Targeted immunotherapy using anti-CD138-interferon α fusion proteins and bortezomib results in synergistic protection against multiple myeloma.

Although recent advances have substantially improved the management of multiple myeloma, it remains an incurable malignancy. We now demonstrate that anti-CD138 molecules genetically fused to type I interferons (IFN) synergize with the approved therapeutic bortezomib in arresting the proliferation of human multiple myeloma cell lines both in vitro and in vivo. The anti-CD138-IFNα14 fusion protein was active in inducing increased expression of signal transducer and activator of transcription 1 (STAT1) and its phosphorylation while the cell death pathway induced by bortezomib included generation of reactive oxygen species. Interferon regulatory factor 4 (IRF4), an important survival factor for myeloma cells, was down regulated following combination treatment. Induction of cell death appeared to be caspase-independent because treatment with inhibitors of caspase activation did not decrease the level of cell death. The observed caspase-independent synergistic cell death involved mitochondrial membrane depolarization, and poly(ADP-ribose) polymerase-1 (PARP-1) cleavage, and resulted in enhanced induction of apoptosis. Importantly, using 2 different in vivo xenograft models, we found that combination therapy of anti-CD138-IFNα14 and bortezomib was able to cure animals with established tumors (7 of 8 using OCI-My5 or 8 of 8 using NCI-H929). Thus, the combination of anti-CD138-IFNα with bortezomib shows great promise as a novel therapeutic approach for the treatment of multiple myeloma, a malignancy for which there are currently no cures.

Anti-CD138-targeted interferon is a potent therapeutic against multiple myeloma.

Multiple myeloma (MM), a plasma cell malignancy, is the second most prevalent hematologic malignancy in the US. Although much effort has been made trying to understand the etiology and the complexities of this disease with the hope of developing effective therapies, MM remains incurable at this time. Because of their antiproliferative and proapoptotic activities, interferons (IFNs) have been used to treat various malignancies, including MM. Although some success has been observed, the inherent toxicities of IFNs limit their efficacy. To address this problem, we produced anti-CD138 antibody fusion proteins containing either IFNα2 or a mutant IFNα2 (IFNα2(YNS)) with the goal of targeting IFN to CD138-expressing cells, thereby achieving effective IFN concentrations at the site of the tumor in the absence of toxicity. The fusion proteins inhibited the proliferation and induced apoptosis of U266, ANBL-6, NCI-H929, and MM1-144 MM cell lines. The fusion proteins decreased the expression of IFN regulatory factor 4 (IRF4) in U266. In addition, the fusion proteins were effective against primary cells from MM patients, and treatment with fusion proteins prolonged survival in the U266 murine model of MM. These studies show that IFNα antibody fusion proteins can be effective novel therapeutics for the treatment of MM.

Characterization of IgA and IgM binding and internalization by surface-expressed human Fcα/µ receptor.

The Fcα/µ receptor (Fcα/µR) is an unusual Fc receptor in that it binds to two different antibody isotypes, IgA and IgM. This receptor is of interest because it is thought to be involved in the capture of IgA- and IgM-immune complexes and antigen presentation. To further characterize this receptor, we were able to stably express human Fcα/µR on the surface of the 293T cell line. Using this system, we determined the affinity of the interactions of the receptor with IgA and IgM, which led to novel insights including the important finding that IgM polymers can bind to human Fcα/µR in the absence of J chain. This is in contrast to the polymeric immunoglobulin receptor (pIgR), which requires the presence of J chain to bind to polymeric IgA and IgM. The dissociation constants (K(d)) of all of the different human IgA isotypes and allotypes for human Fcα/µR were determined, and we show that the N-linked glycans on IgA1 are not required for binding to the receptor. In addition, we demonstrate that IgA can be rapidly internalized by human Fcα/µR in the presence of cross-linking antibody.

Differences in N-glycan structures found on recombinant IgA1 and IgA2 produced in murine myeloma and CHO cell lines.

The development and production of recombinant monoclonal antibodies is well established. Although most of these are IgGs, there is also great interest in producing recombinant IgAs since this isotype plays a critical role in providing immunologic protection at mucosal surfaces. The choice of expression system for production of recombinant antibodies is crucial because they are glycoproteins containing at least one N-linked carbohydrate. These glycans have been shown to contribute to the stability, pharmacokinetics and biologic function of antibodies. We have produced recombinant human IgA1 and all three allotypes of IgA2 in murine myeloma and CHO cell lines to systematically characterize and compare the N-linked glycans. Recombinant IgAs produced in murine myelomas differ significantly from IgA found in humans in that they contain the highly immunogenic Galalpha(1,3)Gal epitope and N-glycolylneuraminic acid residues, indicating that murine myeloma is not the optimal expression system for the production of human IgA. In contrast, IgAs produced in CHO cells contained glycans that were more similar to those found on human IgA. Expression of IgA1 and IgA2 in Lec2 and Lec8 cell lines that are defective in glycan processing resulted in a less complex pool of N-glycans. In addition, the level of sialylation of rIgAs produced in murine and CHO cells was significantly lower than that previously reported for serum IgA1. These data underscore the importance of choosing the appropriate cell line for the production of glycoproteins with therapeutic potential.

Human immunoglobulin G2 (IgG2) and IgG4, but not IgG1 or IgG3, protect mice against Cryptococcus neoformans infection.

The encapsulated yeast Cryptococcus neoformans is a significant cause of meningitis and death in patients with AIDS. Some murine monoclonal antibodies (MAbs) against the glucuronoxylomannan (GXM) component of the C. neoformans capsular polysaccharide can prolong the lives of infected mice, while others have no effect or can even shorten survival. To date, no one has systematically compared the efficacies of antibodies with the same variable regions and different human constant regions with their unique combination of effector functions in providing protection against murine C. neoformans infection. In the present study, we examined the efficacies of anti-GXM MAbs of the four human immunoglobulin G (IgG) subclasses, which have identical variable regions but differ in their capacities to bind the three types of Fc receptors for IgG (FcgammaR), their abilities to activate complement, and their half-lives. IgG2 and IgG4 anti-GXM prolonged the lives of infected BALB/c mice, IgG3 anti-GXM did not affect animal survival, while mice treated with IgG1 anti-GXM died earlier than mice treated with phosphate-buffered saline or irrelevant isotype-matched MAbs. All MAbs decreased serum GXM in infected animals. Effector pathways traditionally believed to be important in defense against microbes, such as opsonophagocytosis and complement binding, negatively correlated with antibody efficacy. It is generally accepted that human IgG1 has the most favorable combination of effector functions for therapeutic use against infections. Therefore, our findings have significant implications for humanization of the mouse IgG1 currently in clinical trials for cryptococcal meningitis and for the design of antibody therapeutics to treat other infectious diseases as well.

IgA: an immune glycoprotein.

IgA is a glycoprotein containing multiple N-linked carbohydrates as well as O-linked glycans in the case of IgA1. Because of the critical role it plays in providing protection at mucosal surfaces, IgA is an ideal candidate for use as a therapeutic or prophylactic agent. The presence or absence of carbohydrates, as well as their structure, has been found to influence effector functions and binding to specific IgA receptors. In addition, changes in IgA glycosylation are associated with immune pathology. A thorough understanding of the contributions of the glycans to IgA immune protection will aid in the design of clinically suitable antibodies.

Human IgG2 can form covalent dimers.

Unlike IgA and IgM, IgG has not yet been shown to form covalent polymers. However in the presence of specific Ag, murine IgG3 has been shown to polymerize through noncovalent interactions. In contrast to the noncovalent oligomers found with murine IgG3, we have detected covalent dimers in three different recombinant human IgG2 Abs produced in myeloma cells. Both IgG2,kappa and IgG2,lambda can form dimers. In addition, analysis of pooled human gamma globulin and several normal sera revealed the presence of IgG2 dimers. The IgG2 dimers are in contrast to the noncovalent IgG dimers found in pooled sera of multiple donors resulting from idiotype/anti-idiotype (Id/anti-Id) interactions. Cyanogen bromide cleavage analysis suggests that one or more Cys residues in the gamma 2 hinge are involved in dimer assembly. The potential role of IgG2 dimers in immunity against carbohydrate Ags is discussed.

Myeloma expression systems.

Myeloma expression systems have been utilized successfully for the production of various recombinant proteins. In particular, myeloma cell lines have been exploited to express a variety of different antibodies for diagnostic applications as well as in the treatment of various human diseases. The use of myeloma cells for antibody production is advantageous because they are professional immunoglobulin-secreting cells and are able to make proper post-translational modifications. Proper glycosylation has been shown to be important for antibody function. Advances in genetic engineering and molecular biology techniques have made it possible to isolate murine and human variable regions of almost any desired specificity. Antibodies and antibody variants produced in myeloma cells have been extremely helpful in elucidating the amino acid residues and structural motifs that contribute to antibody function. Because of their domain nature, immunoglobulin genes can be easily manipulated to produce chimeric or humanized antibodies. These antibodies are less immunogenic in humans and also retain their specificity for antigen and biologic properties. In addition, novel proteins in which antibodies are fused to non-immunoglobulin sequences as well as secretory IgA have been produced in myeloma cells.