Antibody-Drug Conjugate αEGFR-E-P125A Reduces Triple-negative Breast Cancer Vasculogenic Mimicry, Motility, and Metastasis through Inhibition of EGFR, Integrin, and FAK/STAT3 Signaling.

Primary tumor growth and metastasis in triple-negative breast cancer (TNBC) require supporting vasculature, which develop through a combination of endothelial angiogenesis and vasculogenic mimicry (VM), a process associated with aggressive metastatic behavior in which vascular-like structures are lined by tumor cells. We developed αEGFR-E-P125A, an antibody-endostatin fusion protein that delivers a dimeric, mutant endostatin (E-P125A) payload that inhibits TNBC angiogenesis and VM in vitro and in vivo. To characterize the mechanisms associated with induction and inhibition of VM, RNA sequencing (RNA-seq) of MDA-MB-231-4175 TNBC cells grown in a monolayer (two-dimensional) was compared with cells plated on Matrigel undergoing VM [three-dimensional (3D)]. We then compared RNA-seq between TNBC cells in 3D and cells in 3D with VM inhibited by αEGFR-E-P125A (EGFR-E-P125A). Gene set enrichment analysis demonstrated that VM induction activated the IL6-JAK-STAT3 and angiogenesis pathways, which were downregulated by αEGFR-E-P125A treatment.Correlative analysis of the phosphoproteome demonstrated decreased EGFR phosphorylation at Y1069, along with decreased phosphorylation of focal adhesion kinase Y397 and STAT3 Y705 sites downstream of α5ß1 integrin. Suppression of phosphorylation events downstream of EGFR and α5ß1 integrin demonstrated that αEGFR-E-P125A interferes with ligand-receptor activation, inhibits VM, and overcomes oncogenic signaling associated with EGFR and α5ß1 integrin cross-talk. In vivo, αEGFR-E-P125A treatment decreased primary tumor growth and VM, reduced lung metastasis, and confirmed the inhibition of signaling events observed in vitro. Simultaneous inhibition of EGFR and α5ß1 integrin signaling by αEGFR-E-P125A is a promising strategy for the inhibition of VM, tumor growth, motility, and metastasis in TNBC and other EGFR-overexpressing tumors. SIGNIFICANCE: αEGFR-E-P125A reduces VM, angiogenesis, tumor growth, and metastasis by inhibiting EGFR and α5ß1 integrin signaling, and is a promising therapeutic agent for TNBC treatment, used alone or in combination with chemotherapy.

Inhibition of Vasculogenic Mimicry and Angiogenesis by an Anti-EGFR IgG1-Human Endostatin-P125A Fusion Protein Reduces Triple Negative Breast Cancer Metastases.

Triple negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. Metastasis is the major cause of TNBC mortality. Angiogenesis facilitates TNBC metastases. Many TNBCs also form vascular channels lined by tumor cells rather than endothelial cells, known as 'vasculogenic mimicry' (VM). VM has been linked to metastatic TNBC behavior and resistance to anti-angiogenic agents. Epidermal growth factor receptor (EGFR) is frequently expressed on TNBC, but anti-EGFR antibodies have limited efficacy. We synthesized an anti-EGFR antibody-endostatin fusion protein, αEGFR IgG1-huEndo-P125A (αEGFR-E-P125A), designed to deliver a mutant endostatin, huEndo-P125A (E-P125A), to EGFR expressing tumors, and tested its effects on angiogenesis, TNBC VM, and motility in vitro, and on the growth and metastasis of two independent human TNBC xenograft models in vivo. αEGFR-E-P125A completely inhibited the ability of human umbilical vein endothelial cells to form capillary-like structures (CLS) and of TNBC cells to engage in VM and form tubes in vitro. αEGFR-E-P125A treatment reduced endothelial and TNBC motility in vitro more effectively than E-P125A or cetuximab, delivered alone or in combination. Treatment of TNBC with αEGFR-E-P125A was associated with a reduction in cytoplasmic and nuclear ß-catenin and reduced phosphorylation of vimentin. αEGFR-E-P125A treatment of TNBC xenografts in vivo inhibited angiogenesis and VM, reduced primary tumor growth and lung metastasis of orthotopically implanted MDA-MB-468 TNBC cells, and markedly decreased lung metastases following intravenous injection of MDA-MB-231-4175 lung-tropic TNBC cells. Combined inhibition of angiogenesis, VM, and TNBC motility mediated by αEGFR-E-P125A is a promising strategy for the prevention of TNBC metastases.

Anti-CD20-interleukin-21 fusokine targets malignant B cells via direct apoptosis and NK-cell-dependent cytotoxicity.

In spite of newly emerging therapies and the improved survival of patients with non-Hodgkin lymphoma (NHL), relapses or primary refractory disease are commonly observed and associated with dismal prognosis. Although discovery of the anti-CD20 antibody rituximab has markedly improved outcomes in B-cell NHL, rituximab resistance remains an important obstacle to successful treatment of these tumors. To improve the efficacy of CD20-targeted therapy, we fused interleukin 21 (IL-21), which induces direct lymphoma cytotoxicity and activates immune effector cells, to the anti-CD20 antibody (αCD20-IL-21 fusokine). We observed substantially enhanced IL-21R-mediated signaling by the fusokine compared with native IL-21 at equimolar concentrations. Fusokine treatment led to direct apoptosis of lymphoma cell lines and primary tumors that otherwise were resistant to native IL-21 treatment. In addition to direct cytotoxicity, the fusokine enhanced NK cell activation, effector functions, and interferon γ production, resulting in greater antibody-dependent cell-mediated cytotoxicity compared with IL-21 and/or anti-CD20 antibody treatments. Further, the αCD20-IL-21 fusokine stabilizes IL-21 and prolongs its half-life. In vivo αCD20-IL-21 therapy resulted in a significant tumor control in the rituximab-resistant A20-huCD20 tumors. Collectively, the dual functional ability of the αCD20-IL-21 fusokine to induce direct apoptosis and activate immune effector cells may provide benefit over existing treatments for NHL.

Mammary-tumor-educated B cells acquire LAP/TGF-ß and PD-L1 expression and suppress anti-tumor immune responses.

B lymphocytes play a role in inhibiting the immune response against certain tumors, but the underlying mechanisms are poorly understood. EMT-6 mammary tumors grow well in wild-type (WT) mice but show reduced growth in B-cell-deficient µ(-/-) BALB/c mice (BCDM). WT mice demonstrate extensive B-cell infiltration into the tumor bed, reduced CD8(+) T cell and CD49(+) NK cell infiltration, and markedly reduced cytolytic T-cell response relative to BCDM. Expression of LAP/TGF-ß1, CD80, CD86 and PD-L1 is significantly increased in tumor-infiltrating B cells (TIL-B) relative to splenic B cells. LAP/TGF-ß1 expression on TIL-B progressively increased from 5.4±1.7% on day 8 to 43.1±6.1% by day 21 post tumor implantation. Co-culture of EMT-6 tumor cells with Naive-B cells ex vivo generated B cells (EMT6-B) with a similar immunophenotype to TIL-B. Purified TIL-B, or in-vitro-generated EMT6-B suppressed CD4(+), CD8(+) and CD4(+)CD25(-) T-cell proliferation, and Th1 cytokine secretion, and also suppressed purified NK-cell proliferation in response to IL-15, compared to naive splenic B cells. Acquired B regulatory function required direct tumor cell: B-cell contact, and was partially reversed by antibody to TGF-ß or PD-L1, leading to tumor rejection in vivo B-cell acquisition of a suppressive phenotype following tumor infiltration may result in profound inhibition of T-cell anti-tumor responses.

B lymphocyte inhibition of anti-tumor response depends on expansion of Treg but is independent of B-cell IL-10 secretion.

The mechanisms by which B lymphocytes inhibit anti-tumor immunity remain poorly understood. Murine EMT-6 mammary tumors grow readily in immune competent mice (BALB/c), but poorly in B-cell-deficient µ(-/-) BALB/c mice (BCDM). T regulatory cell (Treg) expansion and function were impaired in BCDM compared with BALB/c. In this study, we compared tumor growth, Treg cell proliferation, tumor lymphocyte infiltration and cytolytic T cell activity in BALB/c, BCDM and BCDM partially reconstituted with B cells by adoptive transfer (BCDM+B). Partial reconstitution of BCDM with adoptively transferred B cells restored EMT-6 tumor growth, which was independent of IL-10 secretion by B cells. Instead, high frequencies of intratumoral B cells were associated with increased recruitment and proliferation of Treg cells within the tumor microenvironment. The B-cell-dependent accumulation of Treg within the tumor microenvironment was associated with reduced tumor infiltration by CD49+ NK and CD8+ T cells and reduced cytotoxic T cell activity against EMT-6 targets. Our studies indicate that tumor-dependent immunosuppression of T-cell-mediated anti-tumor immunity is coordinated within the tumor microenvironment by B-cell-dependent cross talk with Treg cells, which does not require production of IL-10 by B cells.

Targeted delivery of an antibody-mutant human endostatin fusion protein results in enhanced antitumor efficacy.

The antiangiogenic protein endostatin showed considerable preclinical antitumor activity, but limited efficacy in phase I/II trials. Prior studies using an anti-HER2 antibody-murine endostatin fusion showed enhanced antitumor activity compared to anti-HER2 antibody or endostatin given alone, or in combination. We have generated two anti-HER2 human endostatin fusion proteins by fusing either wild-type or a mutant human endostatin (huEndo-P125A) to the 3' end of a humanized anti-HER2 IgG3 antibody. Antitumor efficacy was examined in murine and human breast tumor models. HuEndo-P125A antibody fusion protein (αHER2-huEndo-P125A) inhibited VEGF and bFGF induced endothelial cell proliferation, and tube formation in vitro, more efficiently than endostatin alone, wild-type endostatin fusion protein (αHER2-huEndo), or parental anti-HER2 antibody (αHER2 IgG3). Wild-type and mutant human endostatin was rapidly cleared from serum in mice (T½(2) = 2.0-2.1 hours), whereas αHER2-huEndo fusion proteins had a significantly prolonged half-life (T½(2) = 40.7-57.5 hours). Treatment of SK-BR-3 breast cancer xenografts with anti-HER2 IgG3-huEndo-P125A fusion resulted in greater inhibition of tumor growth and improved survival, compared to treatment with either αHER2 IgG3 (P = 0.025), human endostatin (P = 0.034), or anti-HER2 IgG3-huEndo (P = 0.016). αHER2-huEndo-P125A specifically inhibited tumors expressing HER2 in mice simultaneously implanted with murine mammary tumor EMT6 cells and with EMT6 engineered to express HER2 antigen (EMT6-HER2). Targeting of endostatin using antibody fusion proteins could improve antitumor activity of either anti-HER2 antibody and/or endostatin and provides a versatile approach that could be applied to other tumor targets with alternative antibody specificities.

Model for in vivo progression of tumors based on co-evolving cell population and vasculature.

With countless biological details emerging from cancer experiments, there is a growing need for minimal mathematical models which simultaneously advance our understanding of single tumors and metastasis, provide patient-personalized predictions, whilst avoiding excessive hard-to-measure input parameters which complicate simulation, analysis and interpretation. Here we present a model built around a co-evolving resource network and cell population, yielding good agreement with primary tumors in a murine mammary cell line EMT6-HER2 model in BALB/c mice and with clinical metastasis data. Seeding data about the tumor and its vasculature from in vivo images, our model predicts corridors of future tumor growth behavior and intervention response. A scaling relation enables the estimation of a tumor's most likely evolution and pinpoints specific target sites to control growth. Our findings suggest that the clinically separate phenomena of individual tumor growth and metastasis can be viewed as mathematical copies of each other differentiated only by network structure.

Delivery of NKG2D ligand using an anti-HER2 antibody-NKG2D ligand fusion protein results in an enhanced innate and adaptive antitumor response.

NKG2D ligands link the innate and adapative immune response by activating the receptors expressed on effector cells of both the innate (NK) and adaptive immune systems (CD8(+) T cells). In this study, we explored the potential therapeutic utility of this intersection by fusing the murine NKG2D ligand Rae-1ß to the 3' end of an anti-HER2 IgG3 antibody containing an intact Fc domain (anti-HER2 IgG3-Rae-1ß), thereby targeting an NK cell activation signal to HER2+ breast tumor cells. The antitumor efficacy of this anti-HER2-Rae-1ß fusion protein was examined in a mouse mammary tumor model engineered to express HER2 (EMT6-HER2 cells). We observed an enhanced cytotoxic response of NK effectors against EMT-HER2 cells in vitro. Mice implanted on one flank with EMT6-HER2 cells and contralaterally with control EMT6 cells exhibited rapid regression of EMT6-HER2 tumors but delayed regression of contralateral EMT6 tumors. IFNγ was implicated, given a lack of antitumor efficacy in IFNγ(-/-) mice. Depletion of either NK cells or CD8(+) T cells abrogated tumor growth inhibition, suggesting essential roles for each in the observed antitumor activity. Mice rejecting EMT6-HER2 tumors after anti-HER2-Rae-1ß treatment showed markedly decreased tumor growth when rechallenged with EMT6-HER2 or EMT6 cells, whereas both EMT6 and EMT6-HER2 cells grew in control mice, indicating the development of an adaptive memory response. Our findings demonstrate that administration of an antibody-NKG2D ligand fusion protein can enhance innate and adaptive immune antitumor responses, also evoking additional nontargeted antigens to enhance the potential clinical utility of this approach.

Augmentation of anti-tumor responses of adoptively transferred CD8+T cells in the lymphopenic setting by HSV amplicon transduction.

Treatment of cancer with cytotoxic agents may induce lymphopenia. Adoptively transferred T cells have been reported to display enhanced anti-tumor efficacy in the lymphopenic setting. We reasoned that the anti-tumor effects of adoptively transferred cells in the lymphopenic host could be further augmented through local provision of an innate stimulus in the tumor bed. Utilizing a model in which mice were irradiated to induce lymphopenia, with limited shielding to allow tumor growth, we demonstrate that "triple" therapy consisting of radiation-induced lymphopenia, adoptive transfer of naïve CD8+ T cells, and intra-tumoral HSV amplicon injection resulted in reduced tumor growth compared to the combination of any two of the aforementioned interventions. To gain insight into the mechanism underlying this effect we studied the effects of HSV amplicon transduction into tumors on cytokine expression and on anti-tumor specific T cells. HSV amplicon transduction specifically induced several cytokine mRNAs including IFN-gamma, and IP-10. Adoptively transferred transgenic OT-1 T cells directed against Ovalbumin were more effective against Ovalbumin-expressing tumors when combined with intra-tumoral HSV amplicon injections in the lymphopenic host. Following intra-tumoral HSV-amplicon injections, anti-tumor T cells secreted higher levels of interferon-gamma in response to in-vitro re-stimulation with tumor cells, implying that HSV amplicon injection provided a strong signal for T cell activation. Combining adoptive transfer of naïve T cells in the lymphopenic setting with local T cell stimulation may facilitate expansion and activation of anti-tumor T cell populations in vivo, resulting in enhanced anti-tumor responses without the need to resort to prolonged in vitro T cell culture and/or manipulation.

Enhanced inhibition of murine tumor and human breast tumor xenografts using targeted delivery of an antibody-endostatin fusion protein.

Endostatin can inhibit angiogenesis and tumor growth in mice. A potential limitation of endostatin as an antitumor agent in humans is the short serum half-life of the protein that may decrease effective concentration at the site of tumor and necessitate frequent dosing. In an effort to improve antitumor activity, endostatin was fused to an antibody specific for the tumor-selective HER2 antigen to create an antibody-endostatin fusion protein (anti-HER2 IgG3-endostatin). Normal endostatin rapidly cleared from serum in mice (T(1/2)(2), = 0.6-3.8 hours), whereas anti-HER2 IgG3-endostatin had a prolonged half-life (90% intact; T(1/2)(2), 40.2-44.0 hours). Antigen-specific targeting of anti-HER2 IgG3-endostatin was evaluated in BALB/c mice implanted with CT26 tumors or CT26 tumors engineered to express the HER2 antigen (CT26-HER2). Radio-iodinated anti-HER2 IgG3-endostatin preferentially localized to CT26-HER2 tumors relative to CT26 tumors. Administration of anti-HER2 IgG3-endostatin to mice showed preferential inhibition of CT26-HER2 tumor growth compared with CT26. Anti-HER2 IgG3-endostatin also markedly inhibited the growth of human breast cancer SK-BR-3 xenografts in severe combined immunodeficient mice. Anti-HER2 IgG3-endostatin inhibited tumor growth significantly more effectively than endostatin, anti-HER2 IgG3 antibody, or the combination of antibody and endostatin. CT26-HER2 tumors treated with the endostatin fusion protein had decreased blood vessel density and branching compared with untreated CT26-HER2 or CT26 treated with the fusion protein. The enhanced effectiveness of anti-HER2 IgG3-endostatin may be due to a longer half-life, improved serum stability, and selective targeting of endostatin to tumors, resulting in decreased angiogenesis. Linking of an antiangiogenic protein, such as endostatin, to a targeting antibody represents a promising and versatile approach to antitumor therapy.

Increased rejection of primary tumors in mice lacking B cells: inhibition of anti-tumor CTL and TH1 cytokine responses by B cells.

We investigated the role of B cells in tumor immunity by studying immune responses of mice genetically lacking B cells to primary tumors. IgM(-/-) B cell-deficient mice (BCDM) exhibited enhanced resistance to 3 histologically diverse syngeneic tumors as compared to the wild-type (WT) mice. EL4 thymoma and MC38 colon carcinoma grew progressively in WT mice, but regressed spontaneously in BCDM whereas growth of B16 melanoma was slowed significantly in BCDM as compared to the WT mice. BCDM exhibited increased T cell infiltration of tumors, higher T(H)1 cytokine response and, in the case of MC38, a higher anti-tumor CTL response. The increased tumor resistance of BCDM did not seem to result from intrinsic changes in their non-B immunocytes because adoptive transfer of WT splenic B cells to BCDM abrogated tumor rejection and resulted in diminished anti-tumor T(H)1 cytokine and CTL responses. Studies involving BCR-transgenic mice indicated that B cells may inhibit anti-tumor T cell responses by antigen-nonspecific mechanisms since neither tumor-specific antibodies nor cognate T:B interactions were necessary for inhibition of tumor immunity by B cells. IFN-gamma secretion in splenocyte:tumor co-cultures of tumor-challenged BCDM was inhibited by WT but not CD40(-/-) B cells indicating that B cells may inhibit anti-tumor T(H)1 cytokine responses in a CD40-dependent manner. Adoptive transfer of CD40(-/-) B cells into BCDM resulted in restored growth of MC38 suggesting additional factors other than CD40 are involved in dampening anti-tumor responses. The effects of B cells on anti-tumor response warrant further study.

Comparison of the antitumor efficacies of Her-2/neu DNA vaccines inducing contrasting IgG immunity but comparable CTL activity in mice.

The relative importance of CTL and antibodies in rejecting Her-2/neu-expressing tumors was evaluated in preventive and therapeutic models by DNA vaccination. Four human Her-2/neu-expressing plasmids (pNeu(TM), pNeu(ECD), pNeu(TM-gDs), and pNeu(ECD-gDs)) were generated encoding either the transmembrane and extracellular domains or the extracellular domain. Interestingly, these plasmids demonstrated substantial difference in inducing Her-2/neu-specific serum IgG according to their signal sequence when injected in BALB/c mice. pNeu(TM) and pNeu(ECD) induced high serum IgG titers. pNeu(TM-gDs) and pNeu(ECD-gDs) induced low or very low serum IgG titers, respectively. As a result, mice vaccinated with not only pNeu(ECD) but also pNeu(ECD-gDs) exhibited complete eradication of a small number of tumor cells. Nevertheless, when the number of tumor cells was increased in a therapeutic model, only pNeu(ECD) exhibited statistically significant antitumor immunity. These studies demonstrate that strong CTL may be sufficient in tumor prevention, but the collaboration of CTL and antibody may be required in tumor therapy.

Both the epitope specificity and isotype are important in the antitumor effect of monoclonal antibodies against Her-2/neu antigen.

The Her-2/neu oncogene, which encodes a growth factor receptor, was implicated in the malignancy of human adenocarcinomas. Antibodies directed to this molecule have been previously shown to have an antitumor effect in vivo. In an attempt to understand the mechanisms of the antitumor activity, we generated 2 monoclonal antibodies (mAbs), HRO G1 and HRT G1, that recognize different epitopes on Her-2/neu. Both of the mAbs bound HER2/neu on the tumor surface, resulting in phosphorylation of HER2/neu. We also generated IgG2a and IgG2b mAbs from these 2 mAbs, respectively. The results of in vitro studies showed that these anti-Her-2/neu mAbs could not inhibit the growth of the tumor cells that express Her-2/neu molecules by themselves. However, in an antibody-dependent cellular cytotoxicity study using mouse splenocytes as effector cells, HRT mAbs had antitumor activities superior to those of HRO mAbs, indicating that the epitope specificity may also partake in antibody-dependent cellular cytotoxicity with antibody isotype. In a complement-dependent cytotoxicity study, the IgG2a and IgG2b mAbs showed stronger effects than IgG1 isotype mAbs irrespective of the epitope specificities. The results of in vivo studies also showed that HRT mAbs had superior antitumor activity to those of HRO mAbs. The antitumor activity was most prominent in the HRT G2b isotype among HRT mAbs. HRT G1 also showed a moderate antitumor effect, while HRT G2a showed only slight inhibition effect. These data indicate that both the epitope specificity and the differences in Fc region of mAbs could play important roles in the antitumor activities.

An anti-transferrin receptor-avidin fusion protein exhibits both strong proapoptotic activity and the ability to deliver various molecules into cancer cells.

We have developed an antibody fusion protein (anti-rat TfR IgG3-Av) with the ability to deliver different molecules into cancer cells. It consists of avidin genetically fused to the C(H)3 region of a human IgG3 specific for the rat transferrin receptor. It forms strong, noncovalent interactions with biotinylated molecules such as glucose oxidase and beta-galactosidase, and delivers them into the rat myeloma cell line Y3-Ag1.2.3 through receptor-mediated endocytosis. Importantly, the beta-galactosidase retains activity after internalization. Furthermore, we have unexpectedly discovered that anti-rat TfR IgG3-Av, but not a recombinant anti-rat TfR IgG3 or a nonspecific IgG3-Av, possesses proapoptotic activities against Y3-Ag1.2.3 and the rat T cell lymphoma cell line C58 (NT) D.1.G.OVAR.1. These activities were not observed in two rat cell lines of nonhematopoietic lineage (bladder carcinoma BC47 and gliosarcoma 9L). Anti-human TfR IgG3-Av also demonstrated proapoptotic activity against the human erythroleukemia cell line K562. Studies showed that anti-rat TfR IgG3-Av exists as a dimer, suggesting that cross-linking of the surface transferrin receptor may be responsible for the cytotoxic activity. These findings demonstrate that it is possible to transform an antibody specific for a growth factor receptor that does not exhibit inhibitory activity into a drug with significant intrinsic cytotoxic activity against selected cells by fusing it with avidin. The antitumor activity may be enhanced by delivering biotinylated therapeutics into cancer cells. Further development of this technology may lead to effective therapeutics for in vivo eradication of hematological malignancies, and ex vivo purging of cancer cells in autologous transplantation.

Genetically engineered antibodies and their application to brain delivery.

Techniques of genetic engineering and expression have been applied to the production of antibodies in a variety of expression systems. Combinatorial libraries produced in bacteriophage may present an alternative to animal immunization as a source of antigen-binding specificities. Transfectomas which express genetically engineered antibody genes provide one approach to overcoming some of the limitations inherent in classical monoclonal antibodies. Novel antibodies have been produced with a variety of modifications: as chimeric antibodies, as 'humanized' antibodies, with catalytic groups, as bifunctional or fusion proteins and as functional fragments such as Fabs or Fvs. The domain structure of the antibody is favorable to such manipulation; the novel proteins often retain their antibody-derived activity and acquire new properties as well. Chimeric and CDR-grafted antibodies have been effective in immunotherapy, but problems of immunogenicity remain. Careful analysis and comparison of effector functions among immunoglobulin isotypes may be applied to the design of effective therapeutic antibodies. In addition, antibody combining specificities can be joined with non-immunoglobulin sequences thereby providing properties not usually found in antibodies. In particular, antibodies fused with the growth factors insulin-like growth factor(IGF)-1, IGF-2 and transferrin have shown increased uptake into the brain parenchyma. These fusion proteins provide a family of reagents with many potential applications.