Browsing by Subject "Antibodies"

Objective

Background

Methods

Results

Conclusion

Glioblastoma (GBM) is a uniformly lethal cancer with an overall survival of less than 15 months. Aggressive standard of care therapies fail to eradicate these tumors and are non-specific, resulting in incapacitating toxicities. In contrast to such therapies, by virtue of exploiting the inherent specificity and vigilance of the immune system, immunotherapy provides an exquisitely precise approach for safe and effective tumor treatment. Specifically, peptide vaccines offer a promising strategy for inducing potent cytotoxic glioma-specific immune responses. However, they are limited by various mechanisms of glioma-mediated immunosuppression, including low/dysfunctional antigen-presentation, an increased fraction of regulatory T cells, T cell inhibitory pathways, and cytokine dysregulation. Such challenges can be overcome by the combined use of immunomodulatory adjuvants to improve the setting in which T cells recognize and respond to glioma antigens. To this end, a clinically-relevant high-affinity human anti-human CD27 immunomodulatory antibody (αhCD27) that induces potent antitumor T cell responses through engagement of the CD27 T cell costimulatory pathway was recently developed. This antibody is efficacious as a monotherapy in preclinical tumor models and has given rise to significant clinical responses in early phase trials. Given the preliminary success of monotherapy αhCD27 in inducing endogenous antitumor immunity, the overall goal of this dissertation research was to develop a peptide vaccine platform that employs αhCD27 as a vaccine adjuvant for its translation as a novel brain tumor immunotherapeutic.

Chapter 1 provides an overview of brain tumor immunotherapy, including the evolution of the field to date, various genres of treatment modalities, and ongoing progress and challenges. Chapter 2 discusses the approach of T cell immunomodulation, an emerging field in cancer treatment, including the clinical development of various FDA-approved antibodies and their relevance to brain tumors, synergy with current brain tumor standard of care, and emerging immunomodulatory targets. Chapter 3 provides the rationale for targeting the CD27 costimulatory molecule in particular and includes preliminary data that serves as the basis for the preclinical development of αhCD27 as an immunotherapy for brain tumors. Chapter 4 shows the systematic approach for optimizing αhCD27 as a vaccine adjuvant in a murine model of intracranial melanoma alongside a vaccine targeting a model tumor antigen. Lastly, Chapter 5 explores the use of αhCD27 to combat tumor-mediated immunosuppression, an important aspect of its adjuvant activity and the basis for two upcoming phase I clinical trials for malignant glioma.

This dissertation comprises original research as well as figures and illustrations from previously published material used to exemplify distinct concepts in immunotherapy for cancer. These published examples were reproduced with permission in accordance with journal and publisher policies described in the Appendix.

In summary, this work 1) identifies costimulatory T cell immunomodulation as a promising strategy for brain tumor immunotherapy, 2) explores and optimizes the potential for an agonist CD27 to enhance the tumor immune response when combined with a vaccine, 3) has opened up a new line of investigation into the role of CD27 in tumor-mediated immunosuppression, and 4) provides future prospects of utilizing an agonist CD27 antibody as a vaccine adjuvant for the treatment of brain tumors. Together, these studies hold great promise to improve the clinical outlook for brain tumor patients.

Conventional therapy for malignant glioma (MG) fails to specifically target tumor cells. In contrast, immunotherapy offers an exquisitely precise approach, and substantial evidence indicates that if appropriately redirected, T cells can eradicate large, well-established tumors. Even the latest generation of redirected T cell therapies are limited, however, in that they require a centralized manufacturing infrastructure with heavily trained laboratory personnel to genetically modify each patient’s own T cells, use viral transduction which poses uncertain risks, are limited to the initial subset of T cells manipulated and infused, and still face uncertainty as to the optimal T cell phenotype to infuse. This dissertation reports the rational development and clinical translation of a fully-human, bispecific antibody (hEGFRvIII-CD3 bi-scFv) that overcomes these limitations through a recombinant antibody approach that effectively redirects any human T cell to lyse MG cells expressing a tumor-specific mutation of the epidermal growth factor receptor (EGFRvIII).

Chapters one, two and three provide an overview of T cell based immunotherapy of cancer and advances in antibody engineering. Also included is a discussion of the current standard-of-care therapy for MG, other immunotherapeutic approaches for MG, and relevant targets and their therapeutic potential for the treatment of MG.

Chapter four details the rational development of a fully-human, anti-human bispecific antibody, hEGFRvIII-CD3 bi-scFv, for immunotherapy of MG. By generating a panel of fully human bispecific single chain variable fragments (bi-scFvs) and testing their specificity through successive stages of screening and refinement, a highly-expressed and easily purified construct with high-affinity to both CD3 and EGFRvIII target antigens was obtained (hEGFRvIII-CD3 bi-scFv). In vitro, hEGFRvIII-CD3 bi-scFv re-directed naïve human T cells to upregulate cell surface activation markers, secrete pro-inflammatory cytokines, and proliferate in response to antigen-bearing targets. Each of these anti-tumor effects were robust and occurred exclusively in the presence of target antigen, illustrating the specificity of the approach. Using MG cell lines expressing EGFRvIII and patient derived MG with endogenous drivers and levels of EGFRvIII expression, bispecific antibody induced specific lysis was assessed. In each case, hEGFRvIII-CD3 bi-scFv was both potent and antigen-specific, mediating significant target-specific lysis at exceedingly low antibody concentrations. Tumor growth and survival was assessed in xenogenic subcutaneous and orthotopic models of human MG, respectively. In both these models, well-engrafted, patient-derived MG was effectively treated. Intravenous administration of hEGFRvIII-CD3 bi-scFv resulted in significant regression of tumor burden in the subcutaneous models and significantly extended survival in the orthotopic models.

Chapter five discusses challenges associated with intratumoral heterogeneity and details two mechanisms by which bispecific antibodies like hEGFRvIII-CD3 bi-scFv can induce epitope spreading, or an immunological response against tumor antigens other than those initially targeted. These mechanisms include: 1) re-activation of pre-existing T cell clones that have specificity for the tumor but fail to mount an immune response prior to bispecific antibody induced stimulation and 2) tumor cell death that results in release of tumor antigens and subsequent antigen uptake, processing and presentation by antigen presenting cells (APCs) leading to a secondary immune response. The chapter concludes with a discussion of a novel class of recombinant antibody molecules developed as part of this dissertation work, Bispecific Activators of Myeloid Cells (BAMs), that function to enhance phagocytosis and antigen presentation. BAM molecules may be useful in conjunction with other immunotherapeutic modalities to induce epitope spreading and combat intratumoral heterogeneity.

Chapter six describes research examining hEGFRvIII-CD3 bi-scFv in a unique human CD3 transgenic murine model. These studies have furthered the rationale for continued clinical translation of hEGFRvIII-CD3 bi-scFv as a safe and effective therapy for MG and have led to the discovery of a novel mechanism of drug delivery to brain tumors. The transgenic murine model was advantageous given that the CD3 binding portion of the fully-human bispecific antibody binds only to human CD3. Accordingly, the model provides a platform where the same molecule to be advanced to human studies can be tested pre-clinically in a pharmacologically responsive, fully-immunocompetent, syngeneic, murine glioma model. In vitro, hEGFRvIII-CD3 bi-scFv induced potent human CD3 transgenic T cell activation, pro-inflammatory cytokine secretion and proliferation exclusively in the presence of the highly-invasive and aggressive murine glioma, CT-2A, bearing EGFRvIII antigen (CT-2A-EGFRvIII). hEGFRvIII-CD3 bi-scFv mediated significant lysis of CT-2A-EGFRvIII at exceedingly low antibody concentrations. In vivo, hEGFRvIII-CD3 bi-scFv significantly reduced tumor growth in human CD3 transgenic mice with well-established, subcutaneous tumors and extended survival of human CD3 transgenic mice with well-established, orthotopic, MG. In the orthotopic setting, adoptive transfer of pre-activated human CD3 transgenic T cells significantly increased efficacy compared to human CD3 transgenic mice treated with hEGFRvIII-CD3 bi-scFv alone.

This led to the hypothesis that activated T cells, known to cross the blood-brain barrier (BBB) to perform routine immunosurveillance of the central nervous system (CNS), may bind to hEGFRvIII-CD3 bi-scFv intravascularly, via its CD3 receptor, and carry or “hitchhike” the large CD3 binding macromolecule to tumors located behind the BBB. Indeed, studies have revealed that adoptive transfer of activated T cells significantly increases the biodistribution of intravenously administered hEGFRvIII-CD3 bi-scFv to orthotopic glioma. Furthermore, blocking T cell extravasation, using natalizumab, for example, a drug used clinically to prevent the migration of T cells to the CNS in patients with multiple sclerosis, completely abrogates the increase in efficacy observed with the adoptive transfer of activated T cells. This newly uncovered hitchhiking mechanism of drug delivery to the CNS provides an important tool to enhance the immunotherapy of brain tumors and has potentially far-reaching consequences for the treatment of other CNS disorders, such as Alzheimer’s or Parkinson’s disease, where issues regarding drug delivery to the CNS are relevant. To begin to study this mechanism of drug delivery in disorders where the blood-brain barrier is intact, we have developed a novel transgenic murine model that expresses EGFRvIII at very low levels within neurons in the brain and have demonstrated that intravenously administered EGFRvIII-targeted recombinant antibody can accumulate in the CNS parenchyma, even in the presence of an intact BBB.

On the basis of these results, a series of clinical research development activities were conducted that have led to the initiation of a clinical study to test the hitchhiking mechanism of drug delivery in patients and ultimately to translate hEGFRvIII-CD3 bi-scFv therapy as a safe and effective treatment for patients with MG. These activities have resulted in a foundation in pre-clinical toxicology, clinical grade biologic manufacturing, clinical protocol development, and regulatory processes necessary to safely translate hEGFRvIII-CD3 bi-scFv therapy to the clinic.

This has involved conducing an extended single-dose toxicity study of hEGFRvIII-CD3 bi-scFv in animals to support studies in humans, the results of which are detailed in chapter seven. To assess for toxicity, human CD3 transgenic mice were administered hEGFRvIII-CD3 bi-scFv or vehicle as a control. Animals were observed for 14 days post-dosing with an interim necropsy on day two. Endpoints evaluated included clinical sings, body weights, feed consumption, clinical chemistries, hematology, urinalysis, and histopathology. There were no clinical observations, evidence of experimental autoimmune encephalomyelitis (EAE), or change in body weight or feed consumption noted during the study that would be associated with toxicity. Furthermore, no statistical difference was observed between drug- and control-receiving cohorts in hematological parameters or urinalysis and no pathological findings related to EGFRvIII-CD3 bi-scFv administration were observed. Statistical differences were observed between drug-treated and control-treated cohorts for some of the clinical chemistries assessed, such as hematocrit, calcium and phosphorus among the female, 14-day analysis cohorts.

To produce hEGFRvIII-CD3 bi-scFv and autologous activated T cells to be administered to patients for clinical study, chemistry, manufacturing and control protocols for the production of clinical grade hEGFRvIII-CD3 bi-scFv and autologous activated T cells were developed and implemented. The data presented in chapter eight describe optimized manufacturing processes and rationale for the selection and implementation of in-process and release analytical methods. This work includes the generation of a stable Chinese hamster ovary (CHO) cell line that expresses high levels of hEGFRvIII-CD3 bi-scFv, the generation and certification of a current Good Manufacturing Practice (cGMP) master cell bank (MCB), optimization and scale up of upstream and downstream manufacturing procedures, and development of standard operating procedures (SOPs) for the manufacture and assessment of clinical grade hEGFRvIII-CD3 bi-scFv and autologous activated T cells. Together, these have allowed for the production of clinical grade antibody and autologous patient derived cells within Duke University Medical Center. The production of recombinant antibodies for use in the clinic is a complex endeavor often performed in industry with teams of highly skilled scientists who test and optimize manufacturing protocols using a large, well-established manufacturing infrastructure. The successful production of clinical grade recombinant antibody at an academic center, therefore, represents a significant achievement and would likely be of interest to other academic-based researchers and clinicians embarking on similar clinical endeavors.

Chapter nine describes a clinical protocol for a phase 0 study of hEGFRvIII-CD3 bi-scFv in patients with recurrent EGFRvIII-positive glioblastoma (GBM). The protocol details intravenous administration of single doses of radiolabeled hEGFRvIII-CD3 bi-scFv with and without pre-administration of radiolabeled autologous activated T cells in a given patient. This will allow for imaging studies that will reveal the pharmacokinetics of the recombinant antibody both with and without adoptive transfer of autologous activated T cells. Endpoints include an assessment of the: intracerebral tumor localization of 124iodine (I)-labeled hEGFRvIII-CD3 bi-scFv with and without prior administration of 111indium (In)-labeled autologous T cells; percentage of patients with unacceptable toxicity; percentage of patients alive or alive without disease progression six months after study drug infusion; median progression-free survival; 111-In-autologous T cell intracerebral tumor localization; and percentage of patients who are EGFRvIII-positive at recurrence.

Chapter 10 concludes with a discussion of ongoing and anticipated future pre-clinical and clinical research. Together, these data presented in this dissertation have been submitted to the US Food and Drug Administration (FDA) in support of an Investigational New Drug (IND) application permit for clinical studies of hEGFRvIII-CD3 bi-scFv at Duke University Medical Center. This clinical study of the hitchhiking mechanism of drug delivery and the pharmacokinetics of hEGFRvIII-CD3 bi-scFv may have far reaching implications for disorders of the CNS where drug access past the BBB is relevant and will advance our understanding of hEGFRvIII-CD3 bi-scFv therapy in patients, guiding future clinical study of the molecule as a safe and effective form of immunotherapy for patients with EGFRvIII-positive GBM and other cancers.

Background and objectives

Materials and methods

Results

Conclusion

Human cytomegalovirus (HCMV) is the most common cause of congenital infection worldwide, impacting 1 in 150 live-born infants. Children afflicted by congenital HCMV frequently suffer from lifelong, debilitating neurologic sequelae including microcephaly, sensorineural hearing loss, and cognitive impairment. Natural maternal immunity to HCMV reduces the frequency of congenital infection, but does not prevent the disease altogether. Thus, a vaccine to reduce the incidence and severity of infant infection is a public health priority. Employing a nonhuman primate model of congenital CMV transmission as well as clinical samples from a partially-efficacious HCMV vaccine trial, we sought to examine both the attributes of anti-HCMV immune responses that provide protective immunity as well as the impact of vaccine-elicited immunity on the in vivo HCMV viral population.

First, we used a nonhuman primate model of congenital CMV infection to investigate the ability of preexisting antibodies to protect against placental CMV transmission in the setting of primary maternal infection and subsequent viremia, which is required for placental virus exposure. Pregnant, CD4+ T cell-depleted, rhesus CMV (RhCMV)-seronegative rhesus monkeys were treated with either standardly-produced hyperimmune globulin (HIG) from RhCMV-seropositive macaques or dose-optimized, potently RhCMV-neutralizing HIG prior to intravenous challenge with an RhCMV swarm. HIG passive infusion provided complete protection against fetal loss in both groups. The dose-optimized, RhCMV-neutralizing HIG additionally inhibited placental transmission of RhCMV and reduced viral replication and diversity. Our findings suggest that the presence of durable and potently-neutralizing antibodies at the time of primary infection can prevent transmission of systemically-replicating maternal RhCMV to the developing fetus.

Next, we assessed the properties of antibody responses elicited by glycoprotein B (gB) + MF59 adjuvant subunit vaccination – the most successful HCMV vaccine tested clinicaly to-date, which demonstrated approximately 50% efficacy in preventing HCMV acquisition in multiple phase 2 trials. Plasma from 33 gB/MF59 vaccinees at peak immunogenicity was tested for gB epitope specificity as well as neutralizing and non-neutralizing anti-HCMV effector functions, and compared to an HCMV-seropositive cohort. gB/MF59 vaccination elicited IgG responses with gB-binding magnitude and avidity comparable to natural infection. Additionally, IgG subclass distribution was similar with predominant IgG1 and IgG3 responses induced by gB vaccination and HCMV infection. However, vaccine-elicited antibodies exhibited limited neutralization of the autologous virus, negligible neutralization of multiple heterologous strains, and limited binding responses against gB structural motifs targeted by neutralizing antibodies including AD-1, AD-2, and Domain I. Interestingly, vaccinees had high-magnitude IgG responses against AD-3 linear epitopes, demonstrating immunodominance against this non-neutralizing, cytosolic region. Finally, vaccine-elicited IgG robustly bound trimeric, membrane-associated gB on the surface of transfected or HCMV-infected cells and mediated virion phagocytosis, raising the possibility that non-neutralizing antibody effector functions contributed to the partial protection against HCMV acquisition observed in gB/MF59 vaccinees.

Lastly, we evaluated the impact of gB/MF59-elicited immune responses on the population of viruses acquired by trial participants. In this analysis, we employed quantitative PCR as well as two distinct next-generation sequencing strategies (short amplicon and whole gene) to interrogate genetic differences between the HCMV populations infecting gB/MF59 vaccinees and placebo recipients. For the majority of subject-specific viral populations analyzed, we identified 1 or 2 dominant viral variants, as well as a large number of minor variants present at very low frequency. This finding suggests that the intrahost viral population constitutes a heterogeneous swarm of genetically-distinct virus quasi-species. Additionally, we identified several distinctions between the viral populations of acutely-infected vaccinees and placebo recipients. First, there was reduced magnitude viral shedding in the saliva of gB vaccinees compared to placebo. Furthermore, we noted evidence of genetic compartmentalization at the gB locus in 3 of 4 vaccinees, though only in 1 of 7 placebo recipients. Finally, we observed an enrichment of gB1 genotype HCMV variants among placebo recipients compared to vaccinees, and hypothesize that the gB1 genotype vaccine immunogen might have elicited genotype-specific protection that accounts for the efficacy observed in clinical trial.

Thus, we have made several observations that will inform rational design of the next generation of HCMV vaccines. First, our data suggests that preexisting antibodies can protect against congenital CMV transmission in a rhesus monkey model, and thus that antibodies could be a primary target of vaccines to eliminate congenital HCMV infection. Secondly, our analysis of antibody responses elicited by gB/MF59 vaccination indicates that non-neutralizing antibody functions contributed to the observed 50% vaccine protection and therefore should be a consideration in future vaccine design. Finally, our examination of viral populations in gB/MF59 vaccinees indicates that gB-elicited antibodies had a measurable impact on viral intrahost population dynamics and that gB immunogen strain-specific responses may have defined vaccine protection, suggesting that immunogen strain breadth may be an important factor to consider for future vaccine design.

Background

Methods

Results

Conclusions