Efficacy Of mAbs Research Articles

The Molecular Mechanism of Action of the CR6261-Azichromycin Combination Found through Computational Analysis

BackgroundCR6261 was found in 2008 and F10 was found in 2009. In 2010 Friesen et al experimentally showed that Oseltamivir/Zanamivir may improve the therapeutic efficacy of CR6261. As a result, the use of CR6261 combined with a drug to provide an antibody-based therapy against all influenza A viruses was proposed. Although CR8020 may neutralize group 2 influenza viruses and FI6 may neutralize both group 1 and group 2 influenza viruses as determined in 2011, the insight of Friesen et al is still interesting. Here, we address the following questions: how to uncover the molecular mechanism of a drug, which improves the therapeutic efficacy of mAbs and how to find drugs that enable CR6261 (CR8020, F10) to become a universal mAb.Methods and FindingsUsing the 3D structures of 3 gbn, 3 gbm, 3 ztn, 3 ztj, 3 fku and 3 sdy, we separate the 3D structures of CR6261, F10, CR8020 and FI6, and the 3D structures of trimer HAs of H3N2 and H5N1. Based on the experimental result of Friesen et al, we have found many clues, which reveal the molecular mechanism of action for a drug and an HA-mAb complex.ConclusionsOseltamivir/Zanamivir may congruously improve the therapeutic efficacies of CR6261, F10, CR8020 and FI6 by providing an additional affinity to compensate for the loss of affinity between HA and mAb resulting from mutations. However, Oseltamivir or Zanamivir are not expected to generally widen the spectrum of these mAbs. In order to enhance CR6261, CR8020, or for F10 to become universal, we may select Azichromycin, Oseltamivir, or the combination of Azichromycin and Oseltamivir, respectively.

Effect of stimulation of natural killer cells with an anti-CD137 mAb on the efficacy of trastuzumab, cetuximab, and rituximab.

2514 Background: Antibody-dependent cell-mediated cytotoxicity (ADCC), mediated by natural killer (NK) cells, plays an important role in the efficacy of monoclonal antibodies (mAb)s. CD137 is a costimulatory molecule expressed on immune cells following activation, including NK cells. We hypothesize that as the antitumor efficacy of mAbs is due to ADCC, their activity can be enhanced by stimulation of NK cells with an anti-CD137 agonistic mAb. Methods: Upregulation of CD137 on NK cells was assessed using CD20+lymphoma, HER2+breast, and EGFR+head and neck cell lines and primary patient samples. NK cell degranulation, cytokine release and cytotoxicity were assessed by CD107a mobilization, IFN-γ secretion, and chromium release. Mechanism of synergy was explored by cell depletion in an immune competent mouse model. Xenotransplanted models were used to demonstrate anti-tumor activity and sufficiency of an innate immune response. Results: NK cells in human primary patient samples do not express CD137 at baseline, however CD137 is highly upregulated when encountering mAb-coated tumor cells. MAb-induced NK cell degranulation and cytotoxicity are enhanced by anti-CD137 agonistic mAb. In a murine lymphoma model, anti-CD137 mAb significantly enhances anti-tumor activity of anti-CD20 mAb leading to complete tumor resolution and prolonged survival. NK cell depletion completely abrogates the therapeutic effect. In seven xenotransplant models, sequential administration of rituximab, trastuzumab or cetuximab plus anti-CD137 mAb provided superior reduction in tumor burden and prolonged overall survival. In a phase 0 biomarker study, level of CD137 expression on circulating and intratumoral NK cells was influenced by disease burden, prior treatment, FcγRIII polymorphism, and time since mAb therapy. Conclusions: Our results demonstrate the synergy of anti-CD137 mAb and a tumor-targeting mAb by stimulation of mAb-activated NK cells with anti-CD137 mAb to enhance ADCC. These results support a novel, sequential antibody approach against CD20+B cell, HER2+breast, and EGFR+head and neck malignancies by targeting first the tumor and then the host immune system.

Soluble CD80 restores T cell activation and overcomes tumor cell Programmed Death Ligand-1-mediated suppression (46.8)

Abstract One of the mechanisms tumor cells employ to escape anti-tumor immunity is expression of Programmed Death Ligand 1 (PD-L1or B7-H1). PD-L1 protects tumor cells by binding to its receptor Programmed Death 1 (PD-1) on activated T cells, thereby causing T cell apoptosis. We previously reported that transfection of tumor cells to express the costimulatory molecule CD80 prevents PD-1 binding and restores T cell activation, consistent with the concept that CD80 binds to PD-L1 and inhibits PD-L1/PD-1 interaction. Since it is not therapeutically feasible to transfect tumor cells with CD80, we have tested a soluble form of CD80, consisting of the extracellular domains of the molecule, for its ability to overcome PD-L1-mediated suppression. Treatment of human lung adenocarcinoma, mammary carcinoma, and cutaneous melanoma cells with soluble CD80 prevents PD-L1-mediated suppression and restores T cell activation. Because on-going clinical trials have shown that mAbs to PD-1 facilitate tumor rejection, we have compared the efficacy of soluble CD80 vs. mAbs to PD-1 or PD-L1. Soluble CD80 treatment is more effective in restoring T cell activation than treatment with mAbs to either PD-1or PD-L1. Therefore, soluble CD80 may be a therapeutic agent for facilitating tumor-specific immunity by overcoming PD-L1-induced immune suppression, and is potentially more effective than mAbs to either PD-1 or PD-L1.

Abstract LB-138: Stimulation of natural killer cells with an anti-CD137 antibody enhances the efficacy trastuzumab, cetuximab, and rituximab in HER2-expressing breast cancer, EGFR+ head and neck cancer, and CD20+ lymphoma

Abstract Antibody-dependent cell-mediated cytotoxicity (ADCC), largely mediated by natural killer (NK) cells, is thought to play an important role in the efficacy of monoclonal antibodies (mAb)s including rituximab, an anti-CD20 mAb, trastuzumab, an anti-HER2 mAb, and cetuximab, an anti-EGFR mAb used to treat patients with B cell lymphomas, HER2-expressing breast cancer, and EGFR-expressing solid tumors. CD137 is a costimulatory molecule expressed on NK cells following activation. We hypothesize that as the antitumor efficacy of mAbs is due in part to ADCC, the anti-cancer activity of these mAbs can be enhanced with an anti-CD137 agonistic mAb. Induction of CD137 on NK cells was assessed using CD20+lymphoma, HER2+breast, and EGFR+head and neck cell lines and primary patient samples and respective mAbs. In-vitro NK cell degranulation, cytokine release and cytotoxicity were assessed by CD107a mobilization, IFN-γ secretion, and chromium release. A murine lymphoma tumor model was used to assess in-vivo synergy and mechanism was explored by T cell, NK cell, and macrophage depletion. Xenotransplanted models in nude or SCID mice with lymphoma, breast cancer, or head and neck cancer were used to demonstrate efficacy of anti-CD137 mAb and rituximab, trastuzumab or cetuximab, and sufficiency of an innate immune response. NK cells in human primary patient samples do not express CD137 at baseline, however these cells highly upregulate CD137 when encountering mAb-coated tumor cells. Monoclonal antibody-induced NK cell degranulation and cytotoxicity as measured by chromium release are enhanced by anti-CD137 agonistic mAb. In a murine lymphoma model, anti-CD137 mAb significantly enhances anti-tumor activity of anti-CD20 mAb leading to complete tumor resolution and prolonged survival. Sequential administration of anti-CD20 mAb followed by anti-CD137 mAb is required for the synergistic effect. NK cell depletion completely abrogates the therapeutic effect of anti-CD20 plus anti-CD137 mAb combination. In xenotransplant lymphoma, breast, and head and neck models, rituximab, trastuzumab or cetuximab plus anti-CD137 mAb provided superior reduction in tumor burden and prolonged overall survival. In a Phase 0 biomarker study, level of CD137 expression on circulating and intratumoral NK cells was influenced by circulating disease burden, extent of prior treatment, FcγRIII polymorphism, and time since mAb therapy. Our results demonstrate the synergy of anti-CD137 mAb and a tumor-targeting mAb (rituximab, trastuzumab, or cetuximab) by stimulation of mAb-activated NK cells with anti-CD137 mAb to enhance ADCC. These results support a novel, sequential antibody approach against CD20+B cell, HER2+breast, and EGFR+head and neck malignancies by targeting first the tumor and then the host immune system. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-138. doi:1538-7445.AM2012-LB-138

Intracellular Tumor-Associated Antigens Represent Effective Targets for Passive Immunotherapy

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

Antiepidermal growth factor receptor monoclonal antibody improves survival outcomes in the treatment of patients with metastatic colorectal cancer

The aim of this study was to determine whether or not the addition of anti-epidermal growth factor receptor (anti-EGFR) monoclonal antibody (mAb) to standard chemotherapy or best supportive care (BSC), compared with chemotherapy or BSC alone, can improve overall survival (OS) and progression-free survival (PFS) in patients with metastatic colorectal cancer (mCRC), and evaluate the influence of KRAS mutant status on the efficacy of anti-EGFR mAb. Medline, Embase, the Cochrane controlled trials register, and the Science Citation Index were searched. Nine trials were identified, covering a total of 7941 patients. The treatment of mCRC with a combination of anti-EGFR mAb and chemotherapy or BSC, as compared with chemotherapy or BSC alone, improved the OS [hazard ratio (HR), 0.90 (0.84-0.96); P=0.002]. The benefit of anti-EGFR mAb in patients with KRAS wild-type tumors was apparent in relation to a marginal trend toward improved OS [HR, 0.84 (0.70-1.01); P=0.06], and significantly improved PFS [HR, 0.64 (0.51-0.81); P<0.001]. No benefit for the addition of anti-EGFR mAb was detected for any efficacy end-point in patients with KRAS mutant tumors. The summary HRs (anti-EGFR mAb vs control) were 0.98 (0.88-1.08) (P=0.71) for OS and 1.08 (0.94-1.25) (P=0.27) for PFS, respectively. In conclusion, this analysis provides confirmation that, compared with chemotherapy or BSC alone, anti-EGFR mAb with chemotherapy or BSC reduces the risk of progression and death of mCRC and that this benefit is seen only in patients with wild-type KRAS tumors.

Quantification of intracellular nucleotide sugars and formulation of a mathematical model for prediction of their metabolism

Quantification of intracellular nucleotide sugars and formulation of a mathematical model for prediction of their metabolism

Prenyl Transferases Are Involved in the Regulation of CD20 Levels and Influence Anti-CD20 Monoclonal Antibody-Mediated Activation of Complement-Dependent Cytotoxicity,

Abstract 3722Anti-CD20 monoclonal antibodies (mAbs) (rituximab or ofatumumab) are being successfully used in the treatment of non-Hodgkin's lymphomas (NHL) and B-cell chronic lymphocytic leukemia (B-CLL). They exert antitumor effects by triggering indirect effector mechanisms of the immune system, such as activation of complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), or immunophagocytosis. Moreover, upon crosslinking with secondary antibodies, anti-CD20 mAbs can induce cell death. It is frequently underscored that CD20 expression levels in various B-cell tumors is relatively constant. However, accumulating evidence indicates that CD20 can be modulated at transcriptional, posttranscriptional, and even posttranslational levels. Moreover, it has been clearly shown in vitro that CDC efficacy of anti-CD20 mAbs clearly depends on CD20 expression. We have previously observed that statins impair detection of CD20 in non-Hodgkin lymphoma cells and impair rituximab-mediated CDC and ADCC (Winiarska et al. PLoS Med 2008). Statins are inhibitors of cholesterol synthesis and decrease production of prenyl groups (farnesyl and geranylgeranyl PPi), which are necessary for posttranslational modification of approximately 1% of cellular proteins. In experiments aimed at elucidation of the molecular mechanisms of statin-mediated modulation of CD20 we observed that neither geranylgeranyl transferase (GGT) nor farnesyl transferase (FNT) inhibitors could mimic statins effects. On the contrary, prenyltransferase inhibitors improved rituximab-mediated CDC. Therefore, we decided to investigate in more detail the interaction of prenyltransferase inhibitors and anti-CD20 mAbs. In the initial experiments we evaluated the effects of three different farnesyl transferase inhibitors as well as three different geranylgeranyl transferase inhibitors. Among all FNT and GGT inhibitors, L-744,832 seemed to produce the most significant influence on both rituximab-mediated CDC and CD20 levels (Figure). Moreover, L-744,832 significantly increased rituximab-mediated CDC in 3 out of 5 primary tumor cell cultures isolated from patients with NHL or CLL. Therefore, L-744,832 was selected for further more systematic studies. Interestingly, in Raji cells L-744,832 did not improve rituximab-mediated ADCC and only at the highest non-toxic concentrations it sensitized to rituximab+crosslinking antibody-mediated cytotoxicity. In 10 out of 17 (58.8%) primary lymphoma/leukemia cells L-744,832 increased CD20 expression by at least 20% as measured with flow cytometry. Moreover, we observed that upon L-744,832 treatment CD20 is up-regulated in Raji cells at both mRNA as well as protein level. Experiments aimed at investigation of FTI influence on proteasome activity as well as CD20 endocytosis and shedding revealed that L-744,832 influences CD20 levels independently from its posttranslational regulation. To verify whether modulation of CD20 levels by L-744,832 results from specific inhibition of farnesyltransferase or is an off-target effect of this compound we performed FNT B subunit (FNTB) knock-down experiments that resulted in increased CD20 levels by almost 60%. Incubation of Raji cells with a transcription inhibitor cycloheximide completely prevented L-744,832-mediated increase of CD20 levels in WB. Therefore, a chromatin immunoprecipitation assay was performed to determine whether inhibition of FNT activity is associated with binding of transcription factors to the promoter of cd20 gene. These studies revealed that L-744,832 promotes binding of PU.1 and Oct2, but not TFE3 to target DNA sequences within cd20 promoter in Raji cells. To conclude, our studies indicate for the first time that CD20 expression can be modulated by prenyltransferase inhibitors. While inhibition of FNT activity significantly up-regulates expression of CD20, the influence of GGT inhibitors on this protein is more complex, and requires further studies. Furthermore, pre-incubation of NHL and CLL cells with L-744,832, a FNT inhibitor, potentiates anti-CD20 mAb-mediated activation of the complement-mediated cytotoxicity. Therefore, the combination seems to be promising and its efficacy should be determined in patients with NHL or CLL. [Display omitted] Disclosures:Winiarska:Genmab A/S: Research Funding. Golab:Genmab A/S: Research Funding.

Monoclonal antibodies for copper-64 PET dosimetry and radioimmunotherapy

Background: We previously described a two-antibody model of 64Cu radioimmunotherapy to evaluate low-dose, solid-tumor response. This model was designed to test the hypothesis that cellular internalization is critical in causing tumor cell death by mechanisms in addition to radiation damage. The purpose of the present study was to estimate radiation dosimetry for both antibodies (mAbs) using positron emission tomography (PET) imaging, and evaluate the effect of internalization on tumor growth. Results: Dosimetry was similar between therapy groups. Median time to tumor progression to 1 g ranged from 7 to 12 days for control groups and was 32 days for both treatment groups (p < 0.0001). No statistically significant difference existed between any control group or between the treatment groups. Material and methods: In female nude mice bearing LS174T colon carcinoma xenografts, tumor dosimetry was calculated using serial PET images of three mice in each group of either internalizing 64Cu-labeled DOTA-cBR96 (DOTA = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) or non-internalizing 64Cu-labeled DOTA-cT84.66 from 3 to 48 h. For the therapy study, controls (n = 10) received saline, DOTA-cBR96, or DOTA-cT84.66. Treatment animals (n = 9) received 0.890 mCi of 64Cu-labeled DOTA-cBR96 or 0.710 mCi of 64Cu-labeled DOTA-cT84.66. Tumors were measured daily. Conclusions: PET imaging allows the use of 64Cu for pre-therapy calculation of tumor dosimetry. In spite of highly similar tumor dosimetry, an internalizing antibody did not improve the outcome of 64Cu radioimmunotherapy. Radioresistance of this tumor cell line and copper efflux may have confounded the study. Further investigations of the 2 therapeutic efficacy of 64Cu-labeled mAbs will focus on interaction between 64Cu and tumor suppressor genes and copper chaperones.

Abstract 3665: Enzymatic depletion of pericellular HA from tumor cell surface sensitizes Erbitux dependent immune cell-mediated cytotoxicity

Abstract Hyaluronan (HA), a ubiquitous high-molar-mass linear glycosaminoglycan (GAG) plays a critical role in cell adhesion, motility, growth and differentiation. HA accumulation is associated with many human cancers. Human tumor cells can form HA rich pericellular matrix which might favor tumor growth by preventing the access of host immune cells to malignant cells. Monoclonal antibody (MAb) based therapeutics are emerging as an important component of therapy for human malignancies. Despite the very high specificity of monoclonal antibodies, resistance to MAb therapy is common. Antibody Dependent Cell-mediated Cytotoxicity (ADCC) is an important effector for the clinical efficacy of MAbs and relies on effective interaction between the Fc domain of antigen bound antibody and FcγRIII positive immune cells. We hypothesize that pericellular HA coating may contribute to resistance of HA-rich malignant cells to MAb mediated therapy by preventing efficient access of effector cells to their tumor targets. Enzymatic depletion of HA with PEGPH20 hyaluronidase could potentially sensitize such tumors to MAb mediated therapy. remove ell surface and ated therapy by preventing tumor cells contact EGFR positive breast carcinoma cell line MDA-MB-231luc and genetically engineered HAS2 over expressing MDA-MB-231HAS2 were used to investigate the role of HA-rich pericellular matrix in inhibition of ADCC. ADCC assay was performed by incubating Calcein AM labeled monolayer cells with monoclonal antibody (1h at 37°C) followed by co-incubation of recombinant human IL-2 (rhIL-2) activated human peripheral blood mononuclear cells (PBMC, 5h at 37°C, 5% CO2) with or without PEGPH20. Live cells from each treatment were assessed by fluorescence readings at Ex/Em 488nm/537nm (SpectraMax M2e plate reader) at the end of the assay. Percentages of cell killing were calculated against control. The student's t test was used to compare the difference between treatments. Particle exclusion assay demonstrated that HAS2 over expressing MDA-MB-231HAS2 cells formed a pericellular coat that inhibited access of the rhIL-2 activated PBMC to tumor cells. Incubation with PEGPH20 removed the HA-rich pericellular matrix and promoted contact of PBMC to tumor cells. Incubation of Erbitux with rhIL-2 activated PBMC induced a dose dependent killing of both MDA-MB-231luc and MDA-MB-231HAS2 cells. PEGPH20 increased ADCC by 10% on MDA-MB-231luc (HAlow) and 25% on MDA-MB-231HAS2 (HAhigh) tumor cells. Our results show that the HA-rich pericellular matrix on adherent tumor cells inhibits ADCC and PEGPH20 depletion of this coat sensitizes the tumor cells to ADCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3665. doi:10.1158/1538-7445.AM2011-3665

Time-resolved Fluorescence Resonance Energy Transfer (TR-FRET) to Analyze the Disruption of EGFR/HER2 Dimers: A NEW METHOD TO EVALUATE THE EFFICIENCY OF TARGETED THERAPY USING MONOCLONAL ANTIBODIES

In oncology, simultaneous inhibition of epidermal growth factor receptor (EGFR) and HER2 by monoclonal antibodies (mAbs) is an efficient therapeutic strategy but the underlying mechanisms are not fully understood. Here, we describe a time-resolved fluorescence resonance energy transfer (TR-FRET) method to quantify EGFR/HER2 heterodimers on cell surface to shed some light on the mechanism of such therapies. First, we tested this antibody-based TR-FRET assay in NIH/3T3 cell lines that express EGFR and/or HER2 and in various tumor cell lines. Then, we used the antibody-based TR-FRET assay to evaluate in vitro the effect of different targeted therapies on EGFR/HER2 heterodimers in the ovarian carcinoma cell line SKOV-3. A simultaneous incubation with Cetuximab (anti-EGFR) and Trastuzumab (anti-HER2) disturbed EGFR/HER2 heterodimers resulting in a 72% reduction. Cetuximab, Trastuzumab or Pertuzumab (anti-HER2) alone induced a 48, 44, or 24% reduction, respectively. In contrast, the tyrosine kinase inhibitors Erlotinib and Lapatinib had very little effect on EGFR/HER2 dimers concentration. In vivo, the combination of Cetuximab and Trastuzumab showed a better therapeutic effect (median survival and percentage of tumor-free mice) than the single mAbs. These results suggest a correlation between the extent of the mAb-induced EGFR/HER2 heterodimer reduction and the efficacy of such mAbs in targeted therapies. In conclusion, quantifying EGFR/HER2 heterodimers using our antibody-based TR-FRET assay may represent a useful method to predict the efficacy and explain the mechanisms of action of therapeutic mAbs, in addition to other commonly used techniques that focus on antibody-dependent cellular cytotoxicity, phosphorylation, and cell proliferation.

Cetuximab plus FOLFOX-4 in Untreated Patients with Advanced Colorectal Cancer: A Gruppo Oncologico dell’Italia Meridionale Multicenter Phase II Study

Objectives: FOLFOX-4 and FOLFIRI are considered equivalent in terms of activity and efficacy as first-line chemotherapy in metastatic colorectal cancer (mCRC). The monoclonal antibody (mAb) cetuximab showed intrinsic activity as a single agent in mCRC and was approved in combination with CPT-11 for patients who failed previous CPT-11-based treatment. The purpose of this phase II study was to evaluate the activity and safety of FOLFOX-4 plus cetuximab in untreated mCRC patients. Methods: Untreated patients with measurable metastatic disease and expressing epidermal growth factor receptor (EGFR) received cetuximab at a loading dose of 400 mg/m², followed by weekly doses of 250 mg/m², in combination with the FOLFOX-4 regimen every 2 weeks for a maximum of 12 cycles, after which a maintenance program using cetuximab alone was allowed for a maximum of 6 months. Results: Eighty-two unselected patients were screened; 70 were EGFR+ and entered the trial. Of the 67 assessable patients, the objective response rate was 64.2% (95% CI: 52.5–75.5%) and the tumor growth control rate was 94% (95% CI: 88–99%). All the objective responses except 1 were confirmed. In the group of patients with initially unresectable liver disease alone, 7/33 (21%) were resected. The median time to progression (TTP) and overall survival (OS) were 10.0 and 22.0 months, respectively. The treatment was well tolerated, with no treatment-related deaths, while 24.2% of the patients were affected by cutaneous toxicity of grade >2. Mutational analysis of the KRAS and BRAF genes was retrospectively performed on 35 of the 69 patients treated with cetuximab (51%). KRAS was mutated in 13 out of the 35 cases (37%), whereas no mutations were detected in the BRAF gene. A trend toward an association between KRAS mutations and objective response to treatment (p = 0.07) was demonstrated. Analysis of survival showed that patients harboring KRAS mutations had a trend toward worst TTP (p = 0.14) confirmed by age- and sex-adjusted Cox multivariate regression (hazard ratio, HR = 0.62; 95% CI: 0.36–1.06; p = 0.08). Indeed, KRAS mutations were significantly associated with worst OS in both unadjusted analysis (p = 0.047; log rank test) and age- and sex-adjusted Cox multivariate regression (HR = 0.458; 95% CI: 0.248–0.847; p = 0.01). Conclusions: These results suggest that the combination of FOLFOX-4 plus cetuximab is very active and obtains long TTP with an acceptable toxicity profile. Indeed, our results are in line with recent findings from phase II and phase III randomized studies providing strong evidence that the efficacy of anti-EGFR mAb is confined to patients with wild-type KRAS mCRC. Investigation of other predictive biomarkers may be useful to further define the responder population.

The future of anti-CD20 monoclonal antibodies: are we making progress?

AbstractThe anti-CD20 monoclonal antibody (mAb) rituximab has revolutionized the treatment of B-cell malignancies. This unprecedented success has not only substantially changed the mindset of the clinical community about the ability of mAb to improve outcomes but has catalyzed the interest in the pharmaceutical industry to develop the next generation of anti-CD20 mAbs. Since the introduction of rituximab 15 years ago, we have learned much about the potential mechanisms underlying the therapeutic efficacy of anti-CD20 mAbs. In parallel, many novel anti-CD20 mAbs have entered the clinic, each designed with modifications to structure aimed at further improving efficacy. On review of the newer generation of anti-CD20 mAbs entering clinical trials, it appears that the link between the novel mechanistic insights and the development of these next-generation anti-CD20 mAbs is unclear. As we move into an era of personalized medicine, it will become increasingly important for us to develop closer links between the emerging mechanistic insights and the clinical development, to further enhance the potency of anti-CD20 mAbs beyond that achieved with rituximab.

Anti-CD20 antibody-mediated apoptosis of B cells is a lipid raft-dependent process

BackgroundB cell depletion has become a classics in approved conditions and off-label indications. Anti-CD20 have been classified into type-I, such as rituximab (RTX), and type-II monoclonal antibodies (mAbs), such as...

Passive Administration of Monoclonal Antibodies Against &lt;em&gt;H. capsulatum&lt;/em&gt; and Others Fungal Pathogens

The purpose of the use of this methodology is 1) to advance our capacity to protect individuals with antibody or vaccine for preventing or treating histoplasmosis caused by the fungus Histoplasma capsulatum and 2) to examine the role of virulence factors as target for therapy. To generate mAbs, mice are immunized, the immune responses are assessed using a solid phase ELISA system developed in our laboratory, and the best responder mice are selected for isolation of splenocytes for fusion with hybridoma cells. C57BL/6 mice have been extensively used to study H. capsulatum pathogenesis and provide the best model for obtaining the data required. In order to assess the role of the mAbs in infection, mice are intraperitoneally administered with either mAb to H. capsulatum or isotype matched control mAb and then infected by either intravenous (i.v.), intraperitoneal (i.p.), or intranasal (i.n.) routes. In the scientific literature, efficacy of mAbs for fungal infections in mice relies on mortality as an end point, in conjunction with colony forming units (CFU) assessments at earlier time points. Survival (time to death) studies are necessary as they best represent human disease. Thus, efficacy of our intervention would not adequately be established without survival curves. This is also true for establishing efficacy of vaccine or testing of mutants for virulence. With histoplasmosis, the mice often go from being energetic to dead over several hours. The capacity of an intervention such as the administration of a mAb may initially protect an animal from disease, but the disease can relapse which would not be realized in short CFU experiments. In addition to survival and fungal burden assays, we examine the inflammatory responses to infection (histology, cellular recruitment, cytokine responses). For survival/time to death experiments, the mice are infected and monitored at least twice daily for signs of morbidity. To assess fungal burden, histopathology, and cytokine responses, the mice are euthanized at various times after infection. Animal experiments are performed according to the guidelines of the Institute for Animal Studies of the Albert Einstein College of Medicine.

Development of monoclonal antibodies to highly pathogenic avian influenza H5N1 virus and their application to diagnostics, prophylaxis, and therapy

A panel of 17 monoclonal antibodies (MAbs) against highly pathogenic avian influenza virus (HPAIV) A/Duck/Novosibirsk/56/05 A/H5N1 (subclade 2.2) isolated in Russian Federation was developed. Immunoblot analysis showed that 12 MAbs were specific for the hemagglutinin (HA) and 5 MAbs for nucleoprotein (NP). All anti-HA MAbs were reactive in ELISA and immunofluorescence (IF) test and 10 of them were reactive in hemagglutination-inhibition (HI) and neutralization tests. Quantitative competitive ELISA revealed that anti-HA MAbs recognized at least 4 non-overlapping antigenic determinants and anti-NP MAbs recognized at least 3 non-overlapping antigenic determinants. Four sandwich ELISA procedures were developed using the obtained MAbs. These procedures are useful for 1) identification of avian, human, and swine influenza A viruses, 2) differentiation of avian influenza virus (AIV) from human and swine influenza viruses, 3) differentiation of AIV H5 from other AIV subtypes, and 4) differentiation between 2.2 and 2.3.2 subclades of H5N1 influenza viruses. Prophylactic and therapeutic efficacy of anti-HA MAbs with high neutralization activity was tested in BALB/c mice. A complete protection was achieved by single injection of MAbs (20 mg/kg) 24 hrs before challenge with 10 LD50 of HPAIV H5N1. Therapeutic efficacy was 90% that was similar to those of Rimantadine and Tamiflu.

Immunomodulation of NK Cells through 4-1BB (CD137) to Improve the Anti-Lymphoma Activity of Rituximab: Antibody-Based Anti-Lymphoma Synergy

AbstractAbstract 422 Background:Antibody-dependent cell-mediated cytotoxicity (ADCC), largely mediated by natural killer (NK) cells, is thought to play an important role in the efficacy of rituximab, an anti-CD20 monoclonal antibody (mAb) used to treat patients with B cell lymphomas. CD137 is a costimulatory molecule expressed on a variety of immune cells following activation, including NK cells. We hypothesized that the anti-lymphoma activity of rituximab could be enhanced by stimulation of NK cells with an anti-CD137 agonistic mAb. Methods:Rituximab induced upregulation of CD137 on NK cells was assessed using lymphoma cell lines and primary lymphoma patient samples. In-vitro NK cell degranulation and cytotoxicity were assessed by CD107a mobilization and chromium release. A murine lymphoma tumor model targeted by mouse anti-mouse CD20 mAb was used to assess in-vivo synergy of anti-CD20 and anti-CD137 mAbs. Mechanism of synergy was explored by T cell, NK cell, and macrophage depletion in the immune competent mouse model. A xenotransplant model in SCID mice with disseminated, luciferase-labeled lymphoma was used to demonstrate efficacy of anti-CD137 mAb and rituximab, and sufficiency of an innate immune response. Results:NK cells in human primary lymphoma samples do not express CD137 at baseline, however these cells highly upregulate CD137 when encountering rituximab-coated tumor B cells. Rituximab-induced NK cell degranulation and cytotoxicity as measured by CD107a mobilization (p=.006) and chromium release (p=.01) are enhanced by anti-CD137 agonistic mAb. In a murine lymphoma model, anti-CD137 mAb significantly enhances anti-tumor activity of anti-CD20 mAb leading to complete tumor resolution (p<.001) and prolonged survival (p=.048). Sequential administration of anti-CD20 mAb followed by anti-CD137 mAb (p=.027) is required for the synergistic effect. In-vivo administration of anti-CD20 mAb induces upregulation of CD137 on mouse NK cells (p=0.001), allowing subsequent targeting with anti-CD137 mAb. NK cell depletion completely abrogates the therapeutic effect of anti-CD20 plus anti-CD137 mAb combination (p<.001). In a xenotransplant disseminated lymphoma model (Figure 1A), rituximab plus anti-CD137 mAb provided superior reduction in tumor burden, as quantified by bioluminescence (p=.001; Figure 1B), and prolonged overall survival (p=.013; Figure 1C). Conclusions:Our results demonstrate the synergy of anti-CD137 mAb and rituximab by stimulation of rituximab-activated NK cells with anti-CD137 mAb to enhance ADCC. These results support a novel, sequential antibody approach against B cell malignancies by targeting first the tumor and then the host immune system. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Characterization of Gene Use and Efficacy of Mouse Monoclonal Antibodies toStreptococcus pneumoniaeSerotype 8

Streptococcus pneumoniae is the most common cause of community-acquired pneumonia in the United States and globally. Despite the availability of pneumococcal capsular polysaccharide (PPS) and protein conjugate-based vaccines, the prevalence of antibiotic-resistant pneumococcal strains, serotype (ST) replacement in nonconjugate vaccine strains, and uncertainty as to whether the PPS vaccine that is used in adults protects against pneumonia emphasize the need for continued efforts to understand the nature of protective PPS antibody responses. In this study, we generated mouse monoclonal antibodies (MAbs) to a conjugate consisting of the PPS of serotype 8 (PPS8) S. pneumoniae and tetanus toxoid. Thirteen MAbs, including four IgMs that bound to PPS8 and phosphorylcholine (PC) and five IgMs and four IgG1s that bound to PPS8 but not PC, were produced, and their nucleotide sequences, epitope and fine specificity, and efficacy against lethal challenge with ST8 S. pneumoniae were determined. MAbs that bound to PPS8 exhibited gene use that was distinct from that exhibited by MAbs that bound to PC. Only PPS8-binding MAbs that did not bind PC were protective in mice. All 13 MAbs used germ line variable-region heavy (V(H)) and light (V(L)) chain genes, with no evidence of somatic hypermutation. Our data reveal a relationship between PPS specificity and V(H) gene use and MAb efficacy in mice. These findings provide insight into the relationship between antibody molecular structure and function and hold promise for the development of novel surrogates for pneumococcal vaccine efficacy.

Optimizing Tumor-Reactive &amp;#947;&amp;#948;T Cells for Antibody-Based Cancer Immunotherapy

Monoclonal antibodies (mAbs) constitute the most rapidly growing class of human therapeutics and the second largest class of drugs after vaccines. The treatment of B-cell malignancies and HER2/Neu(+) breast cancer has benefited considerably from the use of therapeutic mAbs, either alone or in combination with standard chemotherapy. Frequent relapses, however, demonstrate that the bioactivity of these mAbs is still suboptimal. The concept of improving the anti-tumor activity of mAbs is well established and potentiating the cytotoxicity induced by anticancer mAbs can be achieved by strategies that target the downstream cytolytic effector cells. The recruitment of Fcγ receptor-dependent functions appears well suited in this regard, because several lines of evidence suggest that enhancing antibody-dependent cellular cytotoxicity (ADCC) induced by therapeutic mAbs may directly improve their clinical efficacy. The cytolytic effector cells involved in ADCC are FcγR-expressing natural killer (NK) cells, but also γδ T cells can be amplified and finetuned for stronger ADCC activity. γδ T cells are raising a considerable interest in the immunotherapy community given their intrinsic antitumor activity that can be boosted by stimulation with synthetic phosphoantigens (PAgs), or with drugs that cause their accumulation into target cells, like aminobisphosphonates (N-BPs), and low doses interleukin (IL)-2. The field is interesting, and several papers have already explored this approach in solid and haematological malignancies. Thus, we propose that enhancing the efficacy of mAbs by combination with γδ T cell activation may have considerable therapeutic potential for a variety of malignancies, most especially for patients whose FcγR alleles impair ADCC.

Immuno-Positron Emission Tomography: Shedding Light on Clinical Antibody Therapy

Summation Monoclonal antibodies (mAbs) have been approved for therapeutic use in a broad range of medical indications, especially in oncology, and are forming the most rapidly expanding category of pharmaceuticals. Although engineered mAb fragments and nontraditional antibody-like scaffolds are receiving increasingly more attention, most of the mAb candidates evaluated in past and ongoing clinical trials are full-length mAbs. Immuno-positron emission tomography (PET), the tracking and quantification of mAbs with PET in vivo at superior imaging quality, is an exciting novel option for better understanding the in vivo behavior and efficacy of mAbs in individual patients. This review focuses on immuno-PET with full-length mAbs, and the associated use of the long-lived positron emitters zirconium-89 ((89)Zr) and iodine-124 ((124)I). Very recently, crucial achievements have been obtained to allow broad-scale application of (89)Zr- and (124)I-immuno-PET in clinical mAb development and applications. (89)Zr and (124)I became commercially available worldwide for clinical use. A chelate for facile coupling of (89)Zr to mAbs became commercially available, and generic procedures for labeling of mAbs with (89)Zr and (124)I in a current good manufacturing practice compliant way were established. In this review, critical aspects for the translation of immuno-PET from preclinical investigations to clinical trials will be discussed, as well as the potential clinical applications of immuno-PET. An overview of the results of the first clinical immuno-PET studies will be provided.