune activation was seen suggesting further study of antitumor applications is warranted. Because of cardiac toxicity; 852A should be used cautiously in heavily pretreated patients.

Addition of rituximab to chemotherapy (R-chemo) has been shown to improve overall survival (OS) in patients with diffuse large B cell lymphoma (DLBCL). Germinal center B cell-like (GCB) subtype of DLBCL has a significantly better clinical outcome than those with non-germinal center B cell-like (non-GCB) subtype. Further research is needed to confirm this difference between those two subtypes treated with R-chemo. We searched for randomized controlled trials that compared R-chemo with identical chemotherapy alone in patients with newly diagnosed or relapsed DLBCL. A random versus fixed effects model was selected according to heterogeneity. Six eligible trials involving 748 adult patients were included in this meta-analysis. Fixed-effects analysis showed OS to be superior for the GCB patients treated with R-chemo (relative risk (RR) = 1.16, 95% confidence interval (CI) = 1.03-1.31, P = 0.02). Superiority was also observed for the GCB subtype under R-chemo with respect to disease control (RR = 1.16, 95% CI = 0.99-1.36) and overall response (RR = 1.19, 95% CI = 0.99-1.99). Both subtypes showed an increased OS (RR = 1.30, 95% CI = 1.11-1.51; RR = 1.89, 95% CI = 1.52-2.35, respectively) and disease control rate (RR = 1.27, 95% CI = 1.05-1.54, P = 0.01; RR = 2.21, 95% CI = 1.68-2.90, respectively) following R-chemo. Therefore, treated with R-chemo, GCB patients still has a significantly better clinical outcome than those with non-GCB subtype.

Purpose: Monoclonal antibodies (mAb) are an important and growing class of cancer therapeutics, but pharmacokinetic analyses have in many cases been constrained by the lack of standard and robust pharmacologic assays. The goal of this project was to develop a general method for the production of immunoassays that can measure the levels of therapeutic monoclonal antibodies in biologic samples at relevant concentrations., Methods: Alemtuzumab and rituximab are monoclonal approved for the treatment of B-cell malignancies and were used as a model system. Phage-displayed peptide libraries were screened for peptide sequences recognized by alemtuzumab (anti-CD52) or rituximab (anti-CD20). Synthetic biotinylated peptides were used in enzyme-linked immunosorbent assays (ELISA). Peptides directly synthesized on polymer resin beads were used in an immunofluorescent-based assay., Results: Peptide mimetope sequences were recovered for both mAb and confirmed by competitive staining and kinetic measurements. A peptide-based ELISA method was developed for each. The assay for rituximab had a limit of detection of 4 microg/ml, and the assay for alemtuzumab had a limit of detection of 1 microg/ml. Antibody-specific staining of peptide conjugated beads could be seen in a dose-dependent manner., Conclusion: Phage-displayed peptide libraries can be a source of highly specific mimetopes for therapeutic mAb. The biotinylated forms of those peptides are compatible with conventional ELISA methods with sensitivities comparable to other assay methods and sufficient for pharmacological studies of those mAb given at high dose. The process outlined here can be applied to any mAb to enable improved pharmacokinetic analysis during the development and clinical use of this class of therapies.

Purpose: A new VEGF receptor fusion protein FP3 was shown to have promising antitumor potency better than Bevacizumab. Characterization of its immune response is essential to the safe and effective administration in clinical trials. In this study, both BIACORE and ELISA assays were employed to assess pre-clinical immunogenicity of FP3 in monkeys., Experimental Design: Serum samples from 20 rhesus monkeys were analyzed for the generation of anti-FP3 antibody after intravenous administration of three doses of FP3 (n = 6 per group) or buffer control (n = 2). Sera samples were obtained at 2, 4, 6, 8, 10 weeks after the first administration., Results: It showed BIACORE presented linear correlation with the dilution of anti-FP3 antibody and the results of ELISA. Two weeks after the initial FP3 injection, anti-FP3 antibody was detected in about 20% FP3-treated monkeys. The ratio of positive samples and the titer of antibody increased along with the FP3-treatment time. Six weeks following FP3 injection almost all the samples were anti-FP3 antibody positive. Moreover, the titer of anti-FP3 antibody but not the ratio of positive samples was also enhanced when the dose of FP3 was elevated. Furthermore, the immunoglobulin types and subclasses of anti-FP3 antibody serum components were mainly identified as IgG1 and IgG4, not IgM. Serum antibodies are characterized that they could not block FP3 binding to VEGF and were non-neutralizing., Conclusions: Our data implied that proteins with full human sequences may also have the potential to induce immune response in rhesus monkeys, and BIACORE could be an effective approach to detect the immunogenicity of protein therapeutics in clinic.

Background: Cetuximab and panitumumab are chimeric and fully human monoclonal antibodies, respectively, against epidermal growth factor receptor used in the treatment of metastatic colorectal cancer (mCRC). Incidence of documented infusion reaction (IR) is more common with cetuximab (all grades [g]: 15-21%, g 3/4: 2-5%) than panitumumab (all g: 4%, g 3/4: 1%). Anecdotal reports suggest successful challenge with panitumumab following IR with cetuximab (Saif et al. in Cancer Chemother Pharmacol 63(6):1017-1022, 2009). However, safety of cetuximab after IR with panitumumab is not known. We report two patients successfully desensitized with cetuximab after IR with panitumumab., Patients and Methods: A 42-year-old female with mCRC received panitumumab as a third-line agent. She developed severe chest tightness, pain, and shortness of breath (SOB), 5 min after first panitumumab infusion. A second 70-year-old male with mCRC developed severe facial flushing, back pain, SOB, tachycardia and hypotension, 5 min after second dose of panitumumab plus irinotecan as a second-line therapy. These two patients received desensitization protocol for cetuximab after a test dose of 20 mg IV over 10 min followed by a slow infusion 10% of original rate in 0-2 h, 25% of original rate in 2-2.5 h, 50% reduced rate in 2.5-3 h, and then 100% infusion rate after 3 h. Patients were observed 4 h after completion of infusion., Results: First patient received a total of 12 cycles of cetuximab with stable disease, no recurrence of IR, and grade 1-2 acniform rash that first developed after third cycle. Second patient received a total of eight cycles uneventfully without IR., Conclusions: To our knowledge, this is the first report of two patients with documented IR with panitumumab being desensitized successfully with cetuximab. Though anecdotal reports suggest safety of panitumumab in patients following IR with cetuximab, panitumumab can also cause severe IR. Our experience suggests that in case of limited options, such patients can be successfully challenged with cetuximab in a hospital after appropriate desensitization and premedication. Further studies focusing on desensitization and identifying hypersensitivity profile of different anti-epidermal growth factor receptor antibodies are warranted.

Purpose: The interaction of Fc fragments of antibodies with the Fcgamma receptors is an essential checkpoint in antibody-dependent cellular cytotoxicity (ADCC). Specific polymorphisms at position 158 enhance FcgammaRIIIa affinity for IgG1 and are associated with improved clinical outcome in lymphoma patients treated with IgG1 anti-CD20 antibody. The role of ADCC in the therapeutic effects of the alpha-epidermal growth factor receptor (EGFR) mAb, cetuximab, in patients with squamous cell carcinoma of the head and neck (SCCHN) is poorly defined. We employed three SCCHN cell lines to test two hypotheses: (1) SCCHN is susceptible to cetuximab-mediated ADCC, (2) efficacy of ADCC is associated with polymorphisms at position 158 of FcgammaRIIIa., Experimental Design: FcgammaRIIIa-158 polymorphisms were determined for healthy donors, and their purified NK cells were used as effector cells against three SCCHN cell lines in ADCC assays. Cytotoxicity levels were compared for each polymorphism class. Proliferation and cell cycle assays were done to examine the direct effects of cetuximab., Results: Our results indicate that SCCHN is susceptible to cetuximab-mediated ADCC in vitro. NK cytotoxic efficiency correlates with donor 158-polymorphisms in FcgammaRIIIa. Overall cytotoxicity was greatest for individuals having a single V allele when compared to homozygous F/F individuals; the cumulative percent cytotoxicity for each polymorphism among the cell lines was 58.2% V/V, 50.6% V/F, and 26.1% F/F (P < 0.001). Additionally, the presence of a V allele correlated with superior natural cytotoxicity against NK sensitive targets., Conclusion: These data have both prognostic and therapeutic relevance and support the design of a prospective trial to determine the influence of FcgammaRIIIa polymorphisms on the clinical outcome of patients with SCCHN treated with alpha-EGFR mAbs.

A promising new class of anti-cancer drugs includes antibodies that mediate immune regulatory effects. It has become very clear over the last decade that different types of immune cells and different pathways serve to suppress anti-cancer immunity, particularly in the microenvironment of the tumor. The first examples of immune modulating antibodies are those directed against cytotoxic T lymphocyte antigen-4 (CTLA-4), a molecule present on activated T cells. Human antibodies that abrogate the function of CTLA-4 have been tested in the clinic and found to have clinical activity against melanoma. In this review, we discuss some of the controversies surrounding the potential clinical utility of one of those antibodies, ipilimumab, formerly MDX-010, from Medarex and Bristol Myers Squibb. The optimal dose and schedule of ipilimumab was derived in multiple clinical trials whose latest results are described below. Favorable survival in patients with stage IV melanoma were observed that appear to be associated with unique side effects of the drug called "immune-related adverse events". The management of these side effects is described, and the unusual kinetics of anti-tumor response with ipilimumab as well as a newly proposed schema for assessing anti-tumor responses in patients receiving biologic compounds like ipilimumab, which may supercede RECIST or WHO criteria, are addressed.

The peripheral blood lymphocytes of eight patients with metastatic renal cell carcinoma, and of eight healthy volunteers were analyzed by four-color flow cytometry to characterize the immunophenotypic alterations manifested, determine the prevalence of lymphocyte apoptosis, and detect evidence of the systemic effect of inhaled IL-2. The T, B and NK lymphocytes of untreated patients were found to have undergone profound changes characterized by an increase in susceptibility to both spontaneous and mitogen-induced ex vivo apoptosis, a modified distribution of the main lymphocyte populations in the peripheral blood, and alterations in activation status. An increase in the proportion of regulatory T cells was also seen in these patients. Treatment with inhaled IL-2, however, normalized the rate of apoptosis in all the lymphocyte subpopulations studied, as well as their distribution and activation status. These findings demonstrate that inhaled IL-2 has systemic immunomodulatory effects.

Oxaliplatin is a third generation platinum compound used in patients with advanced colorectal carcinoma. Recently, the mechanism of a rare drug-induced immune thrombocytopenia in patients receiving oxaliplatin has been described. This complication is caused by oxaliplatin-dependent antibodies directed against platelet surface glycoproteins, and is unrelated to myelosuppression. In this report, we describe two patients who developed thrombocytopenia immediately soon after receiving oxaliplatin. Sensitization presumably had occurred after receiving oxaliplatin during preceding courses of multiagent chemotherapy that included oxaliplatin.

Observations show that humans and animals respond immunologically to most cancers. Why does the immune system then fail to control cancer? We argue from the literature that there is a commonality in the regulation of responses against most murine tumors, and that a major mechanism of escape may be deviation of an effective Th1, cytotoxic T lymphocyte response to a less effective response with a Th2 component. We examined this hypothesis with two well-studied murine tumors. We found, following primary tumor implantation, that resistance correlates with Th1 responses and IgG2a antibody production and progression with mixed Th1/Th2 responses and production of IgG1 and IgG2a antibodies. Resistance is associated with a modulation of the anti-tumor response towards the Th1 pole in both systems. We conclude that the immune responses against these two tumors are in accord with our hypothesis, and argue that this is likely to be true of many human and murine tumors. The correlation of IgG isotype of anti-tumor antibody with the Th1/Th2 nature of the anti-tumor response readily allows one to longitudinally monitor the changing nature of the anti-tumor response. We suggest that such monitoring can guide immunotherapy to maximize the effectiveness of the host's immune response against cancer.

Aim: Several lines of evidence suggest that NK cell immunotherapy may represent a successful approach in neuroblastoma (NB) patients refractory to conventional therapy. However, homing properties, safety and therapeutic efficacy of NK cell infusions need to be evaluated in a suitable preclinical murine NB model., Materials and Methods: Here, the therapeutic efficacy of NK cell infusions in the presence or absence of NK-activating cytokines have been evaluated in a NB metastatic model set up in NOD/scid mice, that display reduced functional activity of endogenous NK cells., Results: In NOD/scid mice the injected NB cells rapidly reached all the typical sites of metastatization, including bone marrow. Infusion of polyclonal IL2-activated NK cells was followed by dissemination of these cells into various tissues including those colonized by metastatic NB cells. The early repeated injection of IL2-activated NK cells in NB-bearing NOD/scid mice significantly increased the mean survival time, which was associated with a reduced bone marrow infiltration. The therapeutic effect was further enhanced by low doses of human recombinant IL2 or IL15., Conclusion: Our results indicate that NK-based adoptive immunotherapy can represent a valuable adjuvant in the treatment of properly selected NB patients presenting with metastatic disease, if performed in a minimal residual disease setting.

We have previously reported that the antibody fusion proteins anti-HER2/neu IgG3 fused to IL-12 [(IL-12)-IgG3] or GM-CSF [IgG3-(GM-CSF)] independently or in combination are effective anti-tumor agents against D2F2/E2 murine mammary cancer cells expressing human HER2/neu in the peritoneum. Importantly, the long-term survivors were immune to the subcutaneous challenge with D2F2/E2 and the parental D2F2 not expressing HER2/neu. We now show that these long-term survivors also exhibit significant protection against subsequent subcutaneous challenge with the murine colon carcinoma CT26-HER2/neu, and later against subcutaneous challenge with the parental CT26. These results suggest that the long-term systemic protection against mammary cancer elicited by treatment with antibody-cytokine fusion proteins can be extended to prevent the growth of a tumor from different origin expressing HER2/neu, and that this protection is not limited to this antigen alone, since it also prevented the growth of the parental tumor cells.

Although many multiple myeloma (MM) patients initially respond to cytotoxic therapy, most eventually relapse. Novel therapeutic strategies employing a combination of chemotherapy with targeted biologics may significantly enhance the response of tumor cells to treatment. We tested a fully human anti-IGF-IR antibody (A12) against MM, and showed specific inhibition of IGF-I or serum-induced IGF-IR signaling in MM cells in vitro. The A12 as a single agent was demonstrated to exert modest to significant inhibition of tumor growth in vivo in various subcutaneous xenograft MM models. The A12 was also evaluated in a disseminated xenograft MM.1S NOD/SCID model as monotherapy or in combination with other drugs (bortezomib, melphalan) currently in clinical use. The tumor burden, as determined by luciferase bioimaging, was sharply decreased, and overall survival significantly prolonged when the therapies were combined. Immunohistochemical analysis demonstrated that the A12 treated tumors had significantly decreased vascularization compared to control tumors. Furthermore, most MM lines constitutively secreted significant quantities of VEGF, and this was enhanced following IGF-I treatment. Inhibition of IGF-IR by the A12 in vitro suppressed both constitutive and IGF-I-induced secretion of VEGF, indicating that a putative anti-angiogenic mechanism associated with the A12 treatment may contribute to its anti-tumor effect.

Although melanoma tumors usually express antigens that can be recognized by T cells, immune-mediated tumor rejection is rare. In many cases this is despite the presence of high frequencies of circulating tumor antigen-specific T cells, suggesting that tumor resistance downstream from T cell priming represents a critical barrier. Analyzing T cells directly from the melanoma tumor microenvironment, as well as the nature of the microenvironment itself, is central for understanding the key downstream mechanisms of tumor escape. In the current report we have studied tumor-associated lymphocytes from a patient with metastatic melanoma and large volume malignant ascites. The ascites fluid showed abundant tumor cells that expressed common melanoma antigens and retained expression of class I MHC and antigen processing machinery. The ascites fluid contained the chemokines CCL10, CCL15, and CCL18 which was associated with a large influx of activated T cells, including CD8(+) T cells recognizing HLA-A2 tetramer complexes with peptides from Melan-A and NA17-A. However, several functional defects of these tumor antigen-specific T cells were seen, including poor production of IFN-gamma in response to peptide-pulsed APC or autologous tumor cells, and lack of expression of perforin. Although these defects were T cell intrinsic, we also observed abundant CD4(+)CD25(+)FoxP3(+) T cells, as well as transcripts for FoxP3, IL-10, PD-L1/B7-H1, and indoleamine-2,3-dioxygenase (IDO). Our observations suggest that, despite recruitment of large numbers of activated CD8(+) T cells into the tumor microenvironment, T cell hyporesponsiveness and additional negative regulatory mechanisms can limit the effector phase of the anti-tumor immune response.

Molecules belonging to the Tumor Necrosis Factor (TNF) and TNF receptor superfamilies have explosively expanded through the era of genomics and bioinformatics. Biological investigations of these molecules have explored their potency as attractive targets for cancer therapy. Anti-tumor mechanisms mediated by TNF superfamily molecules (TNFSF) could be classified into direct actions onto tumor cells and indirect effects through immune or non-immune components of tumor-bearing host. In this review, we focus on TRAIL, CD40, 4-1BB (CD137), and LIGHT as promising molecules to mediate powerful and selective anti-tumor responses, and summarize their unique effector mechanisms. In addition, optimal approaches to manipulate these molecules for cancer therapy are also discussed. We try to provide an insight into a role of TNFSF in cancer therapeutics and highlight each of their potency to be an important player in anti-cancer strategies.

Alpha tocopheryl succinate (alpha-TOS) is a non-toxic vitamin E analog under study for its anti-cancer properties. In an earlier study, we showed that alpha-TOS, when used in combination with non-matured dendritic cells (nmDC) to treat pre-established tumors, acts as an effective adjuvant. In this study, we have used vesiculated alpha-TOS (Valpha-TOS), a more soluble form of alpha-TOS that is relevant for clinical use, in combination with dendritic cells to treat pre-established murine tumors. We demonstrate that Valpha-TOS kills tumor cells in vitro and inhibits the growth of pre-established murine lung carcinoma (3LLD122) as effectively as alpha-TOS. The combination of Valpha-TOS plus non-matured or TNF-alpha-matured DC is more effective at inhibiting the growth of established tumors than Valpha-TOS alone. We also observed that Valpha-TOS induces expression of heat shock proteins in tumor cells and that co-incubation of non-matured DC with lysate derived from Valpha-TOS-treated tumor cells leads to DC maturation evidenced by up-regulation of co-stimulatory molecules and secretion of IL-12p70. This study therefore demonstrates the immunomodulatory properties of Valpha-TOS that may account for its adjuvant effect when combined with DC vaccines to treat established tumors.

Although the proinflammatory cytokine interferon-gamma (IFN-gamma) has been generally thought to enhance antitumor immune responses and be involved in antitumor mechanisms of many other immunotherapy molecules, it has also been reported that IFN-gamma could promote tumor immune evasion. In this report, by using an ideal mouse model that expresses IFN-gamma locally in muscle, we demonstrate that sustained low-level expression of IFN-gamma promotes the development of several types of tumor including H22 hepatoma, MA782/5S mammary adenocarcinoma and B16 melanoma. However, transitory expression of IFN-gamma does not have such an effect. On the other hand, sustained high-level expression of IFN-gamma mediates significant antitumor effect on H22 hepatoma. Low level of IFN-gamma upregulates expression of PD-L1, PD-L2, CTLA-4 and Foxp3, which may partly account for the tumor immune evasion promoted by IFN-gamma. Furthermore, blockade of PD-L inhibits IFN-gamma's tumor-promoting effect. Our findings provide a mechanistic link between chronic inflammation and cancer and would have potential implications for cancer prevention and also for the design of cytokine-based cancer immunotherapy.

Cytokine-based vaccines play a major part in tumor immuno-gene therapy. However, down-regulated antigen expression on tumor cells may diminish the immuno-potentiating aspects of cellular vaccines. In this study, we coexpressed a tumor antigen epitope with IFN-gamma in the same gene by replacing the IFN-gamma signal peptide with an antigen epitope-expressing signal peptide. We then investigated the effect of the antigen epitope-incorporated IFN-gamma on the immunotherapy of murine melanoma B16 tumors. Results showed that TRP-2 epitope-expressing IFN-gamma decreased B16 tumorigenicity and enhanced its immunogenicity after gene transfer. Protective immunity against wild type B16 tumors was induced by vaccination with IFN-gamma transiently gene-modified tumor cells. These data suggest that cellular vaccines engineered to express an antigen epitope within an immunostimulatory cytokine could potentiate the immunization effect.

Antibody-targeted chemotherapy with immunoconjugates of calicheamicin is a clinically validated strategy in cancer therapy. This study describes the selection of a murine anti-CD22 mAb, m5/44, as a targeting agent, its conjugation to a derivative of calicheamicin (CalichDM) via either acid-labile or acid-stable linkers, the antitumor activity of CalichDM conjugated to m5/44, and its subsequent humanization by CDR grafting. Murine IgG1 mAb m5/44 was selected based on its subnanomolar affinity for CD22 and ability to be internalized into B cells. CalichDM conjugated to m5/44 caused potent growth inhibition of CD22+ human B-cell lymphomas (BCLs) in vitro. The conjugate of m5/44 with an acid-labile linker was more potent than an acid-stable conjugate, a nonbinding conjugate with a similar acid-labile linker, or unconjugated CalichDMH in inhibiting BCL growth. CalichDM conjugated to m5/44 caused regression of established BCL xenografts in nude mice. In contrast, both unconjugated m5/44 and a nonbinding conjugate were ineffective against these xenografts. Based on the potent antitumor activity of m5/44-CalichDM conjugates, m5/44 was humanized by CDR grafting to create g5/44, an IgG4 anti-CD22 antibody. Both m5/44 and g5/44 bound CD22 with subnanomolar affinity. Competitive blocking with previously characterized murine anti-CD22 mAbs suggested that g5/44 recognizes epitope A located within the first N-terminal Ig-like domain of human CD22. Antitumor efficacy of CalichDM conjugated to g5/44 against BCL xenografts was more potent than its murine counterpart. Based on these results, a calicheamicin conjugate of g5/44, CMC-544, was selected for further development as a targeted chemotherapeutic agent for the treatment of B-cell malignancies.

Unlabelled: Hyaluronidase has been gaining increasing interest as a spreading factor for better penetration of chemotherapeutics into CNS tumours. Five out of 16 patients with CNS tumours treated with hyaluronidase in addition to chemotherapeutic agents developed symptoms of immediate type allergic reactions, therefore we sought to characterize the harmful allergenic proteins of the bovine testes hyaluronidase enzyme preparation (Neopermease). The role of specific IgE for the allergic reaction was investigated. Using an immunoblotting technique, we investigated sera from 16 children treated with Neopermease (5 of them having developed anaphylactic reactions), 5 patients with atopy (atopic eczema) with high total IgE levels and 4 healthy children. SDS-PAGE of hyaluronidase preparation Neopermease revealed two major bands at 73 and 41-43 kDa. In all 5 sera from patients with adverse reactions, binding of specific IgE antibodies to have 73 and 41-43 kDa bands was found. Two patients reacted with the 73 kDa band exclusively, two patients reacted with both bands, one patient displayed IgE only to the 41-43 kDa band. A specific inhibition of IgE-binding to both bands was achieved after preincubation of the sera in four out of five patients with partially purified bovine hyaluronidase. Furthermore preincubation with gelatin, a stabilising agent in the commercial extract, led to a partial inhibition in the sera of three patients. No specific IgE binding was detected either in the sera of atopic patients, or in the control group., Conclusion: IgE mediated allergic reactions to hyaluronidase may occur in paediatric oncological patients treated with hyaluronidase. Whether these children are sensitized by intravenous hyaluronidase treatment or by cross-reactivity of other preformed IgE antibodies, yet to be specified, remains to be elucidated.

Hydroperoxides of d-limonene were shown to be potent contact allergens when studied in guinea-pigs. Limonene-2-hydroperoxide (2-hydroperoxy-p-mentha-6,8-diene, a mixture of trans and cis isomers) was synthesized for the first time. The ratio between the trans and cis forms was 3:1. These two hydroperoxides were identified as the major hydroperoxides in autoxidized d-limonene. In photo-oxidized d-limonene, they constituted a minor part of the hydroperoxide fraction. Hydroperoxides may bind to proteins of the skin to make antigens either via a radical mechanism or after reactions to give epoxides. The cross-reactivity between the epoxide limonene-1,2-oxide, a potent contact allergen, and the hydroperoxides was therefore studied. No significant pattern of cross-reactivity was found. Further studies to identify and test the allergenicity of single hydroperoxides are needed to elucidate the mechanism of the allergenicity.

Ganglioside GD3, which is one of the major gangliosides expressed on the cell surface of human tumors of neuroectodermal origin has been focused on as a target molecule for passive immunotherapy. We have cloned the cDNA encoding the immunoglobulin light and heavy chains of an anti-GD3 monoclonal antibody KM641 (murine IgG3, kappa), and constructed the chimeric genes by linking the cDNA fragments of the murine light and heavy variable regions to cDNA fragments of the human kappa and gamma 1 constant regions, respectively. The transfer of these cDNA constructs into SP2/0 mouse myeloma cells resulted in the production of the chimeric antibody, designated KM871, that retained specific binding activity to GD3. Indirect immunofluorescence revealed the same staining pattern for chimeric KM871 and the mouse counterpart KM641 on GD3-expressing melanoma cells. When human serum and human peripheral blood mononuclear cells were used as effectors in complement-mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity respectively, the chimeric KM871 was more effective in killing GD3-expressing tumor cells than was the mouse counterpart KM641. Intravenous injection of chimeric KM871 markedly suppressed tumor growth in nude mice. The chimeric KM871, having enhanced antitumor activities and less immunogenicity than the mouse counterpart, would be a useful agent for passive immunotherapy of human cancer.

The effect of tumor cells and immunostimulants on the release of cytostatic factors (CF) from Lactobacillus casei YIT 9018 (LC)-, Corynebacterium parvum (CP)- or peptone-elicited peritoneal macrophages (PM) was investigated in vitro and in vivo. Significant release of CF into the culture medium from PM elicited with LC was induced by seven of eight mitomycin C-pretreated tumor cell lines and not by normal spleen cells, while no CF was released extracellularly from peptone-elicited PM given the same stimulus. CF were released from LC-elicited PM (LCEPM) after stimulation with LC, bacille Calmette-Guérin, streptococcal preparation OK-432, fucoidan or lipopolysaccharide, and LC but not CP induced CF production in the peritoneal cavities of LC- or CP-primed mice. The release of CF from LCEPM after stimulation with mitomycin C-pretreated 3T12-3 cells was inhibited by D-mannose and not by L-fucose. L-Rhamnose and mannose 6-phosphate, but not D-mannose or L-fucose, caused the release of CF from the PM. It was suggested that the release of CF from activated PM is caused by stimulation by some tumor cells, sugars, or bacterial immunostimulants, D-Mannose and L-rhamnose on the surface of tumor cells or bacteria, respectively, may plan an important role in the release of CF from activated macrophages.