Enhancing Antibody-Specific Productivity: Unraveling the Impact of XBP1s Overexpression and Glutamine Availability in SP2/0 Cells.

Augmentation of glycoprotein synthesis requirements induces endoplasmic reticulum (ER) stress, activating the unfolded protein response (UPR) and triggering unconventional XBP1 splicing. As a result, XBP1s orchestrates the expression of essential genes to reduce stress and restore homeostasis. When this mechanism fails, chronic stress may lead to apoptosis, which is thought to be associated with exceeding a threshold in XBP1s levels. Glycoprotein assembly is also affected by glutamine (Gln) availability, limiting nucleotide sugars (NS), and preventing compliance with the increased demands. In contrast, increased Gln intake synthesizes ammonia as a by-product, potentially reaching toxic levels. IgA2m(1)-producer mouse myeloma cells (SP2/0) were used as the cellular mammalian model. We explored how IgA2m(1)-specific productivity (qIgA2m(1)) is affected by (i) overexpression of human XBP1s (h-XBP1s) levels and (ii) Gln availability, evaluating the kinetic behavior in batch cultures. The study revealed a two and a five-fold increase in qIgA2m(1) when lower and higher levels of XBP1s were expressed, respectively. High h-XBP1s overexpression mitigated not only ammonia but also lactate accumulation. Moreover, XBP1s overexpressor showed resilience to hydrodynamic stress in serum-free environments. These findings suggest a potential application of h-XBP1s overexpression as a feasible and cost-effective strategy for bioprocess scalability.

89Zr-ImmunoPET Shows Therapeutic Efficacy of Anti-CD20-IFNα Fusion Protein in a Murine B-cell Lymphoma Model.

Antibody-mediated tumor delivery of cytokines can overcome limitations of systemic administration (toxicity, short half-lives). Previous work showed improved antitumor potency of anti-CD20-IFNα fusion proteins in preclinical mouse models of B-cell lymphoma. Although tumor targeting is mediated by the antibody part of the fusion protein, the cytokine component might strongly influence biodistribution and pharmacokinetics, as a result of its affinity, size, valency, and receptor distribution. Here, we used immunoPET to study the in vivo biodistribution and tumor targeting of the anti-CD20 rituximab-murine IFNα1 fusion protein (Rit-mIFNα) and compared it with the parental mAb (rituximab, Rit). Rit-mIFNα and Rit were radiolabeled with zirconium-89 (89Zr, t1/2 78.4 hours) and injected into C3H mice bearing syngeneic B-cell lymphomas (38C13-hCD20). Dynamic [(2 hours post injection (p.i.)] and static (4, 24, and 72 hours) PET scans were acquired. Ex vivo biodistribution was performed after the final scan. Both 89Zr-Rit-mIFNα and 89Zr-Rit specifically target hCD20-expressing B-cell lymphoma in vivo. 89Zr-Rit-mIFNα showed specific uptake in tumors (7.6 ± 1.0 %ID/g at 75 hours p.i.), which was significantly lower than 89Zr-Rit (38.4 ± 9.9 %ID/g, P < 0.0001). ImmunoPET studies also revealed differences in the biodistribution, 89Zr-Rit-mIFNα showed rapid blood clearance and high accumulation in the liver compared with 89Zr-Rit. Importantly, immunoPET clearly revealed a therapeutic effect of the single 89Zr-Rit-mIFNα dose, resulting in smaller tumors and fewer lymph node metastases compared with mice receiving 89Zr-Rit. Mice receiving 89Zr-Rit-mIFNα had enlarged spleens, suggesting that systemic immune activation contributes to therapeutic efficacy in addition to the direct antitumoral activity of IFNα. In conclusion, immunoPET allows the noninvasive tracking and quantification of the antibody-cytokine fusion protein and helps understand the in vivo behavior and therapeutic efficacy.

An HK2 Antisense Oligonucleotide Induces Synthetic Lethality in HK1-HK2+ Multiple Myeloma.

Although the majority of adult tissues express only hexokinase 1 (HK1) for glycolysis, most cancers express hexokinase 2 (HK2) and many coexpress HK1 and HK2. In contrast to HK1+HK2+ cancers, HK1-HK2+ cancer subsets are sensitive to cytostasis induced by HK2shRNA knockdown and are also sensitive to synthetic lethality in response to the combination of HK2shRNA knockdown, an oxidative phosphorylation (OXPHOS) inhibitor diphenyleneiodonium (DPI), and a fatty acid oxidation (FAO) inhibitor perhexiline (PER). The majority of human multiple myeloma cell lines are HK1-HK2+. Here we describe an antisense oligonucleotide (ASO) directed against human HK2 (HK2-ASO1), which suppressed HK2 expression in human multiple myeloma cell cultures and human multiple myeloma mouse xenograft models. The HK2-ASO1/DPI/PER triple-combination achieved synthetic lethality in multiple myeloma cells in culture and prevented HK1-HK2+ multiple myeloma tumor xenograft progression. DPI was replaceable by the FDA-approved OXPHOS inhibitor metformin (MET), both for synthetic lethality in culture and for inhibition of tumor xenograft progression. In addition, we used an ASO targeting murine HK2 (mHK2-ASO1) to validate the safety of mHK2-ASO1/MET/PER combination therapy in mice bearing murine multiple myeloma tumors. HK2-ASO1 is the first agent that shows selective HK2 inhibition and therapeutic efficacy in cell culture and in animal models, supporting clinical development of this synthetically lethal combination as a therapy for HK1-HK2+ multiple myeloma. SIGNIFICANCE: A first-in-class HK2 antisense oligonucleotide suppresses HK2 expression in cell culture and in in vivo, presenting an effective, tolerated combination therapy for preventing progression of HK1-HK2+ multiple myeloma tumors. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/79/10/2748/F1.large.jpg.

Activity of Anti-CD19 Chimeric Antigen Receptor T Cells Against B Cell Lymphoma Is Enhanced by Antibody-Targeted Interferon-Alpha.

An important emerging form of immunotherapy targeting B cell malignancies is chimeric antigen receptor (CAR) T cell therapy. Despite encouraging response rates of anti-CD19 CAR T cell therapy in B cell lymphomas, limited durability of response necessitates further study to potentiate CAR T cell efficacy. Antibody-targeted interferon (IFN) therapy is a novel approach in immunotherapy. Given the ability of IFNs to promote T cell activation and survival, target cell recognition, and cytotoxicity, we asked whether antibody-targeted IFN could enhance the antitumor effects of anti-CD19 CAR T cells. We produced an anti-CD20-IFN fusion protein containing the potent type 1 IFN isoform alpha14 (α14), and demonstrated its ability to suppress proliferation and induce apoptosis of human B cell lymphomas. Indeed, with the combination of anti-CD20-hIFNα14 and CAR T cells, we found enhanced cell killing among B cell lymphoma lines. Importantly, for all cell lines pretreated with anti-CD20-hIFNα14, the subsequent cytokine production by CAR T cells was markedly increased regardless of the degree of cell killing. Thus, several activities of CD19 CAR T cells were enhanced in the presence of anti-CD20-hIFNα14. These data suggest that antibody-targeted IFN may be an important novel approach to improving the efficacy of CAR T cell therapy.

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.

Anti-CD20-interferon-ß fusion protein therapy of murine B-cell lymphomas.

Type I interferons (IFNα/ß) are cytokines with a broad spectrum of antitumor activities including antiproliferative, proapoptotic, and immunostimulatory effects, and are potentially useful in the treatment of B-cell malignancies and other cancers. To improve antitumor potency and diminish the systemic side effects of IFN, we recently developed anti-CD20-IFNα fusion proteins with in vitro and in vivo efficacy against both mouse and human lymphomas expressing CD20. As IFNß binds more tightly to the IFNα/ß receptor (IFNAR) and has more potent antitumor activities, we have now constructed an anti-CD20 fusion protein with murine IFNß (mIFNß). Anti-CD20-mIFNß was more potent than recombinant mIFNß and anti-CD20-mIFNα in inhibiting the proliferation of a mouse B-cell lymphoma expressing human CD20 (38C13-huCD20). Growth inhibition was accompanied by caspase-independent apoptosis and DNA fragmentation. The efficacy of anti-CD20-mIFNß required the physical linkage of mIFNß to anti-CD20 antibody. Importantly, anti-CD20-mIFNß was active against tumor cells expressing low levels of IFNAR (38C13-huCD20 IFNAR). In vivo, established 38C13-huCD20 tumors were largely insensitive to rituximab or a nontargeted mIFNß fusion protein, yet treatment with anti-CD20-mIFNß eradicated 83% of tumors. Anti-CD20-mIFNß was also more potent in vivo against 38C13-huCD20 than anti-CD20-mIFNα, curing 75% versus 25% of tumors (P=0.001). Importantly, although anti-CD20-mIFNα could not eradicate 38C13-huCD20 IFNAR tumors, anti-CD20-mIFNß treatment prolonged survival (P=0.0003), and some animals remained tumor-free. Thus, antibody fusion proteins targeting mIFNß to tumors show promise as therapeutic agents, especially for use against tumors resistant to the effects of mIFNα.

In vitro C3 deposition on Cryptococcus capsule occurs via multiple complement activation pathways.

Complement can be activated via three pathways: classical, alternative, and lectin. Cryptococcus gattii and Cryptococcus neoformans are closely related fungal pathogens possessing a polysaccharide capsule composed mainly of glucuronoxylomannan (GXM), which serves as a site for complement activation and deposition of complement components. We determined C3 deposition on Cryptococcus spp. by flow cytometry and confocal microscopy after incubation with serum from C57BL/6J mice as well as mice deficient in complement components C4, C3, factor B, and mannose binding lectin (MBL). C. gattii and C. neoformans activate complement in EGTA-treated serum indicating that they can activate the alternative pathway. However, complement activation was seen with factor B(-/-) serum suggesting activation could also take place in the absence of a functional alternative pathway. Furthermore, we uncovered a role for C4 in the alternative pathway activation by Cryptococcus spp. We also identified an unexpected and complex role for MBL in complement activation by Cryptococcus spp. No complement activation occurred in the absence of MBL-A and -C proteins although activation took place when the lectin binding activity of MBL was disrupted by calcium chelation. In addition, alternative pathway activation by C. neoformans required both MBL-A and -C, while either MBL-A or -C was sufficient for alternative pathway activation by C. gattii. Thus, complement activation by Cryptococcus spp. can take place through multiple pathways and complement activation via the alternative pathway requires the presence of C4 and MBL proteins.

Role of complement in protection against Cryptococcus gattii infection.

Previous studies have shown that the alternative pathway of complement activation plays an important role in protection against infection with Cryptococcus neoformans. Cryptococcus gattii does not activate the alternative pathway as well as C. neoformans in vitro. The role of complement in C. gattii infection in vivo has not been reported. In this study, we used mice deficient in complement components to investigate the role of complement in protection against a C. gattii isolate from an ongoing outbreak in northwestern North America. While factor B-deficient mice showed an enhanced rate of death, complement component C3-deficient mice died even more rapidly, indicating that the alternative pathway was not the only complement pathway contributing to protection against disease. Both C3- and factor B-deficient mice had increased fungal burdens in comparison to wild-type mice. Histopathology revealed an overwhelming fungal burden in the lungs of these complement-deficient mice, which undoubtedly prevented efficient gas exchange, causing death. Following the fate of radiolabeled organisms showed that both factor B- and C3-deficient mice were less effective than wild-type mice in clearing organisms. However, opsonization of C. gattii with complement components was not sufficient to prolong life in mice deficient in complement. Killing of C. gattii by macrophages in vitro was decreased in the presence of serum from factor B- and C3-deficient versus wild-type mice. In conclusion, we have demonstrated that complement activation is crucial for survival in C. gattii infection. Additionally, we have shown that the alternative pathway of complement activation is not the only complement pathway contributing to protection.