Darren R. Jones

Antibody-antigen kinetics following immunization of rainbow trout (Oncorhynchus mykiss) with a T-cell dependent antigen.

Enhancement of the immune response through affinity maturation of the antibody response is a feature of the mammalian immune system and has important implications with respect to development of vaccination strategies. However, an absence of germinal centres and apparent lack of somatic hypermutation of immunoglobulin V genes suggests that this phenomenon does not occur in fish. We investigated the question of affinity maturation in rainbow trout (Oncorhynchus mykiss) by measuring antibody-antigen binding kinetics using a BIAcore biosensor. Following immunization with a T-cell dependent antigen (FITC-KLH), relative binding affinities of serum and mucosal antibodies were assessed based on their dissociation rate constants (k(diss.)). A detectable serum anti-FITC response developed by 4 weeks post-immunization, and a consistent shift to higher affinity antibody production (i.e. a decrease in k(diss.)) was observed over the ensuing course of the immune response. An average k(diss.) of 3.5 x 10(-4)+/-0.27 x 10(-4)sec(-1) was observed during early stages of the response (4 weeks), while by 6 weeks this decreased significantly (p<0.05). Further reduction in k(diss.) was observed, with a low of 1.2 x 10(-4)+/-0.06 x 10(-4)sec(-1) being observed by week 12. Analysis of the anti-FITC response in skin-derived mucus revealed a similar pattern of decreasing k(diss.) as the immune response progressed. While these data clearly demonstrate a 2-3 fold increase in antibody-antigen binding during the course of the immune response in trout, the magnitude of this increase is much less than that seen in the mammalian immune response. This may reflect differences in the mechanisms underpinning this phenomenon in divergent species.

The ability to interact with cell membranes suggests possible biological roles for free light chain

During antibody synthesis, immunoglobulin light chains are produced in excess of heavy chains and, as a consequence, can be secreted by plasma cells as free light chains (FLC). Thus, FLC were considered to be a by-product of immunoglobulin synthesis, lacking any biological function or relevance. However, mounting evidence suggests that FLC are bioactive molecules. For example, FLC can induce antigen specific type I hypersensitivity and inhibit viral replication in encephalomyocarditis infected mice. We have recently shown that FLC can associate with the outer membrane of certain plasma cells via interaction with saturated phosphocholine lipids such as sphingomyelin. As these lipids are highly abundant in mammalian cell membranes, we set out to determine whether FLCs can bind to membranes from a variety of cell types. We found that FLCs bind to the plasma membrane of cells from a wide range of lineages. Interestingly, the highest level of binding was to monocytes. As these cells are professional antigen presenting cells, we postulate that membrane-associated FLCs may provide a novel mechanism of antigen uptake by these cells.

Free Ig Light Chains Interact with Sphingomyelin and Are Found on the Surface of Myeloma Plasma Cells in an Aggregated Form

Free κ L chains (FκLCs) are expressed on the surface of myeloma cells and are being assessed as a therapeutic target for the treatment of multiple myeloma. Despite its clinical potential, the mechanism by which FκLCs interact with membranes remains unresolved. In this study, we show that FκLCs associate with sphingomyelin on the plasma membrane of myeloma cells. Moreover, membrane-bound FκLCs are aggregated, suggesting that aggregation is required for intercalation with membranes. Finally, we propose a model where the binding of FκLCs with sphingomyelin on secretory vesicle membranes is stabilized by self-aggregation, with aggregated FκLCs exposed on the plasma membrane after exocytosis. Although it is well known that protein aggregates bind membranes, this is only the second example of an aggregate being found on the surface of cells that also secrete the protein in its native form. We postulate that many other aggregation-prone proteins may associate with cell membranes by similar mechanisms.

Selective B cell non-responsiveness in the gut of the rainbow trout (Oncorhynchus mykiss)

In order to assess the potential for the development of economically viable and effective oral vaccines we have examined the humoral immune response resulting from the anal administration of a hapten-carrier conjugate in the posterior intestine of the rainbow trout, Oncorhynchus mykiss. Fish were administered FITC-conjugated KLH either intraperitoneally (ip) or peranally (pa). Fish immunised pa with FITC-KLH developed significant anti-FITC antibodies in the serum by week 8. However, anti-KLH antibodies were not detected in these fish indicating an apparent selective B cell non-responsiveness to KLH in the gut. Fish immunised ip with FITC-KLH developed strong antibody titres to both hapten and carrier indicating that the failure to respond to this antigen via the mucosal route is not reflected in systemic non-responsiveness to KLH. The failure of FITC-KLH to elicit an anti-KLH response in the gut was not a consequence of epitope dominance on the part of the hapten as fish receiving KLH alone via the gut were also non-responsive to this normally immunogenic protein. Finally, fish previously immunised pa with FITC-KLH or unconjugated KLH and found to be non-responsive to KLH developed anti-KLH antibodies when immunised ip with KLH 21 and 11 weeks, respectively, after first receiving pa antigen. Thus the failure to mount an anti-KLH response in these fish is not a result of the induction of systemic tolerance to the carrier. The results suggest that while the intestinal lymphoid tissue of the trout contains KLH reactive T cells, as reflected in the ability to mount a strong anti-hapten response, there is a lack of responsive KLH-specific B cells in this tissue. This may reflect either a restriction in the repertoire of gut-associated B cells compared to those in the peripheral tissues, or the induction of an anergic state in the KLH-specific population.

Formation of assemblies on cell membranes by secreted proteins: molecular studies of free λ light chain aggregates found on the surface of myeloma cells

We have described the presence of cell-membrane-associated κFLCs (free immunoglobulin light chains) on the surface of myeloma cells. Notably, the anti-κFLC mAb (monoclonal antibody) MDX-1097 is being assessed in clinical trials as a therapy for κ light chain isotype multiple myeloma. Despite the clinical potential of anti-FLC mAbs, there have been limited studies on characterizing membrane-associated FLCs at a molecular level. Furthermore, it is not known whether λFLCs can associate with cell membranes of myeloma cells. In the present paper, we describe the presence of λFLCs on the surface of myeloma cells. We found that cell-surface-associated λFLCs are bound directly to the membrane and in an aggregated form. Subsequently, membrane interaction studies revealed that λFLCs interact with saturated zwitterionic lipids such as phosphatidylcholine and phosphatidylethanolamine, and using automated docking, we characterize a potential recognition site for these lipids. Atomic force microscopy confirmed that membrane-associated λFLCs are aggregated. Given the present findings, we propose a model whereby individual FLCs show modest affinity for zwitterionic lipids, with aggregation stabilizing the interaction due to multivalency. Notably, this is the first study to image FLCs bound to phospholipids and provides important insights into the possible mechanisms of membrane association by this unique myeloma surface antigen.

Characterization of a unique conformational epitope on free immunoglobulin kappa light chains that is recognized by an antibody with therapeutic potential

The murine mAb, K-1-21, recognizes a conformational epitope expressed on free Ig kappa light chains (FκLCs) and also on cell membrane-associated FκLCs found on kappa myeloma cells. This has led to the development of a chimeric version of K-1-21, MDX-1097, which is being assessed in a Phase II clinical trial for the treatment of multiple myeloma. The epitope recognized by K-1-21 is of particular interest, especially in the context that it is not expressed on heavy chain-associated light chains such as in an intact Ig molecule. Using epitope excision techniques we have localized the K-1-21 epitope to a region spanning residues 104-110 of FκLC. This short strand of residues links the variable and constant domains, and is a flexible region that adopts different conformations in FκLC and heavy chain-associated light chain. We tested this region using site-directed mutations and found that the reactivity of K-1-21 for FκLC was markedly reduced. Finally, we applied in silico molecular docking to generate a model that satisfied the experimental data. Given the clinical potential of the Ag, this study may aid the development of next generation compounds that target the membrane form of FκLC expressed on the surface of myeloma plasma cells.

Cell membrane associated free kappa light chains are found on a subset of tonsil and in vitro-derived plasmablasts.

The monoclonal antibody, MDX-1097, is currently progressing through clinical trials as a possible therapy for multiple myeloma. MDX-1097 targets a cell membrane bound form of free immunoglobulin kappa light chain (FκLC), termed kappa myeloma antigen (KMA), which is found on the surface of malignant plasma cells. The clinical potential of MDX-1097 highlights the need to characterise the expression of its cognate antigen, KMA, in normal tissue. In this study, we have analysed the expression of KMA on B cell subsets found in tonsils, peripheral blood and bone marrow. We found KMA expression on a small population of tonsillar and in vitro derived plasmablasts. In contrast, no KMA expression was observed on peripheral blood or bone marrow resident B cell subsets. This study yields important insights into the possible subsets of B cells that might be depleted as a result of an immunotherapy targeting KMA.

MDX-1097 induces antibody-dependent cellular cytotoxicity against kappa multiple myeloma cells and its activity is augmented by lenalidomide

MDX‐1097 is an antibody specific for a unique B cell antigen called kappa myeloma antigen (KMA) that consists of cell membrane‐associated free kappa light chain (κFLC). KMA was detected on kappa human multiple myeloma cell lines (κHMCLs), on plasma cells (PCs) from kappa multiple myeloma (κMM) patients and on κPC dyscrasia tissue cryosections. In primary κMM samples, KMA was present on CD38+ cells that were CD138 and CD45 positive and/or negative. MDX‐1097 exhibited a higher affinity for KMA compared to κFLC and the latter did not abrogate binding to KMA. MDX‐1097‐mediated antibody‐dependent cellular cytotoxicity (ADCC) and in vitro exposure of target cells to the immunomodulatory drug lenalidomide resulted in increased KMA expression and ADCC. Also, in vitro exposure of peripheral blood mononuclear cells (PBMCs) to lenalidomide enhanced MDX‐1097‐mediated ADCC. PBMCs obtained from myeloma patients after lenalidomide therapy elicited significantly higher levels of MDX‐1097‐mediated ADCC than cells obtained prior to lenalidomide treatment. These data establish KMA as a relevant cell surface antigen on MM cells that can be targeted by MDX‐1097. The ADCC‐inducing capacity of MDX‐1097 and its potentiation by lenalidomide provide a powerful rationale for clinical evaluation of MDX‐1097 alone and in combination with lenalidomide.

Preclinical and clinical development of an anti-kappa free light chain mAb for multiple myeloma

Monoclonal antibodies (mAb) have had tremendous success in treating a variety of cancers over the past twenty years. Yet despite their widespread clinical use, which includes treatments for haematological malignancies, there are still no approved mAb therapies for multiple myeloma (MM). This is likely to change within the next few years with a number of mAb therapies being assessed in late stage clinical trials, most notably, the anti-CS-1 mAb, elotuzumab, and the anti-CD38 mAb, daratumumab, which are currently being evaluated in Phase III clinical trials for MM. In this review, we will discuss the preclinical and clinical development of MDX-1097, a Phase II candidate which targets cell membrane-associated kappa immunoglobulin free light chains expressed on the surface of MM cells.