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An expression vector (pJW4) for a human epidermal growth factor (hEGF)-CH1 fusion protein was constructed by fusing the gene for hEGF with the gene for CH1 of murine IgG1 with/without a peptide linker sequence [(GGGGS)3] and inserting the recombinant gene into vector pGEX2T. Expression vector pGEX2T was transfected into E. coli (BL-21) and hEGF-CH1 expressed by induction of the lac Iq promotor with 50 microM isopropyl beta-D-thiogalactopyranoside (IPTG). hEGF- CH1 fused to glutathione S-transferase (GST) was isolated and purified by affinity chromatography. GST was cleaved using thrombin. SDS-PAGE demonstrated a protein with the expected M(r) (18 kDa) positive for hEGF by Western blot. hEGF-linker-CH1 exhibited preserved binding to A431 (2-3 x 10(6) EGFR/cell) and MDA-MB-468 breast cancer cells (1-2 x 10(6) EGFR/cell). hEGF-CH1 without the linker exhibited poor receptor binding. hEGF-linker-CH1 also exhibited strong binding to soluble EGFR equivalent to that of hEGF. The tumor and normal tissue distribution of hEGF-linker-CH1 labeled with 123I was compared with 123 I-hEGF at 24 h after i.v. injection to mice implanted with s.c. MDA-MB-468 xenografts. Fusion of hEGF with CH1 increased its retention in the blood 14-fold but did not significantly increase tumor uptake. Tumor/blood ratios were higher for hEGF than for hEGF-linker-CH1. We conclude that hEGF is more attractive than hEGF-linker-CH1 for imaging EGFR-positive tumors.

Recombinant DNA technology has allowed the preparation of chimeric genes encoding proteins with novel properties. This unit describes the construction and subsequent testing of genes encoding immunoglobulin chimeras. The first protocol details fusion of a protein (or protein fragment) of interest onto an immunoglobulin constant region using a modified version of the expression vector pCDM8. The resulting fusion protein generally retains the functional properties of both the protein of interest and the immunoglobulin constant region; this can be demonstrated as described here.

Due to their multispecificity and versatility, bispecific Abs (BsAbs) are promising therapeutic tools in tomorrow's medicine. Especially intermediate-sized BsAbs that combine body retention with tissue penetration are valuable for therapy but necessitate expression systems that favor heterodimerization of the binding sites for large-scale application. To identify heterodimerization domains to which single-chain variable fragments (scFv) can be fused, we compared the efficiency of heterodimerization of CL and CH1 constant domains with complete L and Fd chains in mammalian cells. We found that the isolated CL:CH1 domain interaction was inefficient for secretion of heterodimers. However, when the complete L and Fd chains were used, secretion of L:Fd heterodimers was highly successful. Because these Fab chains contribute a binding moiety, C-terminal fusion of a scFv molecule to the L and/or Fd chains generated BsAbs or trispecific Abs (TsAbs) of intermediate size (75-100 kDa). These disulfide-stabilized bispecific Fab-scFv ("bibody") and trispecific Fab-(scFv)(2) ("tribody") heterodimers represent up to 90% of all secreted Ab fragments in the mammalian expression system and possess fully functional binding moieties. Furthermore, both molecules recruit and activate T cells in a tumor cell-dependent way, whereby the trispecific derivative can exert this activity to two different tumor cells. Thus we propose the use of the disulfide-stabilized L:Fd heterodimer as an efficient platform for production of intermediate-sized BsAbs and TsAbs in mammalian expression systems.

In the present study a novel Ab-avidin fusion protein has been constructed to deliver biotinylated compounds across the blood brain barrier. This fusion molecule consists of an Ab specific for the transferrin receptor genetically fused to avidin. The Ab-avidin fusion protein (anti-TfR IgG3-CH3-Av) expressed in murine myeloma cells was correctly assembled and secreted and showed both Ab- and avidin-related activities. In animal models, it showed much longer serum half-life than the chemical conjugate between OX-26 and avidin. Most importantly, this fusion protein demonstrated superior [3H]biotin uptake into brain parenchyma in comparison with the chemical conjugate. We also delivered a biotinylated 18-mer antisense peptide-nucleic acid specific for the rev gene of HIV-1 to the brain. Brain uptake of the HIV antisense drug was increased at least 15-fold when it was bound to the anti-TfR IgG3-CH3-Av, suggesting its potential use in neurologic AIDS. This novel Ab fusion protein should have general utility as a universal vehicle to effectively deliver biotinylated compounds across the blood-brain barrier for diagnosis and/or therapy of a broad range of CNS disorders such as infectious diseases, brain tumors as well as Parkinson's and Huntington's diseases.

The complex biantennary oligosaccharide at Asn297 of IgG is essential for some effector functions. To investigate the effect of carbohydrate structure on Ab function, we have now expressed mouse-human chimeric IgG1 Abs in Chinese hamster ovary (CHO) cells with defined defects in carbohydrate biosynthesis. We had previously shown that IgG1 Abs produced in the cell line Lec 1, which attaches a high-mannose intermediate carbohydrate, were severely deficient in complement activation, showed a slightly reduced affinity for Fc gammaRI, and had a reduced in vivo half-life. We have extended these studies by producing the same dansyl-specific IgG1 in cell lines deficient in attachment of sialic acid (Lec 2) and galactose (Lec 8). IgG1-Lec 1, IgG1-Lec 2, and IgG1-Lec 8 all showed varying reactivity with a mAb specific for an epitope in the amino terminal region of C(H)2, suggesting that the conformations of these proteins were altered by the different carbohydrate structures. Functionally, IgG1-Lec 2 and IgG1-Lec 8 were comparable to wild type with respect to in vivo half-life, affinity for Fc gammaRI, and capacity for complement-mediated hemolysis. While IgG1-Lec 2 was essentially identical to wild type in its capacity to interact with individual components of the classical complement activation pathway, IgG1-Lec 8 demonstrated equivalent maximal binding at lower concentrations and was preferentially bound by mannose-binding protein. Although IgG1-Lec 1 was deficient in activation of the classical pathway, it had a superior capacity to activate the alternative pathway. These studies demonstrate that Abs bearing C(H)2-linked carbohydrate of differing structures have different functional properties.

A recombinant gene fusion was created and cloned using a previously constructed gene encoding a monodomain IgG Fc binding protein and the gene coding for bacterial alkaline phosphatase. The construct was able to express and secrete a fusion protein that exhibited both IgG binding and alkaline phosphatase enzymatic activities. Greater than 60% of the protein demonstrating both biological activities was detected from periplasmic space preparations. Nanogram concentrations of the Fc binding--alkaline phosphatase fusion protein allowed primary IgG antibody detection without the use of conjugated secondary antibodies. Removal of the domain coding for alkaline phosphatase resulted in decreased resistance of the protein to proteolytic degradation and the loss of IgG Fc binding ability. Using affinity-purified fusion protein, the specificity of binding to IgG, IgM and IgA was examined; binding was strong to IgG and barely detectable against IgM or IgA. Affinity for binding of the fusion protein to IgG (Kd = 6.7 x 10(-8) M) was determined to be equal to or greater than previously reported for protein A.

A gene encoding a bacterial IgG Fc binding domain was designed and synthesized. The synthetic DNA fragment was cloned 3' to an inducible trpE promoter such that expression of the gene in Escherichia coli produced abundant Fc binding protein fused to the first seven amino acids of the trpE protein. The recombinant protein contained a single Fc binding domain and demonstrated efficient binding to human IgG in Western blot analysis. This protein degraded rapidly following cell lysis in the absence of protease inhibitors, but could be effectively protected by the addition of protease inhibitor. After purification of the protein by IgG affinity chromatography, IgG Fc binding ability was retained for at least 24 h at either 23 or 37 degrees C and on heating for 15 min at temperatures up to 65 degrees C. No immunoprecipitation was observed in interactions between the monodomain Fc binding protein and IgG molecules. Unlike staphylococcal protein A, no detectable binding of the monodomain IgG Fc binding protein was observed to either IgM or IgA. Truncated proteins, expressed from a series of 3' deletions of the synthetic gene, were used to estimate the minimum portion of a monodomain Fc binding protein that retained Fc binding ability.

A recombinant chimeric Fab (rcFab), with Fv derived from the monoclonal A5B7 antibody to carcinoembryonic antigen (CEA), and with human CHI and C kappa was cloned into pUC 19 and expressed in Escherichia coli. rcFab (10 to 12 mg per litre) was produced in bacterial culture fluid, and functional purified rcFab was isolated by affinity chromatography (using antibody to human C kappa) and size-exclusion gel filtration. The rcFab did not show reduced affinity for CEA, and reacted with human colorectal tumours showing a typical anti-CEA pattern by immunocytochemistry; it was also stable after iodination. Biodistribution studies in nude mice bearing human tumour xenografts showed no toxicity and good tumour localization. Therapeutic ratios at early time points were better than those obtained with whole murine antibody. The results demonstrate that bacterially produced anti-CEA Fab is of use for tumour targeting.

Genetic engineering and expression techniques have been used to produce antibody growth factor fusion proteins. Insulin-like growth factors (IGFs) 1 and 2 have been fused to mouse-human chimeric IgG3 at the end of CH1, immediately after the hinge, and at the end of CH3. Fusion heavy chains of the expected molecular weight were expressed, assembled with a co-expressed light chain, and secreted. The resulting molecules continued to bind antigen; they also bound the growth factor receptors, albeit with decreased affinity. The molecule with IGF1 attached after CH3 (CH3-IGF1) had reduced ability to carry out complement-mediated cytolysis. In contrast the molecule with IGF2 attached after CH3 (CH3-IGF2) showed an approximately 50-fold increase in its ability to effect complement-mediated cytolysis and so should be an effective cytolytic agent. Both CH3-IGF1 and CH3-IGF2 bound Fc gamma RI with affinity similar to that of IgG3. The growth factor fusion proteins showed small but significant uptake into the brain parenchyma.