Development of an Fc-enhanced anti-B7-H3 monoclonal antibody with potent antitumor activity.

PURPOSE: The goal of this research was to harness a monoclonal antibody (mAb) discovery platform to identify cell-surface antigens highly expressed on cancer and develop, through Fc optimization, potent mAb therapies toward these tumor-specific antigens. EXPERIMENTAL DESIGN: Fifty independent mAbs targeting the cell-surface immunoregulatory B7-H3 protein were obtained through independent intact cell-based immunizations using human tissue progenitor cells, cancer cell lines, or cell lines displaying cancer stem cell properties. Binding studies revealed this natively reactive B7-H3 mAb panel to bind a range of independent B7-H3 epitopes. Immunohistochemical analyses showed that a subset displayed strong reactivity to a broad range of human cancers while exhibiting limited binding to normal human tissues. A B7-H3 mAb displaying exquisite tumor/normal differential binding was selected for humanization and incorporation of an Fc domain modified to enhance effector-mediated antitumor function via increased affinity for the activating receptor CD16A and decreased binding to the inhibitory receptor CD32B. RESULTS: MGA271, the resulting engineered anti-B7-H3 mAb, mediates potent antibody-dependent cellular cytotoxicity against a broad range of tumor cell types. Furthermore, in human CD16A-bearing transgenic mice, MGA271 exhibited potent antitumor activity in B7-H3-expressing xenograft models of renal cell and bladder carcinoma. Toxicology studies carried out in cynomolgus monkeys revealed no significant test article-related safety findings. CONCLUSIONS: This data supports evaluation of MGA271 clinical utility in B7-H3-expressing cancer, while validating a combination of a nontarget biased approach of intact cell immunizations and immunohistochemistry to identify novel cancer antigens with Fc-based mAb engineering to enable potent antitumor activity.

CAT-8015: a second-generation pseudomonas exotoxin A-based immunotherapy targeting CD22-expressing hematologic malignancies.

PURPOSE: To compare the in vitro and in vivo efficacy of CAT-8015, a second-generation recombinant immunotoxin composed of disulfide-linked affinity matured V(H) and V(L) chains of the mouse anti-CD22 monoclonal antibody RFB4 fused to PE38, to the parental compound CAT-3888. EXPERIMENTAL DESIGN: The biological activity of CAT-8015 was examined in vitro using B-cell tumor lines and in vivo in a JD38-based s.c. tumor model in NCr athymic mice. Pharmacokinetics and interspecies scaling of CAT-8015 were evaluated in mice, rats, and cynomolgus monkeys. The potential toxicity of CAT-8015 was assessed in monkeys in a toxicologic study and compared with CAT-3888. RESULTS: The IC50 values of CAT-8015 in vitro using the EHEB, MEC1, Daudi, CA46, and JD38 cell lines ranged from 0.3 to 8.6 ng/mL. Pharmacokinetic studies with CAT-8015 were conducted in mouse, rat, and cynomolgus monkey. The t1/2 was calculated to be 0.42, 0.61, and 0.79 hours and the Vss was 1.37, 5.57, and 140.3 mL in mouse, rat, and monkey, respectively. In vivo, when JD38 tumor-bearing animals were treated with CAT-8015 at doses > or =75 microg/kg at 48-hour intervals for a total of three doses, a rapid reduction in tumor volume and in some cases complete remission in tumor growth was observed. The comparative toxicologic study showed comparable clinical and anatomic pathology changes for CAT-8015 and CAT-3888. CONCLUSIONS: CAT-8015 is a CD22-targeting immunotoxin that, in preclinical studies, has greatly improved efficacy compared with CAT-3888.

Characterization of a CC49-based single-chain fragment-beta-lactamase fusion protein for antibody-directed enzyme prodrug therapy (ADEPT).

CC49 is a clinically validated antibody with specificity for TAG-72, a carbohydrate epitope that is overexpressed and exposed on the cell surface in a large fraction of solid malignancies. We constructed a single-chain fragment (scFv) based on CC49 and fused it to beta-lactamase (BLA). Following optimization of the scFv domain by combinatorial consensus mutagenesis (CCM) for increased expression and stability, we characterized the protein variant for binding, in vivo pharmacokinetics (PK), and antitumor efficacy. The fusion protein TAB2.5 possessed a similar binding specificity relative to the parent antibody CC49. TAB2.5 also showed prolonged retention (T(1/2) = 36.9 h) in tumor-bearing mice with tumor/plasma ratios of up to 1000. Preliminary evaluation of TAB2.5, in combination with a novel prodrug, GC-Mel, resulted in significant efficacy in a colorectal xenograft tumor model and supports the utility of the protein as an agent for tumor-selective prodrug activation.

Expression of Ciliary Neurotrophic Factor and the Neurotrophins-Nerve Growth Factor, Brain-Derived Neurotrophic Factor and Neurotrophin 3-in Cultured Rat Hippocampal Astrocytes.

Cultured astrocytes are known to possess a range of neurotrophic activities in culture. In order to examine which factors may be responsible for these activities, we have examined the expression of the genes for four known neurotrophic factors-ciliary neurotrophic factor (CNTF), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin 3 (NT3)-in purified astrocyte cultures derived from neonatal rat hippocampus. Hippocampal astrocytes were found to express mRNA for three neurotrophic factors-CNTF, NGF and NT3-at significantly higher levels than other cultured cell types or cell lines examined. BDNF messenger RNA (mRNA), however, was undetectable in these astrocytes. The levels of CNTF, NGF and NT3 mRNA in astrocytes were largely unaffected by their degree of confluency, while serum removal caused only a transient decrease in mRNA levels, which returned to basal levels within 48 h. Astrocyte-derived CNTF was found to comigrate with recombinant rat CNTF at 23 kD on a Western blot. Immunocytochemical analysis revealed strong CNTF immunoreactivity in the cytoplasm of astrocytes, weak staining in the nucleus, but no CNTF at the cell surface. NGF and NT3 were undetectable immunocytochemically. CNTF-like activity, as assessed by bioassay on ciliary ganglion neurons, was found in the extract of cultured astrocytes but not in conditioned medium, whereas astrocyte-conditioned medium supported survival of dorsal root ganglion neurons but not ciliary or nodose ganglion neurons. This conditioned medium activity was neutralized with antibodies to NGF. Astrocyte extract also supported survival of dorsal root ganglion and nodose ganglion neurons, but these activities were not blocked by anti-NGF. Part, but not all, of the activity in astrocyte extracts which sustained nodose ganglion neurons could be attributed to CNTF.