Preclinical Antitumor Efficacy of BAY 1129980-a Novel Auristatin-Based Anti-C4.4A (LYPD3) Antibody-Drug Conjugate for the Treatment of Non-Small Cell Lung Cancer.

C4.4A (LYPD3) has been identified as a cancer- and metastasis-associated internalizing cell surface protein that is expressed in non-small cell lung cancer (NSCLC), with particularly high prevalence in the squamous cell carcinoma (SCC) subtype. With the exception of skin keratinocytes and esophageal endothelial cells, C4.4A expression is scarce in normal tissues, presenting an opportunity to selectively treat cancers with a C4.4A-directed antibody-drug conjugate (ADC). We have generated BAY 1129980 (C4.4A-ADC), an ADC consisting of a fully human C4.4A-targeting mAb conjugated to a novel, highly potent derivative of the microtubule-disrupting cytotoxic drug auristatin via a noncleavable alkyl hydrazide linker. In vitro, C4.4A-ADC demonstrated potent antiproliferative efficacy in cell lines endogenously expressing C4.4A and inhibited proliferation of C4.4A-transfected A549 lung cancer cells showing selectivity compared with a nontargeted control ADC. In vivo, C4.4A-ADC was efficacious in human NSCLC cell line (NCI-H292 and NCI-H322) and patient-derived xenograft (PDX) models (Lu7064, Lu7126, Lu7433, and Lu7466). C4.4A expression level correlated with in vivo efficacy, the most responsive being the models with C4.4A expression in over 50% of the cells. In the NCI-H292 NSCLC model, C4.4A-ADC demonstrated equal or superior efficacy compared to cisplatin, paclitaxel, and vinorelbine. Furthermore, an additive antitumor efficacy in combination with cisplatin was observed. Finally, a repeated dosing with C4.4A-ADC was well tolerated without changing the sensitivity to the treatment. Taken together, C4.4A-ADC is a promising therapeutic candidate for the treatment of NSCLC and other cancers expressing C4.4A. A phase I study (NCT02134197) with the C4.4A-ADC BAY 1129980 is currently ongoing. Mol Cancer Ther; 16(5); 893-904. ©2017 AACR.

Bleomycin-induced hyperpigmentation and hypersensitivity reactions to etoposide and vinblastine in a child with endodermal sinus tumor.

We report a pediatric case who developed bleomycin-induced hyperpigmentation and hypersensitivity reactions to both etoposide and vinblastine while receiving chemotherapy for germ cell tumor. Skin hyperpigmentation related to chemotherapeutic agents has been reported only rarely in pediatric patients. This patient developed a characteristic skin hyperpigmentation which was "flagellate" in appearance. Two features of the hyperpigmentation were noteworthy: development at a low cumulative dose of bleomycin and persistence after cessation of chemotherapy. Additive effect of cisplatinum-induced hyperpigmentation was suggested. Although hypersensitivity reactions to etoposide have been previously reported, hypersensitivity reactions to vinblastine are almost unknown. To our knowledge, this is the first report of hypersensitivity reaction to vinblastine in a child in English literature.

Reduction of toxicity of a vinca alkaloid by an anti-vinca alkaloid antibody.

Inadvertent oncolytic overdoses occur rarely, but can have serious consequences. We have investigated the possibility of using an antibody, 27.8.1A, reactive with vinca alkaloids, as a means of reducing the toxicity associated with overdose situations. In vitro cytotoxicity of a vinca derivative, 4-desacetyl- vinblastine-3-carbox-hydrazide (DAVLBHYD), with and without the addition of 27.8.1A, was determined. Using CCRF-CEM, a human acute lymphoblastic leukemia cell line, as a target in this assay, we observed a greater than 90% increase in cell viability using 100 micrograms/ml 27.8.1A with a 0.1 microgram/ml concentration of DAVLBHYD. 27.8.1A had no effect on cell viability when doxorubicin was used as a control drug in this assay. Similarly, the addition of an irrelevant antibody. EGFrL11, had no effect on the toxicity of DAVLBHYD. In an in vivo survival experiment, nude mice were injected with a toxic dose of DAVLBHYD and subsequently given four doses of 27.8.1A. All anti vinca antibody-treated mice survived, in contrast to the untreated group or irrelevant antibody-treated group in which only 25% and 10% of the mice survived, respectively.

Induction of immunogenicity of monoclonal antibodies by conjugation with drugs.

Human anti-mouse antibody has been a nearly consistent result of human clinical trials utilizing murine antibodies. It is generally anticipated that the problem of human anti-mouse antibody will be reduced as genetically engineered, more human ("humanized") antibodies become available. It is not clear, however, what effect chemical modification of such "humanized" antibodies will have on their immunogenicity. The present studies utilize a mouse antibody and rat host model to explore aspects of this question. Rats injected with unmodified mouse monoclonal antibodies failed to mount anti-mouse immune responses, presumably due to their phylogenetic relatedness. In contrast, rats injected with a Vinca immunoconjugate mounted strong anticonjugate antibody responses that were directed primarily against the linker portion of the conjugate. The in vivo serum pharmacokinetics of 125I-labeled antibody and conjugates were evaluated in rats with existing anticonjugate antibody. The peak serum level attained was inversely correlated with the level of reactivity of the anticonjugate antibody with the injected compound. This model provides a potentially useful tool for exploration of the immunogenicity of drug, toxin, or radionuclide monoclonal antibody conjugates.

Oral administration of [3H]navelbine in patients: comparative pharmacokinetics using radioactive and radioimmunologic determination methods.

[3H]Navelbine (NVB) was administered orally to two patients. Drug levels in biological fluids were monitored by radioimmunoassay (RIA) and direct radioactivity (RA) determinations. NVB absorption was rapid: maximum plasma concentrations appeared in the first 2 h after oral administrations. Pharmacokinetic parameters estimated from RIA data were in complete accordance with those obtained from i.v. injections. Bioavailability (i.v./po) estimated from RIA and RA data averaged 40.6 and 93.0%, respectively. NVB urine excretion was low. Fecal excretion remained its main elimination route. Moreover, large differences were observed in area under NVB plasma concentration-time curve (AUC) values obtained by the two methods, implying intense drug bio-transformations.

The human immune response to KS1/4-desacetylvinblastine (LY256787) and KS1/4-desacetylvinblastine hydrazide (LY203728) in single and multiple dose clinical studies.

Monoclonal antibody (MoAb) conjugates have been used to treat a variety of malignancies. The majority of the MoAbs which have been used therapeutically are from murine sources. The infusion of these foreign proteins into humans can be expected to elicit anti-murine antibodies and may be one of the major limitations to the clinical use of murine MoAbs. In these studies, we report on the nature and specificity of the human anti-murine antibody (HAMA) response in patients given single and multiple infusions of the two Vinca alkaloid conjugates of the MoAb KS1/4, which recognizes tumor-associated antigens in a variety of adenocarcinomas. A HAMA response was induced in a majority of the patients receiving infusions of KS1/4 conjugates, regardless of the specific conjugate used or the number of infusions. The magnitude of the response did not appear to be dose related. Antibodies directed to the drug moieties of these conjugates, anti-Vinca alkaloids, were also induced in patients with HAMA responses. The magnitude of the anti-Vinca response paralleled that of the HAMA.

Monoclonal antibodies to bis-indole alkaloids of Catharanthus roseus and their use in enzyme-linked immuno-sorbent-assays.

High affinity monoclonal antibodies directed against bis-indole alkaloids from Catharanthus roseus were produced. Once characterized, they were used to develop sensitive enzyme-linked-immuno-sorbent-assays, enabling us to determine minute quantities of alkaloids related to vinblastine in plant material.

Monoclonal antibodies to antitumor Vinca alkaloids: thermodynamics and kinetics.

Spleen cells from a mouse and a rat immunized with vinblastine coupled to bovine serum albumin were fused in two independent experiments with P3 X 63-Ag8 (non-secreting variant) mouse myeloma cells. Three mouse X mouse (Vinca 1-3) and two rat X mouse (Vinca 4 and 5) hybrids were selected for production of Vinca alkaloid binding monoclonal antibodies. Each antibody had characteristic cross-reactivities with alkaloids structurally related to vinblastine: Vinca 1 reacted preferentially with deacetylated alkaloids (deacetyl vinblastine and vindesine) and Vinca 2 had a higher affinity for vinblastine and vincristine. Vinca 3-5 recognized equally vinblastine, vincristine and vindesine but differed with respect to their affinities for other analogues. No significant cross-reactivity of the monomeric alkaloids vindoline or catharanthine was observed with any antibody, and dimeric alkaloids modified in the catharanthine moiety had reduced immunoreactivity. Mouse monoclonal antibodies (Vinca 1 and 3) showed moderate affinity (2.2 X 10(-7) and 5.8 X 10(-9) M) for their respective best ligands and fast kinetics (dissociation rate constants greater than 3 X 10(-3) sec-1). Vinca 4 and 5, derived from the rat X mouse hybrids, had much higher affinities (1.5 X 10(-11) and 1.1 X 10(-11) M) and slower kinetics (dissociation rate constants: 2.4 X 10(-5) and 7.2 X 10(-6) sec-1). The major difference between these two antibodies was that Vinca 4 binds and releases the antigen more rapidly than Vinca 5 does. Somatic hybridization techniques thus generated monoclonal antibodies recognizing a given class of low mol. wt antigens with variable specificity, affinity and kinetic behavior, allowing the selection of reagents most appropriate for particular immunochemical applications.

Experience in the preparation and experimental use of immunocytostatics.

Vindesine (VDS) was coupled directly to a monoclonal antibody (791T/36) raised against a human osteogenic sarcoma cell line, and methotrexate (MTX) was coupled to 791T/36 via an intervening human serum albumin (HSA) bridge. Both the VDS-791T/36 and MTX-HSA-791T/36 conjugates were cytotoxic in vitro specifically for tumour target cells expressing the 791T/36-defined antigen, while the free drug in each case was indiscriminately toxic to all target cells. The VDS-791T/36 conjugate retarded growth of osteogenic sarcoma xenografts in immunodeprived mice when administered in multiple doses. Free 791T/36 did not significantly affect tumour growth. VDS was tumour inhibitory, but was toxic to the mice at a total dose of 20 micrograms per kg body weight, while VDS-791T/36 conjugate was not toxic at total doses incorporating VDS at up to 45 mg per kg. It is suggested that this is due to selectivity conferred upon the conjugate by the antibody moiety, and that such conjugates may offer considerable potential as anti-cancer agents.

Induction in mice of cell-mediated immunity to Pseudomonas aeruginosa by high molecular weight polysaccharide and vinblastine.

The effect of the cytotoxic drug vinblastine on the development of immunity to high m.w. polysaccharide (PS) isolated from culture supernates of Pseudomonas aeruginosa was investigated. One microgram of PS, a normally nonimmunogenic, nonprotective dose, plus 75 micrograms of vinblastine were administered to BALB/c mice, and afforded protection to live organism challenge with the homologous strain. The kinetics and serotype specificity of the immune response indicated an active immunization had occurred. Analyses of serum antibody levels of mice given the PS-drug regimen in a sensitive, radioactive antigen-binding assay (RABA) failed to show development of antibody to the immunizing PS. Immunity could be passively transferred with spleen cells but not by serum from PS-drug-immunized animals, and the effector cell was removed by antisera to the Thy-1.2 antigen. Nu/nu mice were also protected against challenge after immunization with PS and vinblastine, but this protection was observed in association with the development of serum antibody to PS in these mice, as measured in the RABA. Protective immunity could not be elicited in the BALB/c mice by PS plus cyclophosphamide. These data suggest that under certain conditions, PS antigens can elicit T cell-dependent immune phenomena, and this T cell-dependent immunity can protect mice from live organism challenge against an extracellular bacterial pathogen.

Radioimmunoassay of vinblastine and vincristine.

1 The cytotoxic agent, vinblastine, was conjugated to albumin, using the Mannich reaction. Rabbits immunized with two conjugates, containing differing amounts of hapten, produce antibodies which bound [3H]-vinblastine. 2 Antisera from one rabbit cross-reacted with both vinblastine and vincristine and were used to develop radioimmunoassays for measuring their concentration in plasma. 3 The antisera showed no cross-reactivity with other alkaloids or cytotoxic drugs and provided assays sensitive to a concentration of 2.1 ng vinblastine or 3.8 ng vincristine/ml of plasma added direct to the assay tubes. 4 This is sufficiently sensitive to permit the measurement of plasma vinblastine levels for up to 24 h after the intravenous administration of 15 mg of the drug.