Humanization of SLCO2B1 in Rats Increases rCYP3A1 Protein Expression but Not the Metabolism of Erlotinib to OSI-420.

The organic anion transporting polypeptide (OATP)2B1 [(gene: solute carrier organic anion transporter family member 2B1 (SLCO2B1)] is an uptake transporter that facilitates cellular accumulation of its substrates. Comparison of SLCO2B1+/+ knockin and rSlco2b1-/- knockout rats showed a higher expression of rCYP3A1 in the humanized animals. We hypothesize that humanization of OATP2B1 not only affects cellular uptake but also metabolic activity. To further investigate this hypothesis, we used SLCO2B1+/+ and rSlco2b1-/ - rats and the OATP2B1 and rCYP3A1 substrate erlotinib, which is metabolized to OSI-420, for in vivo and ex vivo experiments. One hour after administration of a single dose of erlotinib, the knockin rats exhibited significantly lower erlotinib serum levels, but no change was observed in metabolite concentration or the OSI-420/erlotinib ratio. Similar results were obtained for liver tissue levels comparing SLCO2B1+/+ and rSlco2b1-/- rats. Liver microsomes isolated from the erlotinib-treated animals were characterized ex vivo for rCYP3A activity using testosterone, showing higher activity in the knockin rats. The contrary was observed when microsomes isolated from treatment-naïve animals were assessed for the metabolism of erlotinib to OSI-420. The latter is in contrast to the higher rCYP3A1 protein amount observed by western blot analysis in rat liver lysates and liver microsomes isolated from untreated rats. In summary, rats humanized for OATP2B1 showed higher expression of rCYP3A1 in liver and reduced serum levels of erlotinib but no change in the OSI-420/erlotinib ratio despite a lower OSI-420 formation in isolated liver microsomes. Studies with CYP3A-specific substrates are warranted to evaluate whether humanization affects not only rCYP3A1 expression but also metabolic activity in vivo. SIGNIFICANCE STATEMENT: Humanization of rats for the organic anion transporting polypeptide (OATP)2B1 increases rCYP3A1 expression and activity in liver. Using the OATP2B1/CYP3A-substrate erlotinib to assess the resulting phenotype, we observed lower erlotinib serum and liver concentrations but no impact on the liver/serum ratio. Moreover, there was no difference in the OSI-420/erlotinib ratio comparing humanized and knockout rats, suggesting that OSI-420 is not applicable to monitor differences in rCYP3A1 expression as supported by data from ex vivo experiments with rat liver microsomes.

Tetra- and Hexavalent Siglec-8 Ligands Modulate Immune Cell Activation.

Carbohydrate-binding proteins are generally characterized by poor affinities for their natural glycan ligands, predominantly due to the shallow and solvent-exposed binding sites. To overcome this drawback, nature has exploited multivalency to strengthen the binding by establishing multiple interactions simultaneously. The development of oligovalent structures frequently proved to be successful, not only for proteins with multiple binding sites, but also for proteins that possess a single recognition domain. Herein we present the syntheses of a number of oligovalent ligands for Siglec-8, a monomeric I-type lectin found on eosinophils and mast cells, alongside the thermodynamic characterization of their binding. While the enthalpic contribution of each binding epitope was within a narrow range to that of the monomeric ligand, the entropy penalty increased steadily with growing valency. Additionally, we observed a successful agonistic binding of the tetra- and hexavalent and, to an even larger extent, multivalent ligands to Siglec-8 on immune cells and modulation of immune cell activation. Thus, triggering a biological effect is not restricted to multivalent ligands but could be induced by low oligovalent ligands as well, whereas a monovalent ligand, despite binding with similar affinity, showed an antagonistic effect.

Simultaneous quantification of atorvastatin, erlotinib and OSI-420 in rat serum and liver microsomes using a novel liquid chromatography-mass spectrometry method.

Erlotinib is an epidermal growth factor receptor tyrosine kinase inhibitor used in the treatment of cancer. Atorvastatin is a statin commonly applied to treat hypercholesterolemia. In humans, both compounds are metabolized by CYP3A4 and are transported by OATP2B1, ABCB1 and ABCG2. We aimed to generate and validate a bioanalytical method for simultaneous determination of atorvastatin, erlotinib and its major metabolite OSI-420 applicable to biological samples. Quantification of erlotinib, OSI-420, and atorvastatin was achieved with an Agilent high-performance liquid chromatography system 1100/1200 coupled to a triple quadrupole G6410B. The method involved separation over the column Kinetex C8 (100 × 3 mm, 2.6 µm) using 2 mM ammonium acetate (pH 4.0) and acetonitrile as eluent. The method was assessed for selectivity, accuracy, recovery, matrix effect, and stability over a range from 1 to 4,000 ng/mL according to the respective guidelines. We applied the bioanalytical method to quantify the formation of OSI-420 in liver microsomes isolated from male and female Wistar rats. The optimized experiment revealed slower formation in microsomes of female compared to male rats, in which we observed lower amounts of CYP3A1 by Western blot analysis. Moreover, the presence of atorvastatin inhibited the CYP3A-mediated metabolism of erlotinib. Serum obtained from a drug-drug interaction study performed in male rats was also analyzed using the validated method. Non-compartmental pharmacokinetic analysis revealed a lower clearance of erlotinib when atorvastatin was co-administered. However, for atorvastatin we observed a lower systemic exposure in presence of erlotinib. In summary, we report a method to detect OSI-420, erlotinib and atorvastatin applicable to samples from ex vivo and in vivo studies.

Complement inhibition can decrease the haemostatic response in a microvascular bleeding model at multiple levels.

Background: Haemostasis is a crucial process by which the body stops bleeding. It is achieved by the formation of a platelet plug, which is strengthened by formation of a fibrin mesh mediated by the coagulation cascade. In proinflammatory and prothrombotic conditions, multiple interactions of the complement system and the coagulation cascade are known to aggravate thromboinflammatory processes and increase the risk of arterial and venous thrombosis. Whether those interactions also play a relevant role during the physiological process of haemostasis is not yet completely understood. The aim of this study was to investigate the potential role of complement components and activation during the haemostatic response to mechanical vessel injury. Methods: We used a microvascular bleeding model that simulates a blood vessel, featuring human endothelial cells, perfusion with fresh human whole blood, and an inducible mechanical injury to the vessel. We studied the effects of complement inhibitors against components of the lectin (MASP-1, MASP-2), classical (C1s), alternative (FD) and common pathways (C3, C5), as well as a novel triple fusion inhibitor of all three complement pathways (TriFu). Effects on clot formation were analysed by recording of fibrin deposition and the platelet activation marker CD62P at the injury site in real time using a confocal microscope. Results: With the inhibitors targeting MASP-2 or C1s, no significant reduction of fibrin formation was observed, while platelet activation was significantly reduced in the presence of the FD inhibitor. Both common pathway inhibitors targeting C3 or C5, respectively, were associated with a substantial reduction of fibrin formation, and platelet activation was also reduced in the presence of the C3 inhibitor. Triple inhibition of all three activation pathways at the C3-convertase level by TriFu reduced both fibrin formation and platelet activation. When several complement inhibitors were directly compared in two individual donors, TriFu and the inhibitors of MASP-1 and C3 had the strongest effects on clot formation. Conclusion: The observed impact of complement inhibition on reducing fibrin clot formation and platelet activation suggests a role of the complement system in haemostasis, with modulators of complement initiation, amplification or effector functions showing distinct profiles. While the interactions between complement and coagulation might have evolved to support haemostasis and protect against bleeding in case of vessel injury, they can turn harmful in pathological conditions when aggravating thromboinflammation and promoting thrombosis.

Optimized synthesis, polymer conjugation, and proof-of-concept studies of the gd-IgA1 epitope for antibody-scavenging therapies in IgA nephropathy.

IgA nephropathy (IgAN) is the most common glomerular autoimmune disease and has severe long-term consequences for patients, with 40% of the patients eventually progressing to end-stage renal disease. Despite the severity, no causal treatment is currently available. While the pathogenesis of IgAN is complex, disease severity is linked to autoantibodies against the gd-IgA1 epitope, a stretch in the hinge region of IgA1 that lacks O-glycans and is found in the characteristic immune complexes deposited in the kidneys of IgAN patients. One elegant, causal approach would be to remove the anti-gd-IgA1 autoantibodies and consequently reduce the immune complex burden on the kidneys. The administration of synthetic polymers that present autoantigens in a multivalent manner have been established as promising therapeutic strategies in other autoimmune diseases and may be applied to IgAN. We here present an improved protocol for the synthesis of the gd-IgA1 epitope, its successful coupling to a poly-L-lysine polymer and proof-of-concept experiments that the polymer-bound synthetic glycopeptide is able to capture the IgAN autoantibodies, making this approach a promising way forward for developing a targeted treatment option for IgAN patients.

Complement-targeted therapeutics: An emerging field enabled by academic drug discovery.

Within a short few years, the number of complement inhibitors that are either approved for therapeutic application or evaluated in late-stage clinical trials has expanded remarkably. The sudden emergence of this target area in the pipelines of many biotech start-ups and even large pharmaceutical companies appears even more surprising when considering that the involvement of the complement system in various clinical conditions had long been recognized. In many aspects, however, the complement system is far from being a traditional drug target, which may explain the delayed breakthrough of this therapeutic strategy. While complement modulation is now considered an attractive "platform technology" with applications in a wide spectrum of disorders, the broad yet heterogeneous disease involvement of the complement system has long restricted its placement in traditional drug discovery programs. Concerns about the safety of complement-targeted interventions, the large number and high plasma concentrations of target proteins, and the complexity of the complement system's engagement in biological processes are among other factors that kept complement off the drug discovery radar for decades. Alongside technical advances and financial incentives, the innovation and persistence of academic and clinical researchers have been the critical driving force to navigate complement therapeutics out of the shadow into the spotlight. In this commentary, we document this remarkable development using select examples and aim to venture some predictions where this promising field may be headed to.

Complement-regulatory biomaterial coatings: Activity and selectivity profile of the factor H-binding peptide 5C6.

The use of biomaterials in modern medicine has enabled advanced drug delivery strategies and led to reduced morbidity and mortality in a variety of interventions such as transplantation or hemodialysis. However, immune-mediated reactions still present a serious complication of these applications. One of the drivers of such reactions is the complement system, a central part of humoral innate immunity that acts as a first-in-line defense system in its own right but also coordinates other host defense responses. A major regulator of the complement system is the abundant plasma protein factor H (FH), which impairs the amplification of complement responses. Previously, we could show that it is possible to recruit FH to biomedical surfaces using the phage display-derived cyclic peptide 5C6 and, consequently, reduce deposition of C3b, an activation product of the complement system. However, the optimal orientation of 5C6 on surfaces, structural determinants within the peptide for the binding, and the exact binding region on FH remained unknown. Here, we show that the cyclic core and C-terminal region of 5C6 are essential for its interaction with FH and that coating through its N-terminus strongly increases FH recruitment and reduces C3-mediated opsonization in a microparticle-based assay. Furthermore, we could demonstrate that 5C6 selectively binds to FH but not to related proteins. The observation that 5C6 also binds murine FH raises the potential for translational evaluation in animal models. This work provides important insight for the future development of 5C6 as a probe or therapeutic entity to reduce complement activation on biomaterials. STATEMENT OF SIGNIFICANCE: Biomaterials have evolved into core technologies critical to biomedical and drug delivery applications alike, yet their safe and efficient use may be adversely impacted by immune responses to the foreign materials. Taking inspiration from microbial immune evasion strategies, our group developed a peptide-based surface coating that recruits factor H (FH), a host regulator of the complement system, from plasma to the material surface and prevents unwanted activation of this innate immunity pathway. In this study, we identified the molecular determinants that define the interaction between FH and the coated peptide, developed tethering strategies with largely enhanced binding capacity and provided important insight into the target selectivity and species specificity of the FH-binding peptide, thereby paving the way for preclinical development steps.

Insight into mode-of-action and structural determinants of the compstatin family of clinical complement inhibitors.

With the addition of the compstatin-based complement C3 inhibitor pegcetacoplan, another class of complement targeted therapeutics have recently been approved. Moreover, compstatin derivatives with enhanced pharmacodynamic and pharmacokinetic profiles are in clinical development (e.g., Cp40/AMY-101). Despite this progress, the target binding and inhibitory modes of the compstatin family remain incompletely described. Here, we present the crystal structure of Cp40 complexed with its target C3b at 2.0-Å resolution. Structure-activity-relationship studies rationalize the picomolar affinity and long target residence achieved by lead optimization, and reveal a role for structural water in inhibitor binding. We provide explanations for the narrow species specificity of this drug class and demonstrate distinct target selection modes between clinical compstatin derivatives. Functional studies provide further insight into physiological complement activation and corroborate the mechanism of its compstatin-mediated inhibition. Our study may thereby guide the application of existing and development of next-generation compstatin analogs.

Exploring the function of factor XIII free B subunit: Interactions with complement factors and a novel approach to identify potential binding partners.

Background: The factor XIII (FXIII)-B subunit has a critical function as a carrier protein to stabilize FXIII-A in plasma and supply it to its main substrate, fibrinogen. However, the function of the excess free FXIII-B circulating in plasma is still elusive. Objectives: In the present study, we explored potential interactions of free FXIII-B with complement factors and searched for novel binding partners. Methods: We tested for cofactor activity in the degradation of complement C3b and C4b and used ELISA- and surface plasmon resonance-based binding assays to investigate interactions between FXIII-B and complement components. We performed immunoprecipitation and mass spectrometry analysis to identify potential binding partners of free FXIII-B in freshly drawn plasma samples. Results: FXIII-B did not exhibit cofactor activity in the degradation of C3b and C4b similar to factor H and C4b-binding protein, nor did it bind to complement factors to a relevant extent. Identification of proteins potentially binding to free FXIII-B revealed high interindividual variation. We confirmed α2-macroglobulin (α2MG) as a candidate, although direct interactions or functional effects remain to be validated. Conclusions: Our study reveals that free FXIII-B has no direct role in regulating the complement system, despite a structural similarity to major complement regulators. Further studies are needed to validate α2MG as a binding partner and explore potential functional consequences of this binding.

Compstatins: the dawn of clinical C3-targeted complement inhibition.

Despite the growing recognition of the complement system as a major contributor to a variety of clinical conditions, the therapeutic arsenal has remained scarce. The introduction of an anti-C5 antibody in 2007 raised confidence in complement-targeted therapy. However, it became apparent that inhibition of late-stage effector generation might not be sufficient in multifactorial complement disorders. Upstream intervention at the level of C3 activation has therefore been considered promising. The approval of pegcetacoplan, a C3 inhibitor of the compstatin family, in 2021 served as critical validation of C3-targeted treatment. This review delineates the evolution of the compstatin family from its academic origins to the clinic and highlights current and potential future applications of this promising drug class in complement diseases.

From discovery to approval: A brief history of the compstatin family of complement C3 inhibitors.

The FDA approval of pegcetacoplan (Empaveli), a PEGylated compstatin-based C3 therapeutic, as a new treatment for paroxysmal nocturnal hemoglobinuria (PNH) marks a milestone in the history of complement drug discovery. Almost 15 years after the approval of the first complement-specific drug for PNH, the anti-C5 antibody eculizumab, a novel class of complement inhibitors with a distinct mechanism of action finally enters the clinic. This landmark decision broadens the spectrum of available complement therapeutics, offering patients with unmet clinical needs or insufficient responses to anti-C5 therapy an alternative treatment option with a broad activity profile. Here we present a brief historical account of this newly approved complement drug, consolidating its approval within the long research record of the compstatin family of peptidic C3 inhibitors.

Selective Monovalent Galectin-8 Ligands Based on 3-Lactoylgalactoside.

Galectin-8 has gained attention as a potential new pharmacological target for the treatment of various diseases, including cancer, inflammation, and disorders associated with bone mass reduction. To that end, new molecular probes are needed in order to better understand its role and its functions. Herein we aimed to improve the affinity and target selectivity of a recently published galectin-8 ligand, 3-O-[1-carboxyethyl]-ß-d-galactopyranoside, by introducing modifications at positions 1 and 3 of the galactose. Affinity data measured by fluorescence polarization show that the most potent compound reached a KD of 12 µM. Furthermore, reasonable selectivity versus other galectins was achieved, making the highlighted compound a promising lead for the development of new selective and potent ligands for galectin-8 as molecular probes to examine the protein's role in cell-based and in vivo studies.

Tipping the balance: intricate roles of the complement system in disease and therapy.

The ability of the complement system to rapidly and broadly react to microbial intruders, apoptotic cells and other threats by inducing forceful elimination responses is indispensable for its role as host defense and surveillance system. However, the danger sensing versatility of complement may come at a steep price for patients suffering from various immune, inflammatory, age-related, or biomaterial-induced conditions. Misguided recognition of cell debris or transplants, excessive activation by microbial or damaged host cells, autoimmune events, and dysregulation of the complement response may all induce effector functions that damage rather than protect host tissue. Although complement has long been associated with disease, the prevalence, impact and complexity of complement's involvement in pathological processes is only now becoming fully recognized. While complement rarely constitutes the sole driver of disease, it acts as initiator, contributor, and/or exacerbator in numerous disorders. Identifying the factors that tip complement's balance from protective to damaging effects in a particular disease continues to prove challenging. Fortunately, however, molecular insight into complement functions, improved disease models, and growing clinical experience has led to a greatly improved understanding of complement's pathological side. The identification of novel complement-mediated indications and the clinical availability of the first therapeutic complement inhibitors has also sparked a renewed interest in developing complement-targeted drugs, which meanwhile led to new approvals and promising candidates in late-stage evaluation. More than a century after its description, complement now has truly reached the clinic and the recent developments hold great promise for diagnosis and therapy alike.

Therapeutic Peptides as Emerging Options to Restore Misguided Host Defence and Homeostasis: From Teaching to Concept to Clinic.

Among the many molecular entities suitable for therapeutic use, peptides have emerged as a particularly attractive option for academic drug discovery and development. Their modular structure and extendibility, the availability of powerful and affordable screening platforms, and the relative ease-of-synthesis render therapeutic peptides highly approachable for teaching and research alike. With a strong focus on the therapeutic modulation of host defence pathways, including the complement and renin-angiotensin systems, the Molecular Pharmacy group at the University of Basel strongly relies on peptides to introduce students to practical aspects of modern drug design, to discover novel therapeutics for immune and inflammatory diseases, and to expand on options for the preclinical development of a promising drug class. Current projects reach from student-driven iterative design of peptidic angiotensin-converting enzyme inhibitors and the use of phage display technology to discover novel immune modulators to the development of protective peptide coatings for biomaterials and transplants and the structure-activity-relationship-guided optimization of therapeutic peptide drug candidates in late-stage clinical trials. Even at the current stage, peptides allow for a perfect circle between pharmaceutical research and education, and the recent spark of clinical applications for peptide-based drugs may only increase the value and relevance of this versatile drug class.

C1q binding to surface-bound IgG is stabilized by C1r2s2 proteases.

Complement is an important effector mechanism for antibody-mediated clearance of infections and tumor cells. Upon binding to target cells, the antibody's constant (Fc) domain recruits complement component C1 to initiate a proteolytic cascade that generates lytic pores and stimulates phagocytosis. The C1 complex (C1qr2s2) consists of the large recognition protein C1q and a heterotetramer of proteases C1r and C1s (C1r2s2). While interactions between C1 and IgG-Fc are believed to be mediated by the globular heads of C1q, we here find that C1r2s2 proteases affect the capacity of C1q to form an avid complex with surface-bound IgG molecules (on various 2,4-dinitrophenol [DNP]-coated surfaces and pathogenic Staphylococcus aureus). The extent to which C1r2s2 contributes to C1q-IgG stability strongly differs between human IgG subclasses. Using antibody engineering of monoclonal IgG, we reveal that hexamer-enhancing mutations improve C1q-IgG stability, both in the absence and presence of C1r2s2 In addition, hexamer-enhanced IgGs targeting S. aureus mediate improved complement-dependent phagocytosis by human neutrophils. Altogether, these molecular insights into complement binding to surface-bound IgGs could be important for optimal design of antibody therapies.

The Promiscuous Profile of Complement Receptor 3 in Ligand Binding, Immune Modulation, and Pathophysiology.

The ß2-integrin receptor family has a broad spectrum of physiological functions ranging from leukocyte adhesion, cell migration, activation, and communication to the phagocytic uptake of cells and particles. Among the members of this family, complement receptor 3 (CR3; CD11b/CD18, Mac-1, αMß2) is particularly promiscuous in its functional profile and ligand selectivity. There are close to 100 reported structurally unrelated ligands for CR3, and while many ligands appear to cluster at the αMI domain, molecular details about binding modes remain largely elusive. The versatility of CR3 is reflected in its functional portfolio, which includes prominent roles in the removal of invaders and cell debris, induction of tolerance and synaptic pruning, and involvement in the pathogenesis of numerous autoimmune and chronic inflammatory pathologies. While CR3 is an interesting therapeutic target for immune modulation due to these known pathophysiological associations, drug development efforts are limited by concerns of potential interference with host defense functions and, most importantly, an insufficient molecular understanding of the interplay between ligand binding and functional impact. Here, we provide a systematic summary of the various interaction partners of CR3 with a focus on binding mechanisms and functional implications. We also discuss the roles of CR3 as an immune receptor in health and disease, as an activation marker in research and diagnostics, and as a therapeutic target.

Deregulation of Factor H by Factor H-Related Protein 1 Depends on Sialylation of Host Surfaces.

To discriminate between self and non-self surfaces and facilitate immune surveillance, the complement system relies on the interplay between surface-directed activators and regulators. The dimeric modulator FHR-1 is hypothesized to competitively remove the complement regulator FH from surfaces that strongly fix opsonic C3b molecules-a process known as "deregulation." The C-terminal regions of FH and FHR-1 provide the basis of this competition. They contain binding sites for C3b and host surface markers and are identical except for two substitutions: S1191L and V1197A (i.e., FH "SV"; FHR-1 "LA"). Intriguingly, an FHR-1 variant featuring the "SV" combination of FH predisposes to atypical hemolytic uremic syndrome (aHUS). The functional impact of these mutations on complement (de)regulation, and their pathophysiological consequences, have largely remained elusive. We have addressed these questions using recombinantly expressed wildtype, mutated, and truncated versions of FHR-1 and FH. The "SV" to "LA" substitutions did not affect glycosaminoglycan recognition and had only a small effect on C3b binding. In contrast, the two amino acids substantially affected the binding of FH and FHR-1 to α2,3-linked sialic acids as host surfaces markers, with the S-to-L substitution causing an almost complete loss of recognition. Even with sialic acid-binding constructs, notable deregulation was only detected on host and not foreign cells. The aHUS-associated "SV" mutation converts FHR-1 into a sialic acid binder which, supported by its dimeric nature, enables excessive FH deregulation and, thus, complement activation on host surfaces. While we also observed inhibitory activities of FHR-1 on C3 and C5 convertases, the high concentrations required render the physiological impact uncertain. In conclusion, the SV-to-LA substitution in the C-terminal regions of FH and FHR-1 diminishes its sialic acid-binding ability and results in an FHR-1 molecule that only moderately deregulates FH. Such FH deregulation by FHR-1 only occurs on host/host-like surfaces that recruit FH. Conversion of FHR-1 into a sialic acid binder potentiates the deregulatory capacity of FHR-1 and thus explains the pathophysiology of the aHUS-associated FHR-1 "SV" variant.

Sweet turning bitter: Carbohydrate sensing of complement in host defence and disease.

The complement system plays a major role in threat recognition and in orchestrating responses to microbial intruders and accumulating debris. This immune surveillance is largely driven by lectins that sense carbohydrate signatures on foreign, diseased and healthy host cells and act as complement activators, regulators or receptors to shape appropriate immune responses. While carbohydrate sensing protects our bodies, misguided or impaired recognition can contribute to disease. Moreover, pathogenic microbes have evolved to evade complement by mimicking host signatures. While complement is recognized as a disease factor, we only slowly start to appreciate the role of carbohydrate interactions in the underlying processes. A better understanding of complement's sweet side will contribute to a better description of disease mechanisms and enhanced diagnostic and therapeutic options. This review introduces the key components in complement-mediated carbohydrate sensing, discusses their role in health and disease, and touches on the potential effects of carbohydrate-related disease intervention. LINKED ARTICLES: This article is part of a themed issue on Canonical and non-canonical functions of the complement system in health and disease. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.14/issuetoc.

Intracellular C4BPA Levels Regulate NF-κB-Dependent Apoptosis.

The importance of innate immunity in cancer is increasingly being recognized with recent reports suggesting tumor cell-intrinsic intracellular functions for innate immunity proteins. However, such functions are often poorly understood, and it is unclear whether these are affected by patient-specific mutations. Here, we show that C4b-binding protein alpha chain (C4BPA), typically thought to reside in the extracellular space, is expressed intracellularly in cancer cells, where it interacts with the NF-κB family member RelA and regulates apoptosis. Interestingly, intracellular C4BPA expression is regulated in a stress- and mutation-dependent manner and C4BPA mutations are associated with improved cancer survival outcome. Using cell lines harboring patient-specific C4BPA mutations, we show that increasing intracellular C4BPA levels correlate with sensitivity to oxaliplatin-induced apoptosis in vitro and in vivo. Mechanistically, sensitive C4BPA mutants display increased IκBα expression and increased inhibitory IκBα-RelA complex stability. These data suggest a non-canonical intracellular role for C4BPA in regulating NF-κB-dependent apoptosis.