Chemical Synthesis and Antigenicity Evaluation of an Aminoglycoside Trisaccharide Repeating Unit of Pseudomonas aeruginosa Serotype O5 O-Antigen Containing a Rare Dimeric-ManpN3NA.

Pseudomonas aeruginosa bacteria are becoming increasingly resistant against multiple antibiotics. Therefore, the development of vaccines to prevent infections with these bacteria is an urgent medical need. While the immunological activity of lipopolysaccharide O-antigens in P. aeruginosa is well-known, the specific protective epitopes remain unidentified. Herein, we present the first chemical synthesis of highly functionalized aminoglycoside trisaccharide 1 and its acetamido derivative 2 found in the P. aeruginosa serotype O5 O-antigen. The synthesis of the trisaccharide targets is based on balancing the reactivity of disaccharide acceptors and monosaccharide donors. Glycosylations were analyzed by quantifying the reactivity of the hydroxyl group of the disaccharide acceptor using the orbital-weighted Fukui function and dual descriptor. The stereoselective formation of 1,2-cis-α-fucosylamine linkages was achieved through a combination of remote acyl participation and reagent modulation. The simultaneous SN2 substitution of azide groups at C2' and C2″ enabled the efficient synthesis of 1,2-cis-ß-linkages for both 2,3-diamino-D-mannuronic acids. Through a strategic orthogonal modification, the five amino groups on target trisaccharide 1 were equipped with a rare acetamidino (Am) and four acetyl (Ac) groups. Glycan microarray analyses of sera from patients infected with P. aeruginosa indicated that trisaccharides 1 and 2 are key antigenic epitopes of the serotype O5 O-antigen. The acetamidino group is not an essential determinant of antibody binding. The ß-D-ManpNAc3NAcA residue is a key motif for the antigenicity of serotype O5 O-antigen. These findings serve as a foundation for the development of glycoconjugate vaccines targeting P. aeruginosa serotype O5.

Cutting Edge: The Use of Topical Aminoglycosides as an Effective Pull in "Prime and Pull" Vaccine Strategy.

The presence of tissue-resident memory T cells at barrier tissues is critical for long-lasting protective immune responses. Previous work has shown that tissue-resident memory T cells can be established by "pulling" virus-specific effector T cells from circulation to the genital mucosa via topical vaginal application of chemokines in mice. Once established, these cells protect hosts against genital herpes infection. We recently showed that vaginal application of aminoglycoside antibiotics induces robust activation of the IFN signaling pathway, including upregulation of chemokine expression within the tissue in mice. In this study, we show that a single topical application of neomycin, an inexpensive and vaginally nontoxic antibiotic, is sufficient to pull CD8 T cells to the vaginal mucosa and provide protection against genital herpes infection in mice.

Characterization of CD33/CD3 Tetravalent Bispecific Tandem Diabodies (TandAbs) for the Treatment of Acute Myeloid Leukemia.

PURPOSE: Randomized studies with gemtuzumab ozogamicin have validated CD33 as a target for antigen-specific immunotherapy of acute myelogenous leukemia (AML). Here, we investigated the potential of CD33/CD3-directed tandem diabodies (TandAbs) as novel treatment approach for AML. These tetravalent bispecific antibodies provide two binding sites for each antigen to maintain the avidity of a bivalent antibody and have a molecular weight exceeding the renal clearance threshold, thus offering a longer half-life compared to smaller antibody constructs. EXPERIMENTAL DESIGN: We constructed a series of TandAbs composed of anti-CD33 and anti-CD3 variable domains of diverse binding affinities and profiled their functional properties in CD33+ human leukemia cell lines, xenograft models, and AML patient samples. RESULTS: Our studies demonstrated that several CD33/CD3 TandAbs could induce potent, dose-dependent cytolysis of CD33+ AML cell lines. This effect was modulated by the effector-to-target cell ratio and strictly required the presence of T cells. Activation and proliferation of T cells and maximal AML cell cytolysis correlated with high avidity to both CD33 and CD3. High-avidity TandAbs were broadly active in primary specimens from patients with newly diagnosed or relapsed/refractory AML in vitro, with cytotoxic properties independent of CD33 receptor density and cytogenetic risk. Tumor growth delay and inhibition were observed in both prophylactic and established HL-60 xenograft models in immunodeficient mice. CONCLUSIONS: Our data show high efficacy of CD33/CD3 TandAbs in various preclinical models of human AML. Together, these findings support further study of CD33/CD3 TandAbs as novel immunotherapeutics for patients with AML. Clin Cancer Res; 22(23); 5829-38. ©2016 AACR.

Antibody-drug conjugates: Intellectual property considerations.

Antibody-drug conjugates are highly complex entities that combine an antibody, a linker and a toxin. This complexity makes them demanding both technically and from a regulatory point of view, and difficult to deal with in their patent aspects. This article discusses different issues of patent protection and freedom to operate with regard to this promising new class of drugs.

Natural products as exquisitely potent cytotoxic payloads for antibody- drug conjugates.

Antibody-drug conjugates (ADCs) are an emerging area of study within medicinal chemistry and are thought of as sophisticated drug delivery systems due to their specificity to a disease-targeted antigen. ADCs have been actively utilized as therapeutics for hematological and solid tumor cancers due to their capability to deliver a cytotoxic compound to a specific cancer cell without affecting normal cells. An antibody drug conjugate has three major constituents: a monoclonal antibody (mAb), a chemical linker, and a potent cytotoxic payload. There has been a continuing effort to optimize antibody-drug conjugates, with the primary focus of design and development directed at either the mAb or the chemical linker, with little effort devoted to the optimization of payload compounds. In fact, among the 114 ongoing or recently completed clinical trials, there is generally a lack of diversity in the cytotoxic payloads that are utilized, with only seven payload compounds reported (four additional trials are ongoing with structures that have not been reported). Six of these seven payload compounds are derived from natural product sources, highlighting the importance of natural products as cytotoxic payloads for ADC.

Antibody-based therapy of acute myeloid leukemia with gemtuzumab ozogamicin.

Antibodies have created high expectations for effective yet tolerated therapeutics in acute myeloid leukemia (AML). Hitherto the most exploited target is CD33, a myeloid differentiation antigen found on AML blasts in most patients and, perhaps, leukemic stem cells in some. Treatment efforts have focused on conjugated antibodies, particularly gemtuzumab ozogamicin (GO), an anti-CD33 antibody carrying a toxic calicheamicin-g 1 derivative that, after intracellular hydrolytic release, induces DNA strand breaks, apoptosis, and cell death. Serving as paradigm for this strategy, GO was the first anti-cancer immunoconjugate to obtain regulatory approval in the U.S. While efficacious as monotherapy in acute promyelocytic leukemia (APL), GO alone induces remissions in less than 25-35% of non-APL AML patients. However, emerging data from well controlled trials now indicate that GO improves survival for many non-APL AML patients, supporting the conclusion that CD33 is a clinically relevant target for some disease subsets. It is thus unfortunate that GO has become unavailable in many parts of the world, and the drug's usefulness should be reconsidered and selected patients granted access to this immunoconjugate.

Upfront use of gemtuzumab ozogamicin in young children with CD33-positive AML.

Gemtuzumab ozogamicin (GO) is a humanized anti-CD33 antibody used for treating patients with CD33+ acute myeloid leukemia (AML). We report three young children (two infants and one toddler) with AML treated with GO 9 mg/m(2). Two received two doses at diagnosis alone with conventional chemotherapy and one received one dose after relapse. GO was well tolerated and all three achieved remission. All were transplanted: one relapsed after 5 months and died of disease, one died a toxic death in remission due to pulmonary fibrosis, and one survived (41 months from diagnosis). In conclusion, GO was well tolerated in these young patients with evidence for efficacy.

Immunoassays for the rapid detection of gentamicin and micronomicin in swine muscle.

A monoclonal antibody (mAb)-based ELISA and strip test for gentamicin (GEN) and its analogue micronomicin (MIN), are reported in this study. The conjugate gentamicin-glutaraldehyde-bovine serum albumin (GEN-GDA-BSA) was used as an immunogen. The produced anti-GEN mAB exhibited high cross-reactivity with micronomicin (MIN; 131.2%) and slight or negligible crossreactivity with other aminoglycosides. Based on this mAB, an ELISA and a strip test for GEN and MIN were developed and evaluated. The ELISA showed a 50% inhibition concentration (IC50) of 0.75 ng/mL for GEN and 0.58 ng/mL for MIN. For GEN, the average recoveries at 25-200 microg/kg ranged from 73 to 91%, with intraday CVs of 9-16% and interday CVs of 8-15%. For MIN, the average recoveries ranged from 108 to 131%, with intraday CVs of 10-16% and interday CVs of 8-15%. In contrast, the strip test for GEN or MIN had a detection limit of 5 ng/mL in phosphate-buffered saline and 50 microg/kg in muscle (n=24), and the results could be judged within 10 min. The detection results of incurred samples analyzed by the strip test, ELISA, and HPLC indicated that the two immunoassays correlated well with the HPLC method and could be used as convenient tools for the rapid screening of GEN and MIN residues in swine muscle.

[Therapeutic monoclonal antibodies in onco-hematology]. / Anticorps monoclonaux thérapeutiques en oncohématologie.

Rituximab, a chimeric monoclonal anti-CD20 antibody, was introduced into clinical practice in 1997, and has proven to be highly effective in the treatment of B-lymphoproliferative disorders and autoimmune diseases. Despite such success, in vivo mechanisms of action of anti-CD20 have only recently began to be unraveled, pointing to the crucial role of antibody-dependent cellular cytotoxicity response mediated through Fcg receptor signalling. Better understanding of pharmacokinetics and factors influencing individual exposure mediated through anti-CD20 will allow to engineer these molecules to increase their effector responses. Meanwhile, other formats have also been investigated, such as radiolabeled anti-CD20, or coupling of antibodies to cytotoxic drugs such as anti-CD33 used in myeloid leukemia. However these antibodies are used in combination with standard chemotherapy and cannot substitute for cytotoxic drugs. This review summarizes the knowledge acquired through our clinical use of anti-CD20 and authorized monoclonal antibodies in oncohematology and proposes some news areas that will lead to the development of new and more effective therapeutic strategies.

Phase I/II study of humanized anti-CD33 antibody conjugated with calicheamicin, gemtuzumab ozogamicin, in relapsed or refractory acute myeloid leukemia: final results of Japanese multicenter cooperative study.

The primary objective of this study was to investigate the tolerability, efficacy and pharmacokinetic profile of gemtuzumab ozogamicin (GO) in patients with relapsed and/or refractory CD33-positive acute myeloid leukemia (AML). Patients received 2-h infusions of GO twice with an interval of approximately 14 days. Tolerability was assessed using the National Cancer Institute Common Toxicity Criteria Version 2.0. Samples for pharmacokinetics were taken on day 1 and day 8 of the first treatment cycle. The dose was increased stepwise and, in each cohort, patients were treated at the same dose. Forty patients, median age 58 years (range 28-68) were treated; 20 and 20 patients were enrolled to the phase I and II parts, respectively. In the phase I part, dose-limiting toxicities (DLTs) were hepatotoxicities, and the recommended dose was established as 9 mg/m2 given as two intravenous infusions separated by approximately 14 days. The pharmacokinetic study revealed that Cmax and AUC were equivalent to those of non-Japanese patients. In the phase II part, complete remission was observed in 5 patients, and one patient had complete remission without platelet recovery. Four of these 6 in remission and one in the phase I are long-term survivors (alive for at least 44 months). GO is safe and effective as a single agent among Japanese CD33-positive AML patients. Remission lasted longer in a subset of patients than in non-Japanese patients in earlier studies. Further studies of this agent are warranted to establish standard therapy.

Positive patch test reactions to gentamicin show sensitization to aminoglycosides from topical therapies, bone cements, and from systemic medication.

BACKGROUND: A history of prolonged use of topical antimicrobials is common among patients with positive patch test reactions to gentamicin and to aminoglycosides. OBJECTIVES: The aim of this study was to show sources of gentamicin sensitization in patients with positive patch test reactions to gentamicin. PATIENTS AND METHODS: About 7814 patients were patch tested with a baseline patch test series and 620 of them were further tested with gentamicin. The clinical histories, concurrent contact sensitivities, and sources of sensitization are analysed among these patients. RESULTS: Positive patch test reactions to gentamicin were seen in 29/620 patients, most of whom (18/29) also reacted to neomycin and to kanamycin (7/29). Mean age of the gentamicin-positive patients was 62 years, but three young operating room nurses with hand dermatitis had a history of gentamicin exposure from bone cement. Among the 11/29 neomycin-negative patients, a history of exposure to different aminoglycosides was apparent, and one patient had a history of systemic netilmicin-medication-associated exanthema. CONCLUSIONS: Positive patch test reactions to gentamicin reflect sensitization to different aminoglycosides for which gentamicin seems to represent a sensitive indicator. Gentamicin sensitization may result from occupational exposure to gentamicin containing bone cements or from systemic medication with aminoglycosides.

[The role of gemtuzumab ozogamicin in the treatment of acute myeloid leukemia patients].

Gemtuzumab Ozogamicin (GO) targets leukemia cells expressing CD33 by means of a monoclonal antibody conjugated to a cytotoxic agent, calicheamicin. GO has been approved in Japan as monotherapy for the treatment of patients with relapsed/refractory acute myeloid leukemia (AML)since 2005. GO administered as a single agent has resulted in overall response rates of about 30% in previously relapsed adult AML. Preliminary data indicate a potential role for GO also as a component of induction or consolidation regimen. Although caution is advised when administering GO within 115 days of a stem cell transplantation (SCT) procedure because of veno-occlusive disease, recent clinical studies overseas suggest that GO can be integrated into reduced-intensity conditioning therapy before allogeneic SCT in patients with relapsed AML. In order to reduce toxicity and improve efficacy, its optimal dose and schedule should be defined by large clinical trials.

[Reinduction therapy with gemtuzumab ozogamicin for relapsed or refractory CD33-positive acute myelogeneous leukemia].

The efficacy and safety of reinduction therapy with gemtuzumab ozogamicin (GO)were investigated in 7 patients with relapsed or refractory CD33-positive acute myelogeneous leukemia. As the administration method, intravenous drip infusion of 9 mg/m(2) was conducted on day 1 and 15. Though CR was attained in 3 patients, the remaining 4 patients were assessed as PD. Grade 3-4 neutropenia and thrombopenia occurred in all patients, 4 of whom were complicated with febrile neutropenia and 1 with new pneumonia. On the other hand, except for grade 1 digestive symptoms and grade 1 GPT increase, none of the patients had serious complications. Though the treatment with GO is considered comparatively safe, sufficient supportive therapy as in the case of conventional chemotherapy is necessary against hematological toxicity. The effect of monotherapy with GO in reinduction is limited. It is necessary to appropriately select the cases and to investigate an effective administration method including the concomitant use of antitumor agent.

Selection of reaction additives used in the preparation of monomeric antibody-calicheamicin conjugates.

The formation of protein aggregates can be a major problem during the preparation of antibody-drug conjugates. Herein is described the methods by which reaction additives were selected, which reduce the tendency of antibodies to aggregate during the attachment of the cytotoxic agent calicheamicin to form an immunoconjugate. Reaction conditions were delineated that produced optimized yields of monomeric conjugates. These conditions were used in the preclinical preparations of gemtuzumab ozogamicin (Mylotarg), the first commercially available chemotherapeutic immunoconjugate.

The role of Gemtuzumab Ozogamicin in the treatment of acute myeloid leukemia patients.

Gemtuzumab Ozogamicin (GO) is an antibody-targeted chemotherapy agent consisting of the humanized murine CD33 antibody (clone P67.6) to which the calicheamicin-g1 derivative is attached via a hydrolysable bifunctional linker. GO is able to induce apoptosis in vitro in CD33-expressing cells and it has been approved in USA and in Europe as monotherapy for the treatment of elderly patients (older than 60 years) with relapsed acute myeloid leukemia (AML). GO administered as a single agent has resulted in overall response rates of about 30% in previously relapsed adults AML patients (including also with incomplete platelet recovery). Preliminary data indicate a potential role for GO also as a component of induction or consolidation regimens in adults and children. As for adverse events, veno-occlusive syndrome characterizes its tolerability profile, but GO is comparatively well tolerated by most patients.

CD20-specific antibody-targeted chemotherapy of non-Hodgkin's B-cell lymphoma using calicheamicin-conjugated rituximab.

Tumor-targeted delivery of a potent cytotoxic agent, calicheamicin, using its immunoconjugates is a clinically validated therapeutic strategy. Rituximab is a human CD20-specific chimeric antibody extensively used in B-NHL therapy. We investigated whether conjugation to calicheamicin can improve the anti-tumor activity of rituximab against human B-cell lymphoma (BCL) xenografts in preclinical models. BCL cells were cultured with rituximab or its calicheamicin conjugates and their in vitro growth was monitored. BCL cells were injected s.c. to establish localized xenografts in nude mice or i.v. to establish disseminated BCL in severe combined immunodeficient (scid) mice. I.p. treatment with rituximab or its calicheamicin conjugates was initiated and its effect on s.c. BCL growth or survival of mice with disseminated BCL was monitored. Conjugation of calicheamicin to rituximab vastly enhanced its growth inhibitory activity against BCL in vitro. Conjugation to calicheamicin had no deleterious effect on the effector functional activity of rituximab. Calicheamicin conjugated to rituximab with an acid-labile linker exhibited greater anti-tumor activity against s.c. BCL xenografts and improved survival of mice with disseminated BCL over that of unconjugated rituximab. Anti-tumor activities of rituximab conjugated to calicheamicin via an acid-stable linker were similar to that of unconjugated rituximab. Superior anti-tumor efficacy exhibited by a calicheamicin immunoconjugate of rituximab with an acid-labile linker over that of rituximab demonstrates the therapeutic potential of CD20-specific antibody-targeted chemotherapy strategy in the treatment of B-NHL.

Fixed exanthema from systemic tobramycin.

Eye drops contain several ophthalmic medications which can produce allergic reactions. We report the case of a patient with contact dermatitis from neomycin and a probable fixed exanthema after parenteral administration of tobramycin who tolerated topical tobramycin and other aminoglycosides.

Antitumor efficacy of a combination of CMC-544 (inotuzumab ozogamicin), a CD22-targeted cytotoxic immunoconjugate of calicheamicin, and rituximab against non-Hodgkin's B-cell lymphoma.

PURPOSE: CMC-544 is a CD22-targeted cytotoxic immunoconjugate, currently being evaluated in B-cell non-Hodgkin's lymphoma (B-NHL) patients. Rituximab is a CD20-targeted antibody commonly used in B-NHL therapy. Here, we describe antitumor efficacy of a combination of CMC-544 and rituximab against B-cell lymphoma (BCL) in preclinical models. EXPERIMENTAL DESIGN: BCLs were cultured in vitro with CMC-544, rituximab, or their combination. BCLs were injected either s.c. or i.v. to establish localized s.c. BCL in nude mice or disseminated BCL in severe combined immunodeficient mice, respectively. I.p. treatment with CMC-544 or rituximab was initiated at various times either alone or in combination and its effect on s.c. BCL growth or survival of mice with disseminated BCL was monitored. RESULTS: In vitro growth-inhibitory activity of CMC-544 combined with rituximab was additive. Rituximab but not CMC-544 exhibited effector functions, such as antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. Rituximab was less effective in inhibiting growth of established BCL xenografts than developing xenografts. In contrast, CMC-544 was equally effective against both developing and established BCL xenografts. Although CMC-544 and rituximab individually caused partial inhibition of the growth of BCL xenografts at suboptimal doses examined, their combination suppressed xenograft growth by >90%. In a disseminated BCL model, 60% of CMC-544-treated mice and 20% of rituximab-treated mice survived for 125 days. In contrast, 90% of mice treated with the combination of CMC-544 and rituximab survived for longer than 125 days. CONCLUSION: The demonstration of superior antitumor activity of a combination of CMC-544 and rituximab described here provides the preclinical basis for its clinical evaluation as a treatment option for B-NHL.

Antibody-targeted chemotherapy of B-cell lymphoma using calicheamicin conjugated to murine or humanized antibody against CD22.

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.