Ligand Pharmaceuticals (United States)

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer. Bacterial L-asparaginase has played an important role in ALL treatment for several decades; however, hypersensitivity reactions to Escherichia coli-derived asparaginases often preclude their use. Inability to receive asparaginase due to hypersensitivities is associated with poor patient outcomes. Erwinia chrysanthemi-derived asparaginase (ERW) is an effective, non-cross-reactive treatment option, but is limited in supply. Consequently, alternative asparaginase preparations are needed to ensure asparaginase availability for patients with hypersensitivities. Recombinant technology can potentially address this unmet need by programming cells to produce recombinant asparaginase. JZP-458, a recombinant Erwinia asparaginase derived from a novel Pseudomonas fluorescens expression platform with no immunologic cross-reactivity to E. coli-derived asparaginases, has the same primary amino acid sequence as ERW, with comparable activity based on in vitro measurements. The efficient manufacturing of JZP-458 would provide an additional asparaginase preparation for patients with hypersensitivities.

Techniques to obtain large quantities of antigen-specific monoclonal antibodies (mAbs) were first established in the 1970s when Georges Köhler and César Milstein immortalized antibody-producing mouse B-lymphocytes by fusion with myeloma cells (http://www.whatisbiotechnology.org/exhibitions/milstein). Combined with the expression of human antibodies in transgenic animals, this technique allowed upon immunization the generation of highly specific fully human mAbs for therapeutic applications. Apart from being extremely beneficial, mAbs are a huge success commercially. However, despite cell fusion generating many useful mAbs questions have been asked about which types of cells are prone to fuse and whether other methods may identify a wider range of binders. The discovery that expression libraries, using Escherichia coli or yeast, produced different specificities was intriguing and more recently Next-Generation Sequencing has identified wide-ranging usage with highly diverse and unique repertoires. Another strategy is the combination of flow cytometry sorting of antigen-binding B lymphocytes and single-cell reverse transcription polymerase chain reaction followed by reexpression, which has identified many high-affinity mAbs.

BackgroundThere is an ever-increasing need of monoclonal antibodies (mAbs) for biomedical applications and fully human binders are particularly desirable due to their reduced immunogenicity in patients. We have applied a strategy for the isolation of antigen-specific B cells using tetramerized proteins and single-cell sorting followed by reconstruction of human mAbs by RT-PCR and expression cloning.ResultsThis strategy, using human peripheral blood B cells, enabled the production of low affinity human mAbs against major histocompatibility complex molecules loaded with peptides (pMHC). We then implemented this technology using human immunoglobulin transgenic rats, which after immunization with an antigen of interest express high affinity-matured antibodies with human idiotypes. Using rapid immunization, followed by tetramer-based B-cell sorting and expression cloning, we generated several fully humanized mAbs with strong affinities, which could discriminate between highly homologous proteins (eg. different pMHC complexes).ConclusionsTherefore, we describe a versatile and more effective approach as compared to hybridoma generation or phage or yeast display technologies for the generation of highly specific and discriminative fully human mAbs that could be useful both for basic research and immunotherapeutic purposes.

BACKGROUND AND PURPOSEThe chemokine receptor CXCR3 directs migration of T-cells in response to the ligands CXCL9/Mig, CXCL10/IP-10 and CXCL11/I-TAC. Both ligands and receptors are implicated in the pathogenesis of inflammatory disorders, including atherosclerosis and rheumatoid arthritis. Here, we describe the molecular mechanism by which two synthetic small molecule agonists activate CXCR3.EXPERIMENTAL APPROACHAs both small molecules are basic, we hypothesized that they formed electrostatic interactions with acidic residues within CXCR3. Nine point mutants of CXCR3 were generated in which an acidic residue was mutated to its amide counterpart. Following transient expression, the ability of the constructs to bind and signal in response to natural and synthetic ligands was examined.KEY RESULTSThe CXCR3 mutants D112N, D195N and E196Q were efficiently expressed and responsive in chemotaxis assays to CXCL11 but not to CXCL10 or to either of the synthetic agonists, confirmed with radioligand binding assays. Molecular modelling of both CXCL10 and CXCR3 suggests that the small molecule agonists mimic a region of the ‘30s loop’ (residues 30–40 of CXCL10) which interacts with the intrahelical CXCR3 residue D112, leading to receptor activation. D195 and E196 are located in the second extracellular loop and form putative intramolecular salt bridges required for a CXCR3 conformation that recognizes CXCL10. In contrast, CXCL11 recognition by CXCR3 is largely independent of these residues.CONCLUSION AND IMPLICATIONSWe provide here a molecular basis for the observation that CXCL10 and CXCL11 are allosteric ligands of CXCR3. Such findings may have implications for the design of CXCR3 antagonists.LINKED ARTICLEThis article is commented on by O'Boyle, pp. 895–897 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2011.01759.x

Abstract Background: Breast cancer prevention is now possible using anti-estrogen drugs such as tamoxifen and raloxifene. However, the anti-estrogen drugs do not prevent all types of breast cancer. These drugs are useful for the prevention of estrogen receptor (ER)-positive breast cancers, but are unable to prevent the development of ER-negative breast cancer. ER-negative breast cancers are highly aggressive and are particularly difficult to treat. Thus, there is an urgent need to develop ways to prevent these life-threatening cancers. Using a transgenic mouse model of ER-negative breast cancer, our laboratory has previously demonstrated that LGD100268, a ligand to the RXR nuclear hormone receptor, prevents up to 90% of the ER-negative breast cancers in these mice. However, because this drug does not prevent all breast cancers in these mice, we have attempted to achieve more effective cancer prevention by combining two cancer preventive drugs. We hypothesized that the combination of the rexinoid LG100268 in combination with tamoxifen will more effectively prevent the development of ER-negative breast cancer. To test this hypothesis, we used the p53-null mouse model that develops both ER-positive and ER-negative mammary tumors to determine whether the combination of a rexinoid and an anti-estrogen is more effective than either treatment alone in preventing ER-positive and ERnegative mammary tumors. Methods: Using our standard transplant protocol, p53-null Balb/C donor mice was transplanted into both cleared inguinal mammary fat pads of Balb/C p53 wild-type mice. Mice were separated into 5 treatment groups (15 mice each). 1) Sham Control: 2) Vehicle Control: 3) Tamoxifen + Vehicle: 4) Sham pellet (5mg) + Rexinoid: (at 50mg/kg), 5) Tamoxifen + Rexinoid: Mice were treated for 60 days with each drug sequentially. Mice were observed daily for tumor formation and the percentage of tumor free mice were recorded. Tumor incidence and time to tumor formation was analyzed. The expression of mammary tissue biomarkers was done using immunohistochemistry and real time QRT-PCR. Results: 38% of sham and 41% vehicle control animals developed tumors. Sequential treatment with tamoxifen and LGD100268 prevented more (8% incidence) than tamoxifen (24% incidence) or LGD100268 (20% incidence) alone. Proliferation markers Ki67 and cyclin D1 were markedly reduced in the mammary tissue from the mice treated with the combination therapy. We have also observed induction of rexinoid target genes ABCA1 and ABCG1, in the retinoid group and in the combination group. Conclusions: The combination treatment of rexinoid LG100268 and tamoxifen is more effective at preventing mammary tumors than either agent alone. Based on these studies, clinical trials using the combination of tamoxifen and rexinoids should be considered for the prevention of breast cancer in high-risk patients. Supported by NIH grants P50 CA58183 (PB, DM) and R01 CA101211 (PB, DM). Citation Information: Cancer Prev Res 2010;3(12 Suppl):B56.