CD20-directed small modular immunopharmaceutical, TRU-015, depletes normal and malignant B cells.

PURPOSE: CD20-directed therapy with rituximab is effective in many patients with malignant lymphoma or follicular lymphoma. However, relapse frequently occurs within 1 year, and patients become increasingly refractory to retreatment. Our purpose was to produce a compact, single-chain CD20-targeting immunotherapeutic that could offer therapeutic advantages in the treatment of B-cell lymphoma. EXPERIMENTAL DESIGN: Rituximab is a chimeric antibody containing two heavy chains and two light chains. Here, we describe the properties of TRU-015, a small modular immunopharmaceutical specific for CD20, encoded by a single-chain construct containing a single-chain Fv specific for CD20 linked to human IgG1 hinge, CH2, and CH3 domains but devoid of CH1 and CL domains. RESULTS: TRU-015 mediates potent direct signaling and antibody-dependent cellular cytotoxicity but has reduced size and complement-mediated cytotoxicity activity compared with rituximab. TRU-015 is a compact dimer of 104 kDa that comigrates with albumin in size exclusion chromatography and retains a long half-life in vivo. TRU-015 induced growth arrest in multiple B lymphoma cell lines in vitro and showed effective antitumor activity against large, established subcutaneous Ramos or Daudi xenograft tumors in nude mice. TRU-015 also showed rapid, dose-dependent, and durable depletion of peripheral blood B cells following single-dose administration to nonhuman primates. CONCLUSION: These results indicate that TRU-015 may improve CD20-directed therapy by effectively depleting embedded malignant B cells and nonmalignant pathogenic B cells and do so with reduced complement activation.

Synthesis and structure-activity relationships of 4-alkynyloxy phenyl sulfanyl, sulfinyl, and sulfonyl alkyl hydroxamates as tumor necrosis factor-alpha converting enzyme and matrix metalloproteinase inhibitors.

A series of 4-alkynyloxy phenyl sulfanyl, sulfinyl and sulfony alkyl and piperidine-4-carboxylic acid hydroxamides were synthesized. Their structure-activity relationships, against tumor necrosis factor-alpha (TACE) and matrix metalloproteinase (MMP) inhibitor activities, are presented by investigating the oxidation state on sulfur and altering the P1' substituent. The sulfonyl derivatives 20-24 carrying a 4-butynyloxy moiety were selective TACE inhibitors over the MMPs tested. The sulfinyl derivatives showed a preference for a specific oxidation on sulfur as in compounds 25-28. The selectivity over MMPs was also demonstrated in the sulfonyl series. The enhanced cellular activity was achieved upon incorporating a butynyloxy substituent in the piperidene series. Compounds 64 and 65 were potent inhibitors of TNF-alpha release in the mouse at 100 mg/kg po.

Synthesis and SAR of diazepine and thiazepine TACE and MMP inhibitors.

Potent and selective TACE and MMP inhibitors utilizing the diazepine and thiazepine ring systems were synthesized and evaluated for biological activity in in vitro and in vivo models of TNF-alpha release. Oral activity in the mouse LPS model of TNF-alpha release was seen. Efficacy in the mouse collagen induced arthritis model was achieved with diazepine 20.

Periosteal hyperostosis (exostosis) in DBA/1 male mice.

Periosteal hyperostosis (exostosis) was identified in 5.9% (11/188) of DBA/1 male mice 10-14 weeks old used for collagen-induced arthritis (CIA) efficacy testing of immunomodulatory biologics. Mice with and without CIA in the affected limb, and also control and treated groups, were involved, with bilateral lesions in one mouse. Hyperostosis was characterized by circumferential and raised masses of variable location, length, and laterality, generally external to but occasionally breaching the periosteum of the metatarsals, metacarpals, tibia, femur, and humerus. Proportionally, the hyperostotic foci consisted of cancellous and woven bone, followed by osteoid, cartilage, and fibrous connective tissue and rarely inflammatory cells. A displaced, presumably pathological fracture with callus formation was a concurrent lesion in only one case. Tartrate-resistant acid phosphatase-positive cells were frequent at bony interfaces, indicating an active resorptive process. Periosteal hyperostosis is an incidental and potentially common finding in DBA/1 mice. Underreporting may occur due to the male bias in disease expression of this CIA model, sampling bias (generally paws only), tissue obliteration in the presence of CIA, and lack of comprehensive historical data on the background and aging lesions in this strain of mouse. Identification of such confounding bony lesions is important to the interpretation of efficacy studies, and suggests the need to further examine the biology of bone development in this strain of mouse.

Benzodiazepine inhibitors of the MMPs and TACE. Part 2.

A series of benzodiazepine MMP/TACE inhibitors bearing polar moieties has been synthesized in an effort to optimize inhibitory activity against LPS-stimulated TNF production in human monocytes and oral activity in a murine LPS model.

Identification and characterization of 4-[[4-(2-butynyloxy)phenyl]sulfonyl]-N-hydroxy-2,2-dimethyl-(3S)thiomorpholinecarboxamide (TMI-1), a novel dual tumor necrosis factor-alpha-converting enzyme/matrix metalloprotease inhibitor for the treatment of rheumatoid arthritis.

Tumor necrosis factor (TNF)-alpha is a well validated therapeutic target for the treatment of rheumatoid arthritis. TNF-alpha is initially synthesized as a 26-kDa membrane-bound form (pro-TNF) that is cleaved by a Zn-metalloprotease named TNF-alpha-converting enzyme (TACE) to generate the 17-kDa, soluble, mature TNF-alpha. TACE inhibitors that prevent the secretion of soluble TNF-alpha may be effective in treating rheumatoid arthritis (RA) patients. Using a structure-based design approach, we have identified a novel dual TACE/matrix metalloprotease (MMP) inhibitor 4-[[4-(2-butynyloxy)phenyl]sulfonyl]-N-hydroxy-2,2-dimethyl-(3S)thiomorpholinecarboxamide (TMI-1). This molecule inhibits TACE and several MMPs with nanomolar IC(50) values in vitro. In cell-based assays such as monocyte cell lines, human primary monocytes, and human whole blood, it inhibits lipopolysaccharide (LPS)-induced TNF-alpha secretion at submicromolar concentrations, whereas there is no effect on the TNF-alpha mRNA level as judged by RNase protection assay. The inhibition of LPS-induced TNF-alpha secretion is selective because TMI-1 has no effect on the secretion of other proinflammatory cytokines such as interleukin (IL)-1beta, IL-6, and IL-8. Importantly, TMI-1 potently inhibits TNF-alpha secretion by human synovium tissue explants of RA patients. In vivo, TMI-1 is highly effective in reducing clinical severity scores in mouse prophylactic collagen-induced arthritis (CIA) at 5, 10, and 20 mg/kg p.o. b.i.d. and therapeutic CIA model at 100 mg/kg p.o. b.i.d. In summary, TMI-1, a dual TACE/MMP inhibitor, represents a unique class of orally bioavailable small molecule TNF inhibitors that may be effective and beneficial for treating RA.