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Follicular CD8+CXCR5+ T cells are a specialized CD8+ T cell subset with unique follicular-homing capabilities that have been reported to display effector functions in viral immunity, tumor immunity, and autoimmunity. CD8+CXCR5+ T cells exhibit B cell helper functions and express CD40L, ICOS, programmed cell death protein 1 (PD-1), and BCL-6, the transcriptional regulator of CD4+CXCR5+ T follicular helper cells and of germinal center B cells. HIV is known to be sequestered in lymphoid follicles, and CD8+CXCR5+ T cell frequency is a marker for disease severity, given that HIV-infected patients with lower numbers of circulating CD8+CXCR5+ T cells display lower CD4+ T cell counts. Likewise, several groups have reported a direct correlation between the quantity of CD8+CXCR5+ T cells and suppression of HIV viral load. In this study, we observed elevated absolute numbers of CD8+CXCR5+ and CD8+CXCR5+BCL-6+PD-1+ T cells in the blood of HIV-infected participants of the Multicenter AIDS Cohort Study. We further demonstrated in vitro that activated human CD8+CXCR5+ T cells isolated from peripheral blood and tonsil from healthy donors show increased CD40L expression and induce the production of PD ligand 1 (PD-L1)+IgG+ B cells. Moreover, absolute numbers of CD8+CXCR5+ T cells significantly and positively correlated with numbers of PD-L1+ B cells found in blood of HIV-infected individuals. Altogether, these results show that activated CD8+CXCR5+ T cells have the ability to activate B cells and increase the percentage of PD-L1+ and PD-L1+IgG+ B cells, which provides insights into the early events of B cell activation and differentiation and may play a role in disease progression and lymphomagenesis in HIV-infected individuals.

Transferrin receptor 1 (TfR1), also known as CD71, is a transmembrane protein involved in the cellular uptake of iron and the regulation of cell growth. This receptor is expressed at low levels on a variety of normal cells, but is upregulated on cells with a high rate of proliferation, including malignant cells and activated immune cells. Infection with the human immunodeficiency virus (HIV) leads to the chronic activation of B cells, resulting in high expression of TfR1, B-cell dysfunction, and ultimately the development of acquired immunodeficiency syndrome-related B-cell non-Hodgkin lymphoma (AIDS-NHL). Importantly, TfR1 expression is correlated with the stage and prognosis of NHL. Thus, it is a meaningful target for antibody-based NHL therapy. We previously developed a mouse/human chimeric IgG3 specific for TfR1 (ch128.1/IgG3) and showed that this antibody exhibits antitumor activity in an in vivo model of AIDS-NHL using NOD-SCID mice challenged intraperitoneally with 2F7 human Burkitt lymphoma (BL) cells that harbor the Epstein-Barr virus (EBV). We have also developed an IgG1 version of ch128.1 that shows significant antitumor activity in SCID-Beige mouse models of disseminated multiple myeloma, another B-cell malignancy. Here, we aim to explore the utility of ch128.1/IgG1 and its humanized version (hu128.1) in mouse models of AIDS-NHL. To accomplish this goal, we used the 2F7 cell line variant 2F7-BR44, which is more aggressive than the parental cell line and forms metastases in the brain of mice after systemic (intravenous) administration. We also used the human BL cell line JB, which in contrast to 2F7, is EBV-negative, allowing us to study both EBV-infected and non-infected NHL tumors. Treatment with ch128.1/IgG1 or hu128.1 of SCID-Beige mice challenged locally (subcutaneously) with 2F7-BR44 or JB cells results in significant antitumor activity against different stages of disease. Treatment of mice challenged systemically (intravenously) with either 2F7-BR44 or JB cells also showed significant antitumor activity, including long-term survival. Taken together, our results suggest that targeting TfR1 with antibodies, such as ch128.1/IgG1 or hu128.1, has potential as an effective therapy for AIDS-NHL.

Five New World mammarenaviruses (NWMs) cause life-threatening hemorrhagic fever (HF). Cellular entry by these viruses is mediated by human transferrin receptor 1 (hTfR1). Here, we demonstrate that an antibody (ch128.1/IgG1) which binds the apical domain of hTfR1, potently inhibits infection of attenuated and pathogenic NWMs in vitro. Computational docking of the antibody Fab crystal structure onto the known structure of hTfR1 shows an overlapping receptor-binding region shared by the Fab and the viral envelope glycoprotein GP1 subunit that binds hTfR1, and we demonstrate competitive inhibition of NWM GP1 binding by ch128.1/IgG1 as the principal mechanism of action. Importantly, ch128.1/IgG1 protects hTfR1-expressing transgenic mice against lethal NWM challenge. Additionally, the antibody is well-tolerated and only partially reduces ferritin uptake. Our findings provide the basis for the development of a novel, host receptor-targeted antibody therapeutic broadly applicable to the treatment of HF of NWM etiology.

Epstein-Barr virus (EBV) is a human gammaherpesvirus associated with the development of hematopoietic cancers of B-lymphocyte origin, including AIDS-related non-Hodgkin lymphoma (AIDS-NHL). Primary infection of B-cells with EBV results in their polyclonal activation and immortalization. The transferrin receptor 1 (TfR1), also known as CD71, is important for iron uptake and regulation of cellular proliferation. TfR1 is highly expressed in proliferating cells, including activated lymphocytes and malignant cells. We developed a mouse/human chimeric antibody targeting TfR1 (ch128.1/IgG1) that has previously shown significant antitumor activity in immunosuppressed mouse models bearing human malignant B-cells, including multiple myeloma and AIDS-NHL cells. In this article, we examined the effect of targeting TfR1 to inhibit EBV-driven activation and growth of human B-cells in vivo using an immunodeficient NOD.Cg-Prkdcscid Il2rgtm1Wjl /SzJ [NOD/SCID gamma (NSG)] mouse model. Mice were implanted with T-cell-depleted, human peripheral blood mononuclear cells (PBMCs), either without EBV (EBV-), or exposed to EBV in vitro (EBV+), intravenously via the tail vein. Mice implanted with EBV+ cells and treated with an IgG1 control antibody (400 μg/mouse) developed lymphoma-like growths of human B-cell origin that were EBV+, whereas mice implanted with EBV+ cells and treated with ch128.1/IgG1 (400 μg/mouse) showed increased survival and significantly reduced inflammation and B-cell activation. These results indicate that ch128.1/IgG1 is effective at preventing the growth of EBV+ human B-cell tumors in vivo, thus, indicating that there is significant potential for agents targeting TfR1 as therapeutic strategies to prevent the development of EBV-associated B-cell malignancies. SIGNIFICANCE: An anti-TfR1 antibody, ch128.1/IgG1, effectively inhibits the activation, growth, and immortalization of EBV+ human B-cells in vivo, as well as the development of these cells into lymphoma-like tumors in immunodeficient mice.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Therapeutic blockade of PD-1/PD-L1 signaling with monoclonal antibodies (mAbs) has shown clinical success and activity across a broad set of cancer subtypes. However, monotherapy with PD-1/PD-L1 inhibitors are only effective in a subset of patients and ongoing studies show efficacy of treatment depends on a combinatorial approach. Contrary to mAbs chimeric B-cell cancer vaccines incorporating a "promiscuous" T-cell epitope have the advantage of producing a polyclonal B-cell antibody that can potentially induce memory B- and T-cell responses, while reducing immune evasion and suppression. Here, we describe a novel PD-1 B-cell peptide epitope vaccine (amino acid 92-110; PD1-Vaxx) linked to a measles virus fusion peptide (MVF) amino acid 288-302 via a four amino acid residue (GPSL) emulsified in Montanide ISA 720VG that aims to induce the production of polyclonal antibodies that block PD-1 signaling and thus trigger anticancer effects similar to nivolumab. In preclinical studies, the PD1-Vaxx outperformed the standard anti-mouse PD-1 antibody (mAb 29F.1A12) in a mouse model of human HER-2 expressing colon carcinoma. Furthermore, the combination of PD1-Vaxx with combo HER-2 peptide vaccine (B-Vaxx) showed enhanced inhibition of tumor growth in colon carcinoma BALB/c model challenged with CT26/HER-2 cells. The PD-1 or combined vaccines were safe with no evidence of toxicity or autoimmunity.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

An antibody-cytokine fusion protein, composed of the murine single-chain cytokine interleukin-12 (IL-12) genetically fused to a human IgG3 specific for the human tumor-associated antigen HER2/neu maintains antigen binding, cytokine bioactivity, and IL-12 heparin-binding activity. This latter property is responsible for the binding of the cytokine to glycosaminoglycans (GAGs) on the cell surface and the extracellular matrix and has been implicated in modulating IL-12 bioactivity. Previous studies indicate that the p40 subunit of human and murine IL-12 is responsible for the heparin-binding activity of this heterodimeric cytokine. In the present study we used bioinformatic analysis and site-directed mutagenesis to develop a version of the antibody-(IL-12) fusion protein without heparin-binding activity. This was accomplished by replacing the basic arginine (R) and lysine (K) residues in the cluster of amino acids 254-260 (RKKEKMK) of the murine IL-12 p40 subunit by the neutral non-polar amino acid alanine (A), generating an AAAEAMA mutant fusion protein. ELISA and flow cytometry demonstrated that the antibody fusion protein lacks heparin-binding activity but retains antigen binding. A T-cell proliferation assay showed IL-12 bioactivity in this construct. However, the IL-12 bioactivity is decreased compared to its non-mutated counterpart, which is consistent with an ancillary role of the heparin-binding site of IL-12 in modulating its activity. Thus, we have defined a cluster of amino acid residues with a crucial role in the heparin-binding activity of murine IL-12 in the context of an antibody-cytokine fusion protein.

The risk for non-Hodgkin lymphoma (NHL) is markedly increased in persons living with human immunodeficiency virus (HIV) infection, and remains elevated in those on anti-retroviral therapy (cART). Both the loss of immunoregulation of Epstein-Barr virus (EBV) infected cells, as well as chronic B-cell activation, are believed to contribute to the genesis of AIDS-related NHL (AIDS-NHL). However, the mechanisms that lead to AIDS-NHL have not been completely defined. A subset of B cells that is characterized by the secretion of IL10, as well as the expression of the programmed cell death ligand-1 (PD-L1/CD274), was recently described. These PD-L1+ B cells can exert regulatory function, including the dampening of T-cell activation, by interacting with the program cell death protein (PD1) on target cells. The role of PD-L1+ B cells in the development of AIDS-NHL has not been explored. We assessed B cell PD-L1 expression on B cells preceding AIDS-NHL diagnosis in a nested case-control study of HIV+ subjects who went on to develop AIDS-NHL, as well as HIV+ subjects who did not, using multi-color flow cytometry. Archival frozen viable PBMC were obtained from the UCLA Multicenter AIDS Cohort Study (MACS). It was seen that the number of CD19+CD24++CD38++and CD19+PD-L1+cells was significantly elevated in cases 1-4 years prior to AIDS-NHL diagnosis, compared to controls, raising the possibility that these cells may play a role in the etiology of AIDS-NHL. Interestingly, most PD-L1+ expression on CD19+ cells was seen on CD19+CD24++CD38++ cells. In addition, we showed that HIV can directly induce PD-L1 expression on B cells through interaction of virion-associated CD40L with CD40 on B cells.

There is an unmet need for the treatment of glioblastoma multiforme (GBM). The extracellular matrix, including laminins, in the tumor microenvironment is important for tumor invasion and progression. In a panel of 226 patient brain glioma samples, we found a clinical correlation between the expression of tumor vascular laminin-411 (α4β1γ1) with higher tumor grade and with expression of cancer stem cell (CSC) markers, including Notch pathway members, CD133, Nestin, and c-Myc. Laminin-411 overexpression also correlated with higher recurrence rate and shorter survival of GBM patients. We also showed that depletion of laminin-411 α4 and β1 chains with CRISPR/Cas9 in human GBM cells led to reduced growth of resultant intracranial tumors in mice and significantly increased survival of host animals compared with mice with untreated cells. Inhibition of laminin-411 suppressed Notch pathway in normal and malignant human brain cell types. A nanobioconjugate potentially suitable for clinical use and capable of crossing blood-brain barrier was designed to block laminin-411 expression. Nanobioconjugate treatment of mice carrying intracranial GBM significantly increased animal survival and inhibited multiple CSC markers, including the Notch axis. This study describes an efficient strategy for GBM treatment via targeting a critical component of the tumor microenvironment largely independent of heterogeneous genetic mutations in glioblastoma.Significance: Laminin-411 expression in the glioma microenvironment correlates with Notch and other cancer stem cell markers and can be targeted by a novel, clinically translatable nanobioconjugate to inhibit glioma growth.

Brain glioma treatment with checkpoint inhibitor antibodies to cytotoxic T-lymphocyte-associated antigen 4 (a-CTLA-4) and programmed cell death-1 (a-PD-1) was largely unsuccessful due to their inability to cross blood-brain barrier (BBB). Here we describe targeted nanoscale immunoconjugates (NICs) on natural biopolymer scaffold, poly(β-L-malic acid), with covalently attached a-CTLA-4 or a-PD-1 for systemic delivery across the BBB and activation of local brain anti-tumor immune response. NIC treatment of mice bearing intracranial GL261 glioblastoma (GBM) results in an increase of CD8+ T cells, NK cells and macrophages with a decrease of regulatory T cells (Tregs) in the brain tumor area. Survival of GBM-bearing mice treated with NIC combination is significantly longer compared to animals treated with single checkpoint inhibitor-bearing NICs or free a-CTLA-4 and a-PD-1. Our study demonstrates trans-BBB delivery of tumor-targeted polymer-conjugated checkpoint inhibitors as an effective GBM treatment via activation of both systemic and local privileged brain tumor immune response.

The transferrin receptor 1 (TfR1) is an attractive target for Ab-mediated cancer therapy. We previously developed a mouse/human chimeric IgG3 Ab (ch128.1) targeting human TfR1, which exhibits direct in vitro cytotoxicity against certain human malignant B cells through TfR1 degradation and iron deprivation. ch128.1 also demonstrates exceptional antitumor activity against the B cell malignancy multiple myeloma (MM) in xenograft models of SCID-Beige mice bearing either disseminated ARH-77 or KMS-11 cells in an early disease setting. Interestingly, this activity is observed even against KMS-11 cells, which show no sensitivity to the direct cytotoxic activity of ch128.1 in vitro. To understand the contributions of the Fc fragment, we generated a ch128.1 mutant with impaired binding to FcγRs and to the complement component C1q, which retains binding to the neonatal Fc receptor. We now report that this mutant Ab does not show antitumor activity in these two MM models, indicating a crucial role of the Fc fragment in the antitumor activity of ch128.1, which can be attributed to effector functions (Ab-dependent cell-mediated cytotoxicity, Ab-dependent cell-mediated phagocytosis, and/or complement-dependent cytotoxicity). Interestingly, in the KMS-11 model, complement depletion does not affect protection, whereas macrophage depletion does. Consistent with this observation, we found that ch128.1 induces Ab-dependent cell-mediated cytotoxicity and Ab-dependent cell-mediated phagocytosis against KMS-11 cells in the presence of murine bone marrow-derived macrophages. Finally, we found that ch128.1 therapy effectively increases survival in a late MM disease setting. Our results suggest that macrophages play a major role in ch128.1-mediated antitumor protection in our models and that ch128.1 can be effective against human B cell malignancies such as MM.

The transferrin receptor 1 (TfR1) is involved in cellular iron uptake and regulation of cell proliferation. The increased expression of TfR1 observed in malignant cells, compared to normal cells, together with its extracellular accessibility, make this receptor an attractive target for antibody-mediated cancer therapy. We have developed a mouse/human chimeric IgG3 specific for human TfR1 (ch128.1), which shows anti-tumor activity against certain malignant B cells in vitro through TfR1 degradation and iron deprivation, and in vivo through a mechanism yet to be defined. To further explore potential mechanisms of action of ch128.1, we examined its ability to induce antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-mediated cytotoxicity (CDC). We now report that ch128.1 is capable of mediating ADCC and CDC against malignant B cells, which is consistent with its ability to bind FcγRI, FcγRIIIa, and the complement component C1q. To delineate the residues involved in these effector functions, we developed a panel of three constructs with mutations in the lower hinge region and CH2 domain: 1) L234A/L235A, 2) P331S, and 3) L234A/L235A/P331S. The triple mutant consistently displayed a significant reduction in ADCC, while the L234A/L235A mutant exhibited less reduction in ADCC, and the P331S mutant did not show reduced ADCC. However, all three mutants exhibited impaired binding to FcγRI and FcγRIIIa. These results suggest that all three residues contribute to ADCC, although to different degrees. The P331S mutant showed drastically decreased C1q binding and abolished CDC, confirming the critical role of this residue in complement activation, while the other residues play a less important role in CDC. Our study provides insights into the effector functions of human IgG3 in the context of an antibody targeting TfR1.

BackgroundWe previously developed an antibody-avidin fusion protein (ch128.1Av) specific for the human transferrin receptor 1 (TfR1; CD71) to be used as a delivery vector for cancer therapy and showed that ch128.1Av delivers the biotinylated plant toxin saporin-6 into malignant B cells. However, as a result of widespread expression of TfR1, delivery of the toxin to normal cells is a concern. Therefore, we explored the potential of a dual targeted lentiviral-mediated gene therapy strategy to restrict gene expression to malignant B cells. Targeting occurs through the use of ch128.1Av or its parental antibody without avidin (ch128.1) and through transcriptional regulation using an immunoglobulin promoter.MethodsFlow cytometry was used to detect the expression of enhanced green fluorescent protein (EGFP) in a panel of cell lines. Cell viability after specific delivery of the therapeutic gene FCU1, a chimeric enzyme consisting of cytosine deaminase genetically fused to uracil phosphoribosyltransferse that converts the 5-fluorocytosine (5-FC) prodrug into toxic metabolites, was monitored using the MTS or WST-1 viability assay.ResultsWe found that EGFP was specifically expressed in a panel of human malignant B-cell lines, but not in human malignant T-cell lines. EGFP expression was observed in all cell lines when a ubiquitous promoter was used. Furthermore, we show the decrease of cell viability in malignant plasma cells in the presence of 5-FC and the FCU1 gene.ConclusionsThe present study demonstrates that gene expression can be restricted to malignant B cells and suggests that this dual targeted gene therapy strategy may help to circumvent the potential side effects of certain TfR1-targeted protein delivery approaches.

Abstract Background Prostate cancer (PCa) is the second leading cause of cancer deaths in men in the United States. The prostate-specific antigen (PSA), often found at high levels in the serum of PCa patients, has been used as a marker for PCa detection and as a target of immunotherapy. The murine IgG1 monoclonal antibody AR47.47, specific for human PSA, has been shown to enhance antigen presentation by human dendritic cells and induce both CD4 and CD8 T-cell activation when complexed with PSA. In this study, we explored the properties of a novel mouse/human chimeric anti-PSA IgE containing the variable regions of AR47.47 as a potential therapy for PCa. Our goal was to take advantage of the unique properties of IgE in order to trigger immune activation against PCa. Methods Binding characteristics of the antibody were determined by ELISA and flow cytometry. In vitro degranulation was determined by the release of β-hexosaminidase from effector cells. In vivo degranulation was monitored in human FcεRIα transgenic mice using the passive cutaneous anaphylaxis assay. These mice were also used for a vaccination study to determine the in vivo anti-cancer effects of this antibody. Significant differences in survival were determined using the Log Rank test. In vitro T-cell activation was studied using human dendritic cells and autologous T cells. Results The anti-PSA IgE, expressed in murine myeloma cells, is properly assembled and secreted, and binds the antigen and FcεRI. In addition, this antibody is capable of triggering effector cell degranulation in vitro and in vivo when artificially cross-linked, but not in the presence of the natural soluble antigen, suggesting that such an interaction will not trigger systemic anaphylaxis. Importantly, the anti-PSA IgE combined with PSA also triggers immune activation in vitro and in vivo and significantly prolongs the survival of human FcεRIα transgenic mice challenged with PSA-expressing tumors in a prophylactic vaccination setting. Conclusions The anti-PSA IgE exhibits the expected biological properties and is capable of triggering immune activation and anti-tumor protection. Further studies on this antibody as a potential PCa therapy are warranted.

Currently, few rodent models of AIDS-associated non-Hodgkin's lymphoma (AIDS-NHL) exist. In these studies, a novel mouse/human xenograft model of AIDS-associated Burkitt lymphoma (AIDS-BL) was created by injecting cells of the human AIDS-BL cell line, 2F7, intraperitoneally into NOD-SCID mice. Mice developed tumors in the peritoneal cavity, with metastases to the spleen, thymus, and mesenteric lymph nodes. Expression of the chemokine receptor, CXCR5, was greatly elevated in vivo on BL tumor cells in this model, as shown by flow cytometry. CXCL13 is the ligand for CXCR5, and serum and ascites levels of murine, but not human, CXCL13 showed a striking elevation in tumor-bearing mice, with levels as high as 200,000 pg/ml in ascites, as measured by ELISA. As shown by immunohistochemistry, murine CXCL13 was associated with macrophage-like tumor-infiltrating cells that appeared to be histiocytes. Blocking CXCR5 on 2F7 cells with neutralizing antibodies prior to injection into the mice substantially delayed tumor formation. The marked elevations in tumor cell CXCR5 expression and in murine CXCL13 levels seen in the model may potentially identify an important link between tumor-interacting histiocytes and tumor cells in AIDS-BL. These results also identify CXCL13 as a potential biomarker for this disease, which is consistent with previous studies showing that serum levels of CXCL13 were elevated in human subjects who developed AIDS-lymphoma. This mouse model may be useful for future studies on the interactions of the innate immune system and AIDS-BL tumor cells, as well as for the assessment of potential tumor biomarkers for this disease.

Abstract Background HER2/neu is an oncogene that facilitates neoplastic transformation due to its ability to transduce growth signals in a ligand-independent manner, is over-expressed in 20-30% of human breast cancers correlating with aggressive disease and has been successfully targeted with trastuzumab (Herceptin®). Because trastuzumab alone achieves only a 15-30% response rate, it is now commonly combined with conventional chemotherapeutic drugs. While the combination of trastuzumab plus chemotherapy has greatly improved response rates and increased survival, these conventional chemotherapy drugs are frequently associated with gastrointestinal and cardiac toxicity, bone marrow and immune suppression. These drawbacks necessitate the development of new, less toxic drugs that can be combined with trastuzumab. Recently, we reported that orally administered alpha-tocopheryloxyacetic acid (α-TEA), a novel ether derivative of alpha-tocopherol, dramatically suppressed primary tumor growth and reduced the incidence of lung metastases both in a transplanted and a spontaneous mouse model of breast cancer without discernable toxicity. Methods In this study we examined the effect of α-TEA plus HER2/neu-specific antibody treatment on HER2/neu-expressing breast cancer cells in vitro and in a HER2/neu positive human xenograft tumor model in vivo. Results We show in vitro that α-TEA plus anti-HER2/neu antibody has an increased cytotoxic effect against murine mammary tumor cells and human breast cancer cells and that the anti-tumor effect of α-TEA is independent of HER2/neu status. More importantly, in a human breast cancer xenograft model, the combination of α-TEA plus trastuzumab resulted in faster tumor regression and more tumor-free animals than trastuzumab alone. Conclusion Due to the cancer cell selectivity of α-TEA, and because α-TEA kills both HER2/neu positive and HER2/neu negative breast cancer cells, it has the potential to be effective and less toxic than existing chemotherapeutic drugs when used in combination with HER2/neu antibody.