Diversity Outbred Mice Reveal the Quantitative Trait Locus and Regulatory Cells of HER2 Immunity.

The genetic basis and mechanisms of disparate antitumor immune response was investigated in Diversity Outbred (DO) F1 mice that express human HER2. DO mouse stock samples nearly the entire genetic repertoire of the species. We crossed DO mice with syngeneic HER2 transgenic mice to study the genetics of an anti-self HER2 response in a healthy outbred population. Anti-HER2 IgG was induced by Ad/E2TM or naked pE2TM, both encoding HER2 extracellular and transmembrane domains. The response of DO F1 HER2 transgenic mice was remarkably variable. Still, immune sera inhibited HER2+ SKBR3 cell survival in a dose-dependent fashion. Using DO quantitative trait locus (QTL) analysis, we mapped the QTL that influences both total IgG and IgG2(a/b/c) Ab response to either Ad/E2TM or pE2TM. QTL from these four datasets identified a region in chromosome 17 that was responsible for regulating the response. A/J and NOD segments of genes in this region drove elevated HER2 Ig levels. This region is rich in MHC-IB genes, several of which interact with inhibitory receptors of NK cells. (B6xA/J)F1 and (B6xNOD)F1 HER2 transgenic mice received Ad/E2TM after NK cell depletion, and they produced less HER2 IgG, demonstrating positive regulatory function of NK cells. Depletion of regulatory T cells enhanced response. Using DO QTL analysis, we show that MHC-IB reactive NK cells exert positive influence on the immunity, countering negative regulation by regulatory T cells. This new, to our knowledge, DO F1 platform is a powerful tool for revealing novel immune regulatory mechanisms and for testing new interventional strategies.

An HER2 DNA vaccine with evolution-selected amino acid substitutions reveals a fundamental principle for cancer vaccine formulation in HER2 transgenic mice.

Enhancement of endogenous immunity to tumor-associated self-antigens and neoantigens is the goal of preventive vaccination. Toward this goal, we compared the efficacy of the following HER2 DNA vaccine constructs: vaccines encoding wild-type HER2, hybrid HER2 vaccines consisting of human HER2 and rat Neu, HER2 vaccines with single residue substitutions and a novel human HER2 DNA vaccine, ph(es)E2TM. ph(es)E2TM was designed to contain five evolution-selected substitutions: M198V, Q398R, F425L, H473R and A622T that occur frequently in 12 primate HER2 sequences. These ph(es)E2TM substitutions score 0 to 1 in blocks substitutions matrix (BLOSUM), indicating minimal biochemical alterations. h(es)E2TM recombinant protein is recognized by a panel of anti-HER2 mAbs, demonstrating the preservation of HER2 protein structure. Compared to native human HER2, electrovaccination of HER2 transgenic mice with ph(es)E2TM induced a threefold increase in HER2-binding antibody (Ab) and elevated levels of IFNγ-producing T cells. ph(es)E2TM, but not pE2TM immune serum, recognized HER2 peptide p95 355LPESFDGDPASNTAP369, suggesting a broadening of epitope recognition induced by the minimally modified HER2 vaccine. ph(es)E2TM vaccination reduced tumor growth more effectively than wild-type HER2 or HER2 vaccines with more extensive modifications. The elevation of tumor immunity by ph(es)E2TM vaccination would create a favorable tumor microenvironment for neoantigen priming, further enhancing the protective immunity. The fundamental principle of exploiting evolution-selected amino acid substitutions is novel, effective and applicable to vaccine development in general.

Identification of actionable targets for breast cancer intervention using a diversity outbred mouse model.

HER2-targeted therapy has improved breast cancer survival, but treatment resistance and disease prevention remain major challenges. Genes that enable HER2/Neu oncogenesis are the next intervention targets. A bioinformatics discovery platform of HER2/Neu-expressing Diversity Outbred (DO) F1 Mice was established to identify cancer-enabling genes. Quantitative Trait Loci (QTL) associated with onset ages and growth rates of spontaneous mammary tumors were sought. Twenty-six genes in 3 QTL contain sequence variations unique to the genetic backgrounds that are linked to aggressive tumors and 21 genes are associated with human breast cancer survival. Concurrent identification of TSC22D3, a transcription factor, and its target gene LILRB4, a myeloid cell checkpoint receptor, suggests an immune axis for regulation, or intervention, of disease. We also investigated TIEG1 gene that impedes tumor immunity but suppresses tumor growth. Although not an actionable target, TIEG1 study revealed genetic regulation of tumor progression, forming the basis of the genetics-based discovery platform.

Induction of proapoptotic antibodies to triple-negative breast cancer by vaccination with TRAIL death receptor DR5 DNA.

TNF-related apoptosis-inducing ligand receptor 2 [TRAIL-R2 or death receptor 5 (DR5)] is expressed at elevated levels in a broad range of solid tumors to mediate apoptotic signals from TRAIL or agonist antibodies. We tested the hypothesis that DR5 DNA vaccination will induce proapoptotic antibody to trigger apoptosis of tumor cells. BALB/c mice were electrovaccinated with DNA-encoding wild-type human DR5 (phDR5) or its derivatives. Resulting immune serum or purified immune IgG induced apoptosis in triple-negative breast cancer (TNBC) cells, which were also TRAIL sensitive. The proapoptotic activity of immune serum at dilutions of 0.5-2% was comparable to that of 1-2 µg/ml of TRAIL. Apoptotic activity of immune serum was enhanced by antibody crosslinking. Apoptotic cell death induced by anti-DR5 antibody was shown by the cleavage of PARP and caspase-3. In contrast, immune serum had no effect on the proliferation of activated human T cells, which expressed low levels of DR5. In vivo, hDR5 reactive immune serum prevented growth of SUM159 TNBC cells in severe combined immune-deficient mice. DR5-specific IFN-γ-secreting T cells were also induced by DNA vaccination. Furthermore, the feasibility to overcome immune tolerance to self DR5 was shown by the induction of mouse DR5-binding antibody after electrovaccination of BALB/c mice with pmDR5ectm-Td1 encoding a fusion protein of mouse DR5 and an immunogenic fragment of tetanus toxin. These findings support DR5 as a promising vaccine target for controlling TNBC and other DR5-positive cancers.

Early mammalian erythropoiesis requires the Dot1L methyltransferase.

Histone methylation is an important regulator of gene expression; its coordinated activity is critical in complex developmental processes such as hematopoiesis. Disruptor of telomere silencing 1-like (DOT1L) is a unique histone methyltransferase that specifically methylates histone H3 at lysine 79. We analyzed Dot1L-mutant mice to determine influence of this enzyme on embryonic hematopoiesis. Mutant mice developed more slowly than wild-type embryos and died between embryonic days 10.5 and 13.5, displaying a striking anemia, especially apparent in small vessels of the yolk sac. Further, a severe, selective defect in erythroid, but not myeloid, differentiation was observed. Erythroid progenitors failed to develop normally, showing retarded progression through the cell cycle, accumulation during G0/G1 stage, and marked increase in apoptosis in response to erythroid growth factors. GATA2, a factor essential for early erythropoiesis, was significantly reduced in Dot1L-deficient cells, whereas expression of PU.1, a transcription factor that inhibits erythropoiesis and promotes myelopoiesis, was increased. These data suggest a model whereby DOT1L-dependent lysine 79 of histone H3 methylation serves as a critical regulator of a differentiation switch during early hematopoiesis, regulating steady-state levels of GATA2 and PU.1 transcription, thus controlling numbers of circulating erythroid and myeloid cells.

Tumor regression following DNA vaccination and regulatory T cell depletion in neu transgenic mice leads to an increased risk for autoimmunity.

Modulation of the immune system to amplify anti-tumor immunity carries the risk of developing autoimmune diseases, including hypothyroidism, as seen with cancer patients undergoing clinical trials for immunotherapeutic regimens. Although there is a tendency to view autoimmunity as a positive indicator for cancer immunotherapy, some autoimmune manifestations can be life-threatening and necessitate prolonged medical intervention or removal from trial. We have established murine test models to assess such risks by monitoring, simultaneously, the immune reactivity to tumor-associated rat erbB-2 (neu) and another self Ag, mouse thyroglobulin (mTg). We previously reported that in wild-type, thyroiditis-resistant BALB/c mice that underwent regression of neu(+) TUBO tumors following regulatory T cell (Treg) depletion, immune responses to rat neu and mTg with resultant autoimmune thyroiditis (EAT) were both enhanced. In this study, we tested the balance between tumor immunity and autoimmunity in neu-transgenic BALB NeuT female mice. First, growth and progression of neu(+) tumor were compared in neu tolerant mice treated with either CD25 mAb to deplete Tregs and/or DNA vaccination. Only Treg depletion followed by neu DNA vaccination abrogated tolerance to neu, resulting in complete regression of neu(+) tumors, as well as long-term protection from spontaneous tumorigenesis in 58% of mice. The risk of developing EAT was then assessed by incorporated mTg immunization with or without LPS as adjuvant. In mice with induced tumor regression, mTg response was enhanced with modest increases in EAT development. Therefore, tumor regression induced by Treg depletion and DNA vaccination can exacerbate autoimmunity, which warrants close monitoring during immunotherapy.

Review of immune checkpoint inhibitors in immuno-oncology.

Tumor cells predominantly express self-antigens and overcoming self-tolerance is the primary challenge to effective immunotherapy. Tumors also express ligands for co-inhibitory molecules on immune cells, in order to suppress anti-tumor immunity. Over a decade ago, the first antibodies generated to block the co-inhibitory molecule CTLA-4 was tested in patients with metastatic melanoma. Results from this landmark trial have informed not only the current landscape of checkpoint blockade but also the way in which immunotherapy trial outcomes are determined. Antibodies targeting PD-1 and its ligand, PD-L1, soon followed and use of these checkpoint inhibitors (ICIs) have expanded exponentially. ICI treatment has shown long-lasting clinical benefit in several tumor types and patients refractory to other treatments can often respond to ICI therapy. On the other hand, in some tumor types, the response to ICI is short-lived and tumors eventually recur. Current clinical trials are focused on enhancing anti-tumor effects through combinations of multiple ICIs with agents which cause tumor death, particularly in solid tumors, in order to enhance antigen presentation. It is also important to define which patients will respond to therapy with ICIs as over half of all patients suffer from immune-related adverse events (irAE), some of which are severe and long-lasting.

Her-2 DNA versus cell vaccine: immunogenicity and anti-tumor activity.

Direct comparison and ranking of vaccine formulations in pre-clinical studies will expedite the identification of cancer vaccines for clinical trials. Two human ErbB-2 (Her-2) vaccines, naked DNA and whole cell vaccine, were tested side-by-side in wild type and Her-2 transgenic mice. Both vaccines can induce humoral and cellular immunity to the entire repertoire of Her-2 epitopes. Mice were electro-vaccinated i.m. with a mixture of pGM-CSF and pE2TM, the latter encodes Her-2 extracellular and transmembrane domains. Alternatively, mice were injected i.p. with human ovarian cancer SKOV3 cells that have amplified Her-2. In wild type mice, comparable levels of Her-2 antibodies (Ab) were induced by these two vaccines. However, T cell immunity and protection against Her-2(+) tumors were superior in DNA vaccinated mice. In BALB Her-2 transgenic (Tg) mice, which were tolerant to Her-2, DNA and cell vaccines were administered after regulatory T cells (Treg) were removed by anti-CD25 mAb. Again, comparable levels of Her-2 Ab were induced, but DNA vaccines rendered greater anti-tumor activity. In B6xDR3 Her-2 Tg mice that expressed the autoimmune prone HLA-DR3 allele, higher levels of Her-2 Ab were induced by SKOV3 cell than by Her-2 DNA. But anti-tumor activity was still more profound in DNA vaccinated mice. Therefore, Her-2 DNA vaccine induced greater anti-tumor immunity than cell vaccine, whether mice were tolerant to Her-2 or susceptible to autoimmunity. Through such side-by-side comparisons in appropriate pre-clinical test systems, the more effective vaccine formulations will emerge as candidates for clinical trials.

Control of Her-2 tumor immunity and thyroid autoimmunity by MHC and regulatory T cells.

Immune reactivity to self-antigens in both cancer and autoimmune diseases can be enhanced by systemic immune modulation, posing a challenge in cancer immunotherapy. To distinguish the genetic and immune regulation of tumor immunity versus autoimmunity, immune responses to human ErbB-2 (Her-2) and mouse thyroglobulin (mTg) were tested in transgenic mice expressing Her-2 that is overexpressed in several cancers, and HLA-DRB1*0301 (DR3) that is associated with susceptibility to several human autoimmune diseases, as well as experimental autoimmune thyroiditis (EAT). To induce Her-2 response, mice were electrovaccinated with pE2TM and pGM-CSF encoding the extracellular and transmembrane domains of Her-2 and the murine granulocyte macrophage colony-stimulating factor, respectively. To induce EAT, mice received mTg i.v. with or without lipopolysaccharide. Depletion of regulatory T cells (Treg) with anti-CD25 monoclonal antibody enhanced immune reactivity to Her-2 as well as mTg, showing control of both Her-2 and mTg responses by Treg. When immunized with, Her-2xDR3 and B6xDR3 mice expressing H2(b)xDR3 haplotype developed more profound mTg response and thyroid pathology than Her-2 or B6 mice that expressed the EAT-resistant H2(b) haplotype. In Her-2xDR3 mice, the response to mTg was further amplified when mice were also immunized with pE2TM and pGM-CSF. On the contrary, Her-2 reactivity was comparable whether mice expressed DR3 or not. Therefore, induction of Her-2 immunity was independent of DR3 but development of EAT was dictated by this allele, whereas Tregs control the responses to both self-antigens. These results warrant close monitoring of autoimmunity during cancer immunotherapy, particularly in patients with susceptible MHC class II alleles.

Concurrent induction of antitumor immunity and autoimmune thyroiditis in CD4+ CD25+ regulatory T cell-depleted mice.

When CD4+ CD25+ regulatory T cells are depleted or inactivated for the purpose of enhancing antitumor immunity, the risk of autoimmune disease may be significantly elevated because these regulatory T cells control both antitumor immunity and autoimmunity. To evaluate the relative benefit and risk of modulating CD4+ CD25+ regulatory T cells, we established a new test system to measure simultaneously the immune reactivity to a tumor-associated antigen, neu, and an unrelated self-antigen, thyroglobulin. BALB/c mice were inoculated with TUBO cells expressing an activated rat neu and treated with anti-CD25 monoclonal antibody to deplete CD25+ cells. The tumors grew, then regressed, and neu-specific antibodies and IFN-gamma-secreting T cells were induced. The same mice were also exposed to mouse thyroglobulin by chronic i.v. injections. These mice produced thyroglobulin-specific antibody and IFN-gamma-secreting T cells with inflammatory infiltration in the thyroids of some mice. The immune responses to neu or thyroglobulin were greater in mice undergoing TUBO tumor rejection and thyroglobulin injection than in those experiencing either alone. To the best of our knowledge, this is the first experimental system to assess the concurrent induction and possible synergy of immune reactivity to defined tumor and self-antigens following reduction of regulatory T cells. These results illustrate the importance of monitoring immune reactivity to self-antigens during cancer immunotherapy that involves immunomodulating agents, and the pressing need for novel strategies to induce antitumor immunity while minimizing autoimmunity.

Combining human and rat sequences in her-2 DNA vaccines blunts immune tolerance and drives antitumor immunity.

Immune tolerance to tumor-associated self-antigens poses a major challenge in the ability to mount an effective cancer vaccine response. To overcome immune tolerance to HER-2, we formulated DNA vaccines that express both human HER-2 and heterologous rat Neu sequences in separate plasmids or as single hybrid constructs that encode HER-2/Neu fusion proteins. Candidate vaccines were tested in Her-2 transgenic (Tg) mice of BALB/c (BALB), BALB/cxC57BL/6 F1 (F1), or C57BL/6 (B6) background, which exhibit decreasing immune responsiveness to HER-2. Analysis of various cocktails or hybrid vaccines defined a requirement for particular combination of HER/2/Neu sequences to effectively prime immune effector cells in HER-2 Tg mice. In B6 HER-2 Tg mice, rejection of HER-2-positive tumors protected mice from HER-2-negative tumors, providing evidence of epitope spreading. Our findings show that a strategy of combining heterologous antigen with self-antigens could produce a potent DNA vaccine that may be applicable to other tumor-associated antigens.

Autoimmune thyroiditis as an indicator of autoimmune sequelae during cancer immunotherapy.

Improving cancer immunotherapy by targeting T cell network also triggers autoimmunity. We disrupted regulatory T cell (Treg) function to probe the balance between breast cancer vaccination and autoimmune thyroiditis (EAT) in four models, with particular attention to MHC-associated susceptibility, EAT induction with mouse thyroglobulin (mTg) without adjuvant, and tolerance to Her-2/neu in transgenic mice. 1) In EAT-resistant BALB/c mice, Treg depletion enhanced tumor regression, and facilitated mild thyroiditis induction. 2) In Her-2 tolerant C57BL/6 mice expressing HLA-DR3, an EAT-susceptibility allele, Her-2 DNA vaccinations must follow Treg depletion for (Her-2xDR3)F(1) mice to resist tumor challenge; thyroiditis incidence was moderated by the EAT-resistant IA(b) allele. 3) In neu tolerant, EAT-resistant BALB/c mice, implanted neu(+) tumor also regressed only after Treg depletion and DNA vaccinations. Tumor immunity was long-term, providing protection from spontaneous tumorigenesis. In all three, immune stimuli from concurrent tumor regression and EAT development have a noticeable, mutually augmenting effect. 4) In Treg-depleted, EAT-susceptible CBA/J mice, strong tumor protection was established by immunization with a cell vaccine. mTg injections led to greater thyroiditis incidence and severity. Combination models with MHC class II diversity should facilitate autoimmunity risk assessment and management while generating tumor immunity.

DNA vaccination controls Her-2+ tumors that are refractory to targeted therapies.

Her-2/neu(+) tumor cells refractory to antibody or receptor tyrosine kinase inhibitors are emerging in treated patients. To investigate if drug resistant tumors can be controlled by active vaccination, gefitinib and antibody sensitivity of four neu(+) BALB/c mouse mammary tumor lines were compared. Significant differences in cell proliferation and Akt phosphorylation were observed. Treatment-induced drug resistance was associated with increased chromosomal aberrations as shown by spectral karyotyping analysis, suggesting changes beyond neu signaling pathways. When mice were immunized with pneuTM encoding the extracellular and transmembrane domains of neu, antibody and T-cell responses were induced, and both drug-sensitive and drug-resistant tumor cells were rejected. In T-cell-depleted mice, drug-sensitive tumors were still rejected by vaccination, but drug-refractory tumors survived in some mice, indicating their resistance to anti-neu antibodies. To further test if T cells alone can mediate tumor rejection, mice were immunized with pcytneu encoding full-length cytoplasmic neu that is rapidly degraded by the proteasome to activate CD8 T cells without inducing antibody response. All test tumors were rejected in pcytneu-immunized mice, regardless of their sensitivity to gefitinib or antibody. Therefore, cytotoxic T lymphocytes activated by the complete repertoire of neu epitopes were effective against all test tumors. These results warrant Her-2 vaccination whether tumor cells are sensitive or resistant to Her-2-targeted drugs or antibody therapy.