Monocyte recruitment by HLA IgG-activated endothelium: the relationship between IgG subclass and FcγRIIa polymorphisms.

It is currently unclear which donor specific HLA antibodies confer the highest risk of antibody-mediated rejection (AMR) and allograft loss. In this study, we hypothesized that two distinct features (HLA IgG subclass and Fcγ receptor [FcγR] polymorphisms) which vary from patient to patient, influence the process of monocyte trafficking to and macrophage accumulation in the allograft during AMR in an interrelated fashion. Here, we investigated the contribution of human IgG subclass and FcγR polymorphisms in monocyte recruitment in vitro by primary human aortic endothelium activated with chimeric anti-HLA I human IgG1 and IgG2. Both subclasses triggered monocyte adhesion to endothelial cells, via a two-step process. First, HLA I crosslinking by antibodies stimulated upregulation of P-selectin on endothelium irrespective of IgG subclass. P-selectin-induced monocyte adhesion was enhanced by secondary interactions of IgG with FcγRs, which was highly dependent upon subclass. IgG1 was more potent than IgG2 through differential engagement of FcγRs. Monocytes homozygous for FcγRIIa-H131 adhered more readily to HLA antibody-activated endothelium compared with FcγRIIa-R131 homozygous. Finally, direct modification of HLA I antibodies with immunomodulatory enzymes EndoS and IdeS dampened recruitment by eliminating antibody-FcγR binding, an approach that may have clinical utility in reducing AMR and other forms of antibody-induced inflammation.

Antibody-cytokine fusion proteins: innovative weapons in the war against cancer.

Cancer patients who are administered therapeutic doses of cytokines (e.g., interleukin-2, granulocyte macrophage colony stimulating factor, interleukin-12, and tumor necrosis factor-alpha) frequently develop devastating toxic side effects that can lead to discontinuation of therapy. This problem has compelled numerous investigators to design innovative strategies that will reduce prolonged systemic cytokine exposure and promote cytokine accumulation at the site of the tumor. One such strategy involves the use of antibody-cytokine fusion proteins consisting of immunoenhancing cytokines genetically fused to antibodies that are able to target specific antigens exclusively expressed or overexpressed on the surface of tumor cells. Preclinical studies examining their therapeutic efficacy demonstrate that they posses potent tumoricidal activity, suggesting that they may be clinically useful as novel cancer therapeutic agents.

Hybrid IgA2/IgG1 antibodies with tailor-made effector functions.

Immunoglobulin (Ig) A and IgG are the principal immune effector molecules at mucosal surfaces and in blood, respectively. Mucosal IgA is polymeric and bound to secretory component, whereas serum IgG is monomeric. We have now produced IgA2/IgG1 hybrid antibodies that combine the properties of IgA and IgG. Antibodies with Calpha3 at the end of the IgG H chain resemble IgA and form polymers with J chain that bind the polymeric Ig receptor. Like IgG, the hybrid proteins activated complement and bound FcgammaRI and protein A. Though the hybrid proteins contained both Cgamma2 and Cgamma3, they have a short in vivo half-life. Surprisingly, this decreased half-life correlated with a higher avidity than that of IgG for murine FcRn. Interestingly, antibodies with Calpha1 replacing Cgamma1 were resistant to extremes of pH, suggesting that Calpha1 increases antibody stability. These results provide insights into engineering antibodies with novel combinations of effector functions.

Mechanism of antitumor activity of a single-chain interleukin-12 IgG3 antibody fusion protein (mscIL-12.her2.IgG3).

We have constructed an antibody interleukin-12 (IL-12) fusion protein (mscIL-12.her2.IgG3) that demonstrates significant antitumor activity against the murine carcinoma CT26-expressing human HER2/neu. We now report that this antitumor activity is dose dependent and comparable to or better than recombinant murine IL-12 (rMuIL-12) using subcutaneous and metastatic models of disease. The antitumor activity of mscIL-12.her2.IgG3 is reduced in Rag2 knockout mice, suggesting that T cells play a role in tumor rejection. In SCID-beige mice, the antitumor activity is further reduced, suggesting that natural killer (NK) cells or macrophages or both are also important. The isotype of the antibody response to HER2/neu is consistent with a switch from a Th2 to a Th1 immune response and the infiltration of mononuclear cell in tumors from mice treated with mscIL-12.her2.IgG3. Immunohistochemistry reveals that mscIL-12.her2.IgG3 is antiangiogenic. Thus, the mechanism of the antitumor activity exhibited by mscIL-12.her2.IgG3 is highly complex and involves a combination of T and NK cell activity, a switch to a Th1 immune response, and antiantiogenic activity. This is the first study comparing the in vivo antitumor activity of an antibody-IL-12 fusion protein and free IL-12. Our results suggest that antibody-IL-12 fusion proteins may be useful for the treatment of human cancer.

A recombinant IgG3-(IL-2) fusion protein for the treatment of human HER2/neu expressing tumors.

Anti-HER2/neu therapy of human HER2/neu expressing malignancies such as breast cancer has shown only partial success in clinical trials. To expand the clinical potential of this approach, we have genetically engineered an anti-HER2/neu human IgG3 fusion protein containing interleukin-2 (IL-2) fused at its carboxyl terminus. Anti-Her2/neu IgG3-(IL-2) retained antibody and cytokine related activity. Treatment of immunocompentent mice with this antibody fusion protein resulted in significant retardation in the subcutaneous (s.c.) growth of CT26-HER2/neu tumors suggesting that anti-HER2/neu IgG3-(IL-2) fusion protein will be useful in the treatment of HER2/neu expressing tumors. We also found that fusing IL-2 to human IgG3 results in a significant enhancement of the murine anti-human antibody (MAHA) response.

Targeting of functional antibody-decay-accelerating factor fusion proteins to a cell surface.

Recombinant soluble complement inhibitors hold promise for the treatment of inflammatory disease and disease states associated with transplantation. Targeting complement inhibitors to the site of complement activation and disease may enhance their efficacy and safety. Data presented show that targeting of decay-accelerating factor (DAF, an inhibitor of complement activation) to a cell surface by means of antibody fragments is feasible and that cell-targeted DAF provides significantly enhanced protection from complement deposition and lysis compared with soluble untargeted DAF. An extracellular region of DAF was joined to an antibody combining site with specificity for the hapten dansyl, at the end of either C(H)1 or C(H)3 Ig regions. The recombinant IgG-DAF chimeric proteins retained antigen specificity and bound to dansylated Chinese hamster ovary cells. Both soluble C(H)1-DAF and C(H)3-DAF were effective at inhibiting complement-mediated lysis of untargeted Chinese hamster ovary cells at molar concentrations within the range reported by others for soluble DAF. However, when targeted to a dansyl-labeled cell membrane, C(H)1-DAF was significantly more potent at inhibiting complement deposition and complement-mediated lysis. Cell-bound C(H)1-DAF also provided cells with protection from complement lysis after removal of unbound C(H)1-DAF. Of further importance, the insertion of a nonfunctional protein domain of DAF (the N-terminal short consensus repeat) between C(H)1 and the functional DAF domain increased activity of the fusion protein. In contrast to C(H)1-DAF, C(H)3-DAF was not significantly better at protecting targeted versus untargeted cells from complement, indicating that a small targeting vehicle is preferable to a large one. We have previously shown that for effective functioning of soluble complement inhibitor CD59, binding of CD59 to the cell surface close to the site of complement activation is required. Significantly, such a constraint did not apply for effective DAF function.

A murine B cell lymphoma expressing human HER2 / neu undergoes spontaneous tumor regression and elicits antitumor immunity.

In the present study we describe a novel murine tumor model in which the highly malignant murine B cell lymphoma 38C13 has been transduced with the cDNA encoding human tumor-associated antigen HER2/neu. This new cell line (38C13-HER2/neu) showed stable surfiace expression but not secretion of human HER2/neu. It also maintained expression of the idiotype (Id) of the surface immunoglobulin of 38C13, which serves as another tumor-associated antigen. Surprisingly, spontaneous tumor regression was observed following s.c. but not i.v. injection of 38C13-HER2/neu cells in immunocompetent syngeneic mice. Regression was more frequently observed with larger tumor cell challenges and was mediated through immunological mechanisms because it was not observed in syngeneic immunodeficient mice. Mice that showed complete tumor regression were immune to challenge with the parental cell line 38C13 and V1, a variant of 38C13 that does not express the Id. Immunity could be transferred with sera, suggesting that an antibody response mediated rejection and immunity. Continuously growing s.c. tumors as well as metastatic tumors obtained after the i.v. injection of 38C13-HER2/neu maintained expression of human HER2/neu, which can serve as a target for active immunotherapy. As spontaneous tumor regression has not been observed in other human murine models expressing human HER2/neu, our results illustrate the enormous differences that can exist among different murine tumors expressing the same antigen. The present model provides a useful tool for the study of the mechanisms of protective immunity to B cell lymphoma and for the evaluation of different therapeutic approaches based on the stimulation or suppression of the immune response.

Antibody-cytokine fusion proteins for the therapy of cancer.

Advances in genetic engineering and expression systems have led to rapid progress in the development of antibodies fused to other proteins. These 'antibody fusion proteins' have novel properties and include antibodies with specificity for tumor associated antigens fused to cytokines such as interleukin-2 (IL2), granulocyte/macrophage colony-stimulating factor (GM-CSF), and interleukin-12 (IL12). The goal of this approach to cancer therapy is to concentrate the cytokine in the tumor microenvironment and in so doing directly enhance the tumoricidal effect of the antibody and/or enhance the host immune response (T-cell, B-cell or NK) against the tumor. In the past decade, multiple antibody-cytokine fusion proteins have been developed with different specificities targeting a broad variety of tumors. These novel molecules retain both antibody and cytokine associated functions. In addition, in animals bearing tumors, antibody-cytokine fusion proteins are able to target the tumor and to elicit a significant anti-tumor response that in some cases results in a complete elimination of the tumor. These results suggest that antibody-cytokine fusion proteins have potential for use in the treatment of human cancer. In the present review, we describe strategies for construction of antibody-cytokine fusion proteins and discuss the properties of several antibody-cytokine fusion proteins with IgG genetically fused to the cytokines IL2, GM-CSF or IL12.

Preparing frozen bacterial stocks.

After bacteria containing plasmids of interest have been produced, it is frequently convenient to store them for later use. This appendix provides instruction for storing bacteria in such a way that they typically remain viable for years.

Transformation of E. coli by electroporation.

Using electroporation to transform Escherichia coli results in transformation efficiencies greater than can be obtained using the best chemical methods. It is easy to obtain transformation efficiencies 10(8) per milligram DNA and efficiencies of 10(10) have been reported. This appendix describes a procedure for electroporation that can be used to transform many different types of bacteria.

Transfection of lymphoid cells.

Using available vectors, a single immunoglobulin chain or both heavy (H) and light (L) chains can be simultaneously transferred to recipient myeloma cells. Several methods can be used to prepare stable transfected lymphoid cells. The Basic Protocol in this unit describes protoplast fusion, which is suitable for certain lymphoid cells but tends to be more cumbersome than electroporation or liposome-mediated transfection, which are described in the two alternate protocols. All three techniques may be adapted for stable transfection of other eukaryotic cell lines.

Treatment of femoral artery pseudoaneurysms with percutaneous thrombin injection.

The objective of this study was to prospectively evaluate the efficacy of ultrasound-guided thrombin injection for the treatment of post-catheterization femoral artery pseudoaneurysms. Between August 1, 1998 and August 31, 1999, 38 patients underwent ultrasound-guided injection of thrombin into 39 femoral false aneurysms. Peripheral pulses and ankle/brachial indices were assessed before and after the injection. Patients were followed with a control duplex scan within 4 weeks. The good results from this study showed that ultrasound-guided thrombin injection is an effective method for the treatment of post-catheterization false aneurysms. In a minority of patients, signs consistent with arterial embolization or vasospasm were identified.

Chondrogenesis of the branchial skeleton in embryonic sea lamprey, Petromyzon marinus.

This study provides concise temporal and spatial characteristics of branchial chondrogenesis in embryonic sea lamprey, Petromyzon marinus, using high resolution light microscopy, transmission electron, and immunoelectron microscopy. Prechondrogenic condensations representing the first branchial arch appeared first in the mid-region of the third pharyngeal arch at 13 days post-fertilization (pf). Cartilage differentiation, defined by the presence of the unique, fibrillar, non-collagenous matrix protein characteristic of branchial cartilage, was first observed at 14 days pf. Development of lamprey branchial cartilage appeared unusual compared to that in jawed fishes, in that precartilage condensations appear as a one-cell wide orderly stack of flattened cells that extend by the addition of one dorsal and one ventral condensation. Development of lamprey gill arches from three condensations that fuse to form a single skeletal element differs from the developing gill arches of jawed fishes, where more than one skeletal element forms from a single condensation. The initial orderly arrangement of cells in the lamprey branchial prechondrogenic condensations remains throughout development. Once chondrification of the condensations begins, the branchial arches start to grow. Initially, growth occurs as a result of matrix secretion and cell migration. Later in development, the arches grow mainly by cell proliferation and enlargement. This study defines the morphology and timing of lamprey branchial chondrogenesis. Studies of lamprey chondrogenesis provide not only insight into the developmental biology of a unique non-collagenous cartilage in a primitive vertebrate but also into the general evolution of the skeletal system in vertebrates.

Site-specific and directional gene replacement mediated by Cre recombinase.

A novel method for the site-specific introduction of genes into eukaryotic cells using the prokaryotic Cre-LoxP recombination system is presented. Cre recombinase catalyzes recombination between two LoxP sites or between two mutant LoxP 511 sites. However, recombination is not catalyzed between a LoxP and a LoxP 511 site. We now demonstrate that it is possible to catalyze accurate exchange between two DNA segments each flanked by a LoxP and a LoxP 511 site. In the example presented, expression of the Cre recombinase resulted in the replacement of a murine IgA constant region gene with a LoxP site at the 5' end and a LoxP 511 site at the 3' end by a human IgA constant region gene flanked by the same wild type and mutant LoxP sites. This method provides a novel approach for the site-specific substitution of specific genes.

Recombinant anti-human HER2/neu IgG3-(GM-CSF) fusion protein retains antigen specificity and cytokine function and demonstrates antitumor activity.

Anti-HER2/neu therapy of human HER2/neu-expressing malignancies such as breast cancer has shown only partial success in clinical trials. To expand the clinical potential of this approach, we have genetically engineered an anti-HER2/neu IgG3 fusion protein containing GM-CSF. Anti-HER2/neu IgG3-(GM-CSF) expressed in myeloma cells was correctly assembled and secreted. It was able to target HER2/neu-expressing cells and to support growth of a GM-CSF-dependent murine myeloid cell line, FDC-P1. The Ab fusion protein activated J774.2 macrophage cells so that they exhibit an enhanced cytotoxic activity and was comparable to the parental Ab in its ability to effect Ab-dependent cellular cytotoxicity-mediated tumor cell lysis. Pharmacokinetic studies showed that anti-HER2/neu IgG3-(GM-CSF) is stable in the blood. Interestingly, the half-life of anti-HER2/neu IgG3-(GM-CSF) depended on the injected dose with longer in vivo persistence observed at higher doses. Biodistribution studies showed that anti-HER2/neu IgG3-(GM-CSF) is mainly localized in the spleen. In addition, anti-HER2/neu IgG3-(GM-CSF) was able to target the HER2/neu-expressing murine tumor CT26-HER2/neu and enhance the immune response against the targeted Ag HER2/neu. Anti-HER2/neu IgG3-(GM-CSF) is able to enhance both Th1- and Th2-mediated immune responses and treatment with this Ab fusion protein resulted in significant retardation in the growth of s.c. CT26-HER2/neu tumors. Our results suggest that anti-HER2/neu IgG3-(GM-CSF) fusion protein is useful in the treatment of HER2/neu-expressing tumors.

The novel human IgE epsilon heavy chain, epsilon tailpiece, is present in plasma as part of a covalent complex.

Several splice variants of the secreted human epsilon heavy chain have previously been identified by reverse transcription-PCR. The heavy chain of one isoform, IgE tailpiece, differs from the originally identified IgE, IgE classic, by the replacement of the 2 carboxy-terminal amino acids by 8 novel amino acids including a carboxy-terminal cysteine residue. Recombinant human epsilon tailpiece and epsilon classic heavy chains were expressed and secreted as H2L2 monomers in Sp2/0 murine myeloma cells. We have investigated the in vitro function and in vivo occurrence of epsilon tailpiece heavy chains using receptor binding assays, granule release assays, flow cytometry, half-life studies, immunoprecipitation, SDS-PAGE, two-dimensional SDS-PAGE, and Western blotting. IgE tailpiece and IgE classic exhibited similar in vivo half-lives in BALB/c mice, bound the human high- and low-affinity IgE receptors with similar affinities and triggered equivalent levels of high affinity IgE receptor induced degranulation. In humans, IgE classic is present as a 190 kD circulating protein in vivo. In contrast, we found that in plasma epsilon tailpiece was primarily present as part of covalent complexes of approximately 300 and 338 kD. Dissociation of the complexes revealed that two species of epsilon tailpiece heavy chains were present therein and surprisingly, these in vivo derived epsilon tailpiece heavy chains were approximately 5 and 10 kD smaller than the recombinant expressed epsilon tailpiece or epsilon classic heavy chains. These results show that epsilon tailpiece is present in novel covalent complexes in humans.

The N-glycans determine the differential blood clearance and hepatic uptake of human immunoglobulin (Ig)A1 and IgA2 isotypes.

Human immunoglobulin (Ig)A exists in blood as two isotypes, IgA1 and IgA2, with IgA2 present as three allotypes: IgA2m(1), IgA2m(2), and IgA2m(n). We now demonstrate that recombinant, chimeric IgA1 and IgA2 differ in their pharmacokinetic properties. The major pathway for the clearance of all IgA2 allotypes is the liver. Liver-mediated uptake is through the asialoglycoprotein receptor (ASGR), since clearance can be blocked by injection of excess galactose-Ficoll ligand and suppressed in ASGR-deficient mice. In contrast, only a small percentage of IgA1 is cleared through this pathway. The clearance of IgA1 lacking the hinge region with its associated O-linked carbohydrate was more rapid than that of wild-type IgA1. IgA1 and IgA2 that are not rapidly eliminated by the ASGR are both removed through an undefined ASGR-independent pathway with half-lives of 14 and 10 h, respectively. The rapid clearance of IgA2 but not IgA1 through the liver may in part explain why the serum levels of IgA1 are greater than those of IgA2. In addition, dysfunction of the ASGR or altered N-linked glycosylation, but not O-glycans, that affects recognition by this receptor may account for the elevated serum IgA seen in liver disease and IgA nephropathy.

Transport across the primate blood-brain barrier of a genetically engineered chimeric monoclonal antibody to the human insulin receptor.

PURPOSE: Brain drug targeting may be achieved by conjugating drugs, that normally do not cross the blood-brain barrier (BBB), to brain drug delivery vectors. The murine 83-14 MAb to the human insulin receptor (HIR) is a potential brain drug targeting vector that could be used in humans, if this MAb was genetically engineered to form a chimeric antibody. where most of the immunogenic murine sequences are replaced by human antibody sequence. METHODS: The present studies describe the production of the gene for the chimeric HIRMAb, expression and characterization of the protein, radiolabeling of the chimeric HIRMAb with 111-indium and 125-iodine, and quantitative autoradiography of living primate brain taken 2 hours after intravenous administration of the [111In]chimeric HIRMAb. RESULTS: The chimeric HIRMAb had identical affinity to the target antigen as the murine HIRMAb based on Western blotting and immunoradiometric assay using partially purified HIR affinity purified from serum free conditioned media produced by a CHO cell line secreting soluble HIR. The [125I]chimeric HIRMAb was avidly bound to isolated human brain capillaries, and this binding was blocked by the murine HIRMAb. The [111In]chimeric HIRMAb was administered intravenously to an anesthetized Rhesus monkey, and the 2 hour brain scan showed robust uptake of the chimeric antibody by the living primate brain. CONCLUSIONS: A genetically engineered chimeric HIRMAb has been produced, and the chimeric antibody has identical reactivity to the human and primate BBB HIR as the original murine antibody. This chimeric HIRMAb may be used in humans for drug targeting through the BBB of neurodiagnostic or neurotherapeutic drugs that normally do not cross the BBB.

Removal of amphipathic epitopes from genetically engineered antibodies: production of modified immunoglobulins with reduced immunogenicity.

Several approaches have been developed to reduce the human immune response to nonhuman antibodies. However, chimeric antibodies and humanized antibodies often have decreased binding affinity. We described a new approach for reducing the immunogenicity of chimeric antibodies while maintaining the affinity. This approach seeks to prevent the recognition of murine immunogenic peptides from the antibody variable region by human lymphocytes. Putative immunogenic epitopes in the variable region are identified and subjected to site directed mutagenesis to make them human and/or to break the amphipathic motifs. The R3 antibody, which blocks the epidermal growth factor (EGF) receptor, was used as a model system to test this approach. Four segments containing possible amphipathic epitopes were found in the heavy variable domain using the program AMPHI. Six amino acids within two of these segments were substituted by the corresponding residues from a homologous human sequence. No mutations were made in the murine light variable domain. Experiments in monkeys suggested that the "detope" R3 antibody was less immunogenic than its chimeric analogue. A search for possible amphipathic epitopes in the Kabat database revealed the presence of conserved patterns in the different families of variable region sequences, suggesting that the proposed method may be of general applicability.