Carl J. Kozlosky

An Essential Role for Ectodomain Shedding in Mammalian Development

The ectodomains of numerous proteins are released from cells by proteolysis to yield soluble intercellular regulators. The responsible protease, tumor necrosis factor-alpha converting enzyme (TACE), has been identified only in the case when tumor necrosis factor-alpha (TNFalpha) is released. Analyses of cells lacking this metalloproteinase-disintegrin revealed an expanded role for TACE in the processing of other cell surface proteins, including a TNF receptor, the L-selectin adhesion molecule, and transforming growth factor-alpha (TGFalpha). The phenotype of mice lacking TACE suggests an essential role for soluble TGFalpha in normal development and emphasizes the importance of protein ectodomain shedding in vivo.

Molecular characterization of a family of ligands for eph-related tyrosine kinase receptors

A family of tyrosine kinase receptors related to the product of the eph gene has been described recently. One of these receptors, elk, has been shown to be expressed only in brain and testes. Using a direct expression cloning technique, a ligand for the elk receptor has been isolated by screening a human placenta cDNA library with a fusion protein containing the extracellular domain of the receptor. This isolated cDNA encodes a transmembrane protein. While the sequence of the ligand cDNA is unique, it is related to a previously described sequence known as B61. Northern blot analysis of human tissue mRNA showed that the elk ligands mRNA is 3.5 kb long and is found in placenta, heart, lung, liver, skeletal muscle, kidney and pancreas. Southern blot analysis showed that the gene is highly conserved in a wide variety of species. Both elk ligand and B61 mRNAs are inducible by tumour necrosis factor in human umbilical vein endothelial cells. In addition, both proteins show promiscuity in binding to the elk and the related hek receptors. Since these two ligand sequences are similar, and since elk and hek are members of a larger family of eph‐related receptor molecules, we refer to these ligands as LERKs (ligands for eph‐related kinases).

Molecular characterization of receptors for human interleukin-8, GRO/melanoma growth-stimulatory activity and neutrophil activating peptide-2

Interleukin-8 (IL-8), neutrophil activating peptide-2 (NAP-2), and growth regulated gene (GRO, also known as melanoma growth stimulatory activity) are members of a family of peptides which are chemotactic agents for inflammatory cells such as neutrophils. Receptors have been identified for IL-8, GRO and NAP-2 on human neutrophils and granulocytic cell lines, and it has been observed that these cytokines can cross-compete for binding to a common receptor. Using the recently characterized rabbit IL-8 receptor as a probe, two classes of cDNAs, termed type 1 and type 2, were isolated from a human neutrophil library. The type 1 receptor binds only IL-8 while the type 2 receptor binds IL-8, GRO and NAP-2 at high affinity when respective cDNAs are expressed in COS-7 cells. The two cDNAs encode proteins that have an amino acid sequence identity of 77% while the type 1 and 2 receptors have an identity of 84 and 74% with the rabbit IL-8 receptor. These receptors also show significant homology with receptors for other chemotactic agents and with potential coding regions from the human cytomegalovirus genome.

Studies of ligand-induced site-specific phosphorylation of epidermal growth factor receptor

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase involved in the regulation of growth in many animal cells, including cancer cells. Phosphorylation of specific tyrosine residues within the cytoplasmic domain of EGFR is part of the initial activation process that occurs upon ligand binding, and these phosphotyrosine residues subsequently serve as docking sites for intracellular signaling molecules. To study the phosphorylation on each individual site, EGFR generated from a human epidermoid carcinoma cell line (A431) was analyzed by mass spectrometry. Liquid chromatography combined with tandem mass spectrometry (LC/MS/MS) was used to identify the tryptic phosphopeptides and their sites of phosphorylation (Y992, Y1045, Y1068, Y1086, S1142, Y1148, and Y1173). Ion intensities for the phosphorylated and unphosphorylated tryptic peptides containing the sites of phosphorylation were measured, and the intensity ratios were used to assess the degree of phosphorylation at each site. Ligand concentrations were varied for epidermal growth factor (EGF) and transforming growth factor alpha (TGFα) as stimuli, and all of the EGFR tyrosine sites were consequently found to exhibit increased levels of phosphorylation, although at different rates and to different extents. Phosphorylation of Y992 appeared to plateau at lower concentrations of ligand, whereas the other sites continued to have increased phosphorylation throughout a wide range of concentrations. Only small differences could be detected between the EGF and the TGFα-induced EGFR phosphorylation. Pretreatment of A431 cells with a small molecule EGFR inhibitor nearly eliminated the ligand-induced phosphorylation on all of the sites except for Y992 and Y1068.

Isolation of LERK-5: A ligand of the eph-related receptor tyrosine kinases

Hek and elk are members of the eph-related family of receptor tyrosine kinases. Recently we isolated four cDNAs encoding membrane-bound ligands to hek and elk [Beckman et al. (1994) EMBO J. 13, 3757-3762; Kozlosky et al. (1995) Oncogene 10, 299-306]. Because of the promiscuous nature of their binding, we have termed these proteins ligands of the eph-related kinases or LERKs. A search of GenBank revealed an expressed sequence tag (EST) with homology to the LERKs. Using this EST as a probe, we have isolated human and murine cDNAs that encode a protein which we call LERK-5. The human and murine cDNAs encode proteins of 333 and 336 amino acids, respectively, with a 97% amino acid identity; LERK-5 has an amino acid identity of 27-59% with the other reported LERKs. LERK-5 is a ligand for both elk and hek and induces receptor phosphorylation. It is expressed in adult lung and kidney and the fetal tissues heart, lung, kidney, and brain. In addition, Southern blot analysis of DNA from interspecific backcross mice indicated that LERK-5 (Eplg5) maps to the proximal region of mouse chromosome 8.

Assignment of the genes encoding human interleukin-8 receptor types 1 and 2 and an interleukin-8 receptor pseudogene to chromosome 2q35.

Two human cDNA clones that encode different interleukin-8 (IL8) receptors have recently been isolated. The interleukin-8 receptor type 1 (IL8R1) binds IL8 only, whereas the interleukin-8 receptor type 2 (IL8R2) (previously designated IL8RA) also binds growth regulated gene (GRO), and neutrophil activating protein-2 (NAP-2) with high affinity. In the process of screening a genomic library with these cDNAs to obtain large clones for use in chromosomal localization studies, we isolated an interleukin-8 receptor pseudogene (IL8RP) that bears greatest similarity to IL8R2. Using Southern hybridization analysis of human x rodent somatic cell hybrid DNAs with cDNA probes for IL8R1 and IL8R2 and probes from the IL8RP locus, we assigned the three loci to chromosome 2; fluorescence in situ hybridization (FISH) to metaphase chromosome preparations using genomic clones from each locus refined this localization to chromosome 2, band q35, for all three. By virtue of their chromosomal location, IL8R1 and IL8R2 may be considered candidate genes for several human disorders in which the involved locus has been mapped to distal 2q or that are associated with structural abnormalities of this segment, including van der Woude syndrome and the neoplastic diseases rhabdomyosarcoma and uterine leiomyomata. In addition, because this region of chromosome 2q is homologous to proximal mouse chromosome 1 in the segment containing the Lsh-Ity-Bcg locus involved in mediating host resistance to infection with intracellular pathogens, examination for abnormalities of the murine homologues of the IL8R genes should be considered in mice affected by mutations of this locus.

Asymmetrical Fc engineering greatly enhances antibody-dependent cellular cytotoxicity (ADCC) effector function and stability of the modified antibodies.

Background: Co-crystal structure of Fc-FcγRIII complex revealed that Fc binds to FcγRIII asymmetrically. Results: We identified a panel of novel Fc heterodimers with enhanced ADCC activity. Conclusion: Asymmetrical Fc engineering is an efficient approach for enhancing ADCC activity and stability of engineered antibodies. Significance: The discovery could be applied in therapeutic antibodies for the treatment of cancers and infectious diseases. Antibody-dependent cellular cytotoxicity (ADCC) is mediated through the engagement of the Fc segment of antibodies with Fcγ receptors (FcγRs) on immune cells upon binding of tumor or viral antigen. The co-crystal structure of FcγRIII in complex with Fc revealed that Fc binds to FcγRIII asymmetrically with two Fc chains contacting separate regions of the FcγRIII by utilizing different residues. To fully explore this asymmetrical nature of the Fc-FcγR interaction, we screened more than 9,000 individual clones in Fc heterodimer format in which different mutations were introduced at the same position of two Fc chains using a high throughput competition AlphaLISA® assay. To this end, we have identified a panel of novel Fc variants with significant binding improvement to FcγRIIIA (both Phe-158 and Val-158 allotypes), increased ADCC activity in vitro, and strong tumor growth inhibition in mice xenograft human tumor models. Compared with previously identified Fc variants in conventional IgG format, Fc heterodimers with asymmetrical mutations can achieve similar or superior potency in ADCC-mediated tumor cell killing and demonstrate improved stability in the CH2 domain. Fc heterodimers also allow more selectivity toward activating FcγRIIA than inhibitory FcγRIIB. Afucosylation of Fc variants further increases the affinity of Fc to FcγRIIIA, leading to much higher ADCC activity. The discovery of these Fc variants will potentially open up new opportunities of building the next generation of therapeutic antibodies with enhanced ADCC effector function for the treatment of cancers and infectious diseases.

Molecular characterization of the interleukin-8 receptor

Recently a rabbit cDNA (F3R) was characterized as binding and causing calcium mobilization induced by the formyl-methionine-leucine-phenylalanine peptide (fMLP). In the study reported here, cloned DNAs were isolated from rabbit genomic DNA by PCR based on the sequence of F3R. The cloned DNAs have several differences in the DNA sequence compared to the reported F3R sequence that alter the predicted protein sequence. COS-7 cells transfected with these clones in a mammalian expression vector bind human IL-8 with high affinity, but do not bind fMLP. We therefore believe that the cDNAs isolated encode the rabbit IL-8 receptor.

AMG 595, an Anti-EGFRvIII Antibody–Drug Conjugate, Induces Potent Antitumor Activity against EGFRvIII-Expressing Glioblastoma

Epidermal growth factor receptor variant III (EGFRvIII) is a cancer-specific deletion mutant observed in approximately 25% to 50% of glioblastoma multiforme (GBM) patients. An antibody drug conjugate, AMG 595, composed of the maytansinoid DM1 attached to a highly selective anti-EGFRvIII antibody via a noncleavable linker, was developed to treat EGFRvIII-positive GBM patients. AMG 595 binds to the cell surface and internalizes into the endo-lysosomal pathway of EGFRvIII-expressing cells. Incubation of AMG 595 with U251 cells expressing EGFRvIII led to potent growth inhibition. AMG 595 treatment induced significant tumor mitotic arrest, as measured by phospho-histone H3, in GBM subcutaneous xenografts expressing EGFRvIII. A single intravenous injection of AMG 595 at 17 mg/kg (250 μg DM1/kg) generated complete tumor regression in the U251vIII subcutaneous xenograft model. AMG 595 mediated tumor regression in the D317 subcutaneous xenograft model that endogenously expresses EGFRvIII. Finally, AMG 595 treatment inhibited the growth of D317 xenografts orthotopically implanted into the brain as determined by magnetic resonance imaging. These results demonstrate that AMG 595 is a promising candidate to evaluate in EGFRvIII-expressing GBM patients. Mol Cancer Ther; 14(7); 1614–24. ©2015 AACR.

Tumor penetration and epidermal growth factor receptor saturation by panitumumab correlate with antitumor activity in a preclinical model of human cancer

BackgroundSuccessful treatment of solid tumors relies on the ability of drugs to penetrate into the tumor tissue.MethodsWe examined the correlation of panitumumab (an anti-epidermal growth factor [EGFR] antibody) tumor penetration and EGFR saturation, a potential obstacle in large molecule drug delivery, using pharmacokinetics, pharmacodynamics, and tumor growth rate in an A431 epidermoid carcinoma xenograft model of human cancer. To determine receptor saturation, receptor occupancy, and levels of proliferation markers, immunohistochemical and flow cytometric methods were used. Pharmacokinetic data and modeling were used to calculate growth characteristics of panitumumab-treated tumors.ResultsTreatment with panitumumab in vivo inhibited pEGFR, Ki67 and pMAPK levels vs control. Tumor penetration and receptor saturation were dose- and time-dependent, reaching 100% and 78%, respectively. Significant tumor inhibition and eradication (p < 0.05) were observed; plasma concentration associated with tumor eradication was estimated to be 0.2 μg/ml. The tumor inhibition model was able to describe the mean tumor growth and death rates.ConclusionsThese data demonstrate that the antitumor activity of panitumumab correlates with its ability to penetrate into tumor tissue, occupy and inhibit activation of EGFR, and inhibit markers of proliferation and MAPK signaling.