Cagan Gurer

Broad CTL response is required to clear latent HIV-1 due to dominance of escape mutations

Despite antiretroviral therapy (ART), human immunodeficiency virus (HIV)-1 persists in a stable latent reservoir, primarily in resting memory CD4+ T cells. This reservoir presents a major barrier to the cure of HIV-1 infection. To purge the reservoir, pharmacological reactivation of latent HIV-1 has been proposed and tested both in vitro and in vivo. A key remaining question is whether virus-specific immune mechanisms, including cytotoxic T lymphocytes (CTLs), can clear infected cells in ART-treated patients after latency is reversed. Here we show that there is a striking all or none pattern for CTL escape mutations in HIV-1 Gag epitopes. Unless ART is started early, the vast majority (>98%) of latent viruses carry CTL escape mutations that render infected cells insensitive to CTLs directed at common epitopes. To solve this problem, we identified CTLs that could recognize epitopes from latent HIV-1 that were unmutated in every chronically infected patient tested. Upon stimulation, these CTLs eliminated target cells infected with autologous virus derived from the latent reservoir, both in vitro and in patient-derived humanized mice. The predominance of CTL-resistant viruses in the latent reservoir poses a major challenge to viral eradication. Our results demonstrate that chronically infected patients retain a broad-spectrum viral-specific CTL response and that appropriate boosting of this response may be required for the elimination of the latent reservoir.

Priming of protective T cell responses against virus-induced tumors in mice with human immune system components

Many pathogens that cause human disease infect only humans. To identify the mechanisms of immune protection against these pathogens and also to evaluate promising vaccine candidates, a small animal model would be desirable. We demonstrate that primary T cell responses in mice with reconstituted human immune system components control infection with the oncogenic and persistent Epstein-Barr virus (EBV). These cytotoxic and interferon-γ–producing T cell responses were human leukocyte antigen (HLA) restricted and specific for EBV-derived peptides. In HLA-A2 transgenic animals and similar to human EBV carriers, T cell responses against lytic EBV antigens dominated over recognition of latent EBV antigens. T cell depletion resulted in elevated viral loads and emergence of EBV-associated lymphoproliferative disease. Both loss of CD4+ and CD8+ T cells abolished immune control. Therefore, this mouse model recapitulates features of symptomatic primary EBV infection and generates T cell–mediated immune control that resists oncogenic transformation.

Targeting the nuclear antigen 1 of Epstein-Barr virus to the human endocytic receptor DEC-205 stimulates protective T-cell responses.

Dendritic cells (DCs) express many endocytic receptors that deliver antigens for major histocompatibility class (MHC) I and II presentation to CD8(+) and CD4(+) T cells, respectively. Here, we show that targeting Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) to one of them, the human multilectin DEC-205 receptor, in the presence of the DC maturation stimulus poly(I:C), expanded EBNA1-specific CD4(+) and CD8(+) memory T cells, and these lymphocytes could control the outgrowth of autologous EBV-infected B cells in vitro. In addition, using a novel mouse model with reconstituted human immune system components, we demonstrated that vaccination with alphaDEC-205-EBNA1 antibodies primed EBNA1-specific IFN-gamma-secreting T cells and also induced anti-EBNA1 antibodies in a subset of immunized mice. Because EBNA1 is the one EBV antigen that is expressed in all proliferating cells infected with this virus, our data suggest that DEC-205 targeting should be explored as a vaccination approach against symptomatic primary EBV infection and against EBV-associated malignancies.

Virus-specific CD4+ T cells: ready for direct attack

CD4+ T cells are classically thought to orchestrate adaptive immune responses. But recent studies demonstrate that they can also kill infected cells directly. A new paper shows that highly efficient processing of Epstein Barr virus (EBV) glycoproteins for presentation on MHC class II makes virus-transformed B cells susceptible to lysis by CD4+ T cells. Thus, antiviral vaccines should aim to stimulate both helper and cytolytic CD4+ T cells.

A novel humanized mouse model with significant improvement of class-switched, antigen-specific antibody production

Humanized mice are a powerful tool for the study of human hematopoiesis and immune function in vivo. However, the existing models cannot support robust adaptive immune responses, especially the generation of class-switched, antigen-specific antibody responses. Here we describe a new mouse strain, in which human interleukin 6 (IL-6) gene encoding the cytokine that is important for B- and T-cell differentiation was knocked into its respective mouse locus. The provision of human IL-6 not only enhanced thymopoiesis and periphery T-cell engraftment, but also significantly increased class switched memory B cells and serum immunoglobulin G (IgG). In addition, immunization with ovalbumin (OVA) induced OVA-specific B cells only in human IL-6 knock-in mice. These OVA-specific antibodies displayed the highest frequency of somatic mutation, further suggesting that human IL-6 is important for efficient B-cell activation and selection. We conclude that human IL-6 knock-in mice represent a novel and improved model for human adaptive immunity without relying on complex surgery to transplant human fetal thymus and liver. These mice can therefore be used to exploit or evaluate immunization regimes that would be unethical or untenable in humans.

Humanized mouse model supports development, function, and tissue residency of human natural killer cells

Significance Humanized mice represent a promising approach to study the human immune system in health and disease. However, insufficient development and function of human lymphocytes limit the applicability of humanized mice for cancer biology and therapy. We demonstrate that human SIRPA and IL15 knock-in (SRG-15) mice support efficient development of circulating and tissue-resident natural killer (NK) cells, intraepithelial lymphocytes, and innate lymphoid cell subsets. In contrast to previous humanized mouse models, human NK cells in SRG-15 mice mediate efficient antibody-dependent cellular cytotoxicity and thereby enable NK cell-targeted cancer immunotherapy of tumor xenografts. As such, SRG-15 humanized mice may facilitate translational research by enabling the development of novel NK and CD8+ T cell-based therapeutic approaches that target human infections and malignancies. Immunodeficient mice reconstituted with a human immune system represent a promising tool for translational research as they may allow modeling and therapy of human diseases in vivo. However, insufficient development and function of human natural killer (NK) cells and T cell subsets limit the applicability of humanized mice for studying cancer biology and therapy. Here, we describe a human interleukin 15 (IL15) and human signal regulatory protein alpha (SIRPA) knock-in mouse on a Rag2−/− Il2rg−/− background (SRG-15). Transplantation of human hematopoietic stem and progenitor cells into SRG-15 mice dramatically improved the development and functional maturation of circulating and tissue-resident human NK and CD8+ T cells and promoted the development of tissue-resident innate lymphoid cell (ILC) subsets. Profiling of human NK cell subsets by mass cytometry revealed a highly similar expression pattern of killer inhibitory receptors and other candidate molecules in NK cell subpopulations between SRG-15 mice and humans. In contrast to nonobese diabetic severe combined immunodeficient Il2rg−/− (NSG) mice, human NK cells in SRG-15 mice did not require preactivation but infiltrated a Burkitt’s lymphoma xenograft and efficiently inhibited tumor growth following treatment with the therapeutic antibody rituximab. Our humanized mouse model may thus be useful for preclinical testing of novel human NK cell-targeted and combinatory cancer immunotherapies and for studying how they elicit human antitumor immune responses in vivo.

Abstract A193: Fully human bispecific antibodies induce potent anti-tumor effects against prostate tumors in mice

Despite multiple new treatment options for patients with castrate-resistant prostate cancer (CRPC), this is still an area of un-met need. Prostate specific membrane antigen (PSMA) is highly expressed in prostate cancer and expression is maintained in CRPC, making it an attractive target for late stage prostate cancer. We have developed a novel full-length human IgG bispecific antibody that binds to human and monkey PSMA and CD3 and results in T cell activation via the CD3 complex in the presence of PSMA-expressing tumors. In cell-based assays, our PSMAxCD3 bispecific resulted in target cell-dependent activation and cytokine release by human T cells, and efficient redirected T cell lysis of prostate tumor cells. Of note, in the absence of PSMA-expressing tumor cells, no T cell activation was seen. Our bispecific antibody also resulted in redirected killing by cynomolgus monkey T cells, enabling pre-clinical toxicity studies. To examine bispecific anti-tumor efficacy in vivo, we developed xenogenic and syngeneic tumor models. In studies with human tumor cells, NOD SCID IL2R gamma deficient (NSG) mice were co-implanted with human peripheral blood mononuclear (PBMC) cells plus human prostate tumor cells (22Rv1 or C4-2), and the mice treated immediately with PSMAxCD3 i.p. Significant inhibition of tumor growth was observed against both human prostate tumors with dose levels as low as 0.004mg/kg. Importantly, PSMAxCD3 bispecific antibodies could also suppress growth of established C4-2 tumors when PBMCs were transferred i.p.. Our PSMAxCD3 bispecific was also tested in mice engrafted with human hematopoietic CD34+ stem cells. Newborn SIRPα BALB/c-Rag2null IL2rγnull (BRG) pups were engrafted with hCD34+ fetal liver cells as hematopoietic progenitor cells, which gave rise to human T, B, and NK cells, as well as granulocytes, monocytes, and DCs. Once engraftment was achieved (3 months later), mice were given C4-2 tumor cells subcutaneously. Eight days after tumor implantation, mice were treated with PSMAxCD3 (0.4mg/kg) followed by twice weekly doses. The PSMAxCD3 bispecific reduced tumor growth in this model. To examine the effects of our lead-candidate bispecifics in an immune-competent model, we engineered mice to express both human CD3 and PSMA in the corresponding murine loci. T cells from these mice were efficiently activated by antibodies to human CD3, and the expression of human PSMA allowed assessment of effects on normal tissues that may express PSMA. T cell activation, proliferation and cytokine production in response to our bispecific antibodies were examined in the absence of tumor as well as the absence of target. To assess anti-tumor efficacy in this model, a murine prostate cancer line expressing human PSMA was generated and tumors cells were implanted s.c. into these mice. Our bispecific antibodies significantly suppressed tumor growth when mice were treated at the time of implantation and also when treatment was delayed until tumors were established. Moreover, T cells were still present at normal levels in the spleen of mice treated with our bispecific at the end of the experiment and the treatment was well-tolerated. Taken together, these studies show potent anti-tumor activity of our PSMAxCD3 bispecific in several different tumor models and also demonstrate that treatment was well-tolerated in mice expressing human PSMA and human CD3. Citation Format: Alison Crawford, Kristin Vazzana, Jeffrey VanValkenburgh, Lauric Haber, Jennifer Principio, Cagan Gurer, Kara Olson, Eric Smith, Gavin Thurston, Jessica R. Kirshner. Fully human bispecific antibodies induce potent anti-tumor effects against prostate tumors in mice. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A193.