Browsing by Subject "Antibody"

Malignant gliomas are the most common primary brain tumor in adults, with an incidence of five cases per 100,000 persons per year. Grade IV glioblastoma is the most aggressive form and prognosis remains poor despite the current gold-standard first-line treatment – maximal safe resection and combination of radiotherapy with temozolomide chemotherapy – resulting in a median survival of approximately 20 months. Tumor recurrence occurs in virtually all glioblastoma patients, and there currently exists no accepted treatment for these patients. Recent advances in novel directed therapeutics are showing efficacy and have entered clinical trials. This work spans the pre-clinical and clinical development of a bispecific antibody – EGFRvIII:CD3 bi-scFv – for the treatment of malignant gliomas.

Chapter 1 reviews current front-line immunotherapy research in the fields of antibodies, including BiTEs and checkpoint inhibitors, and tumor vaccinations, including peptide and dendritic cell vaccinations. Furthermore, challenges specific to high-grade gliomas as well as opportunities for combination therapies are discussed. Chapter 2 introduces the architecture of the novel bispecific antibody EGFRvIII:CD3 bi-scFv and provides an overview of the molecule’s efficacy in various models. EGFRvIII:CD3 bi-scFv is a truncated antibody with dual specificity. One arm targets the epidermal growth factor receptor mutation variant III (EGFRvIII), a tumor-specific antigen found on glioblastoma. The other arm targets the human CD3 receptor on T cells. As an obligate bispecific antibody, simultaneous binding of both receptors by multiple EGFRvIII:CD3 bi-scFv’s results in the crosslinking of CD3 receptor, activation of T cells, and release of perforin/granzyme which lyses the proximal EGFRvIII-expressing tumor cells. EGFRvIII:CD3 bi-scFv effectively treats orthotopic patient-derived malignant glioma and syngeneic glioblastoma.

Chapter 3 outlines the in-house development of a scalable clinical production process using a WAVE (GE) bioreactor and describes the cGMP-compliant clinical production of EGFRvIII:CD3 bi-scFv. The 250-liter cGMP-production run yielded more than four grams of clinical drug material.

Chapter 4 demonstrates that EGFRvIII:CD3 bi-scFv produced using the cGMP development process is efficacious in both in vitro and in vivo models of glioblastoma. The chapter also describes the approach used to calculate the starting dose for the upcoming first-in-human clinical trial. First-in-human clinical trials require careful selection of a safe yet biologically relevant starting dose. Typically, such starting doses are selected based on toxicity studies in a pharmacologically relevant animal model. However, with the advent of target-specific and highly active immunotherapeutics, both the Food and Drug Administration (FDA) and the European Medicines Agency (EMA) have provided guidance that recommend determining a safe starting dose based on a minimum anticipated biological effect level (MABEL) approach. In order to establish a first-in-human dose, as advised by the FDA for bispecific antibodies, this work uses a MABEL approach to select a safe starting dose for EGFRvIII:CD3 bi-scFv, based on a combination of in vitro data, in vivo animal studies, and theoretical human receptor occupancy modeling. Using the most conservative approach to the MABEL assessment, a dose of 57.4 ng EGFRvIII:CD3 bi-scFv/kg body weight was selected as a safe starting dose for a first-in-human clinical study.

Chapter 5 describes the pharmacokinetic properties of EGFRvIII:CD3 bi-scFv, a necessary step in the drug development process. Using microflow liquid chromatography coupled to high resolution parallel reaction monitoring mass spectrometry, and data analysis in Skyline, the chapter first describes the development of a bottom-up proteomic assay for quantification of EGFRvIII:CD3 bi-scFv in both plasma and whole blood. Importantly, a protein calibrator, along with stable isotope-labeled EGFRvIII:CD3 bi-scFv protein, was used for absolute quantification. A PK analysis in a CD3 humanized mouse revealed that EGFRvIII:CD3 bi-scFv in plasma and whole blood has an initial half-life of ~8 minutes and a terminal half-life of ~2.5 hours. These results establish a sensitive, high-throughput assay for direct quantification of EGFRvIII:CD3 bi-scFv without the need for immunoaffinity enrichment. Moreover, these pharmacokinetic parameters will guide drug optimization and dosing regimens in future IND-enabling and Phase I studies of EGFRvIII:CD3 bi-scFv.

Finally, Chapter 6 provides an outlook of the future development of cancer therapeutics for treating malignant gliomas.

Antibodies are important therapeutic agents that can be elicited by vaccination or directly infused. However, the mechanisms by which antibodies achieve protection against pathogens are not fully elucidated, in particular since antibodies mediate multiple functions, including direct neutralization as well as Fc-mediated effector functions. In this dissertation, I focused on one such Fc-mediated effector function, antibody-dependent phagocytosis, and examined it in the context of HIV-1. Firstly, with regard to the antibody Fv interaction with antigen, I used a novel HIV-1 virion phagocytosis assay to identify targets on the HIV-1 virion surface that can be targeted for antibody-mediated phagocytosis in the context of monoclonal and polyclonal antibodies from HIV-1 infection and vaccination settings, and demonstrated that these include both broadly neutralizing and non-neutralizing antibody epitopes. To examine whether antibody-mediated phagocytosis of HIV-infected cells can be an additional potential antiviral mechanism, I also developed an infected cell phagocytosis assay and demonstrated that in addition to virions, HIV-infected cells can also be targeted for antibody-mediated phagocytosis. Secondly, with regard to the antibody Fc interaction with FcR, I utilized recombinant subclass-switched antibodies and demonstrated that antibody isotypes and subclasses differ for antibody-dependent phagocytosis function, with IgG3 being the most potent. Furthermore, by examining the phagocytosis responses of humans and non-human primates, I demonstrated that there exists sufficient cross-reactivity between humans and rhesus macaques to examine human antibody subclass-specific phagocytosis activity in the rhesus macaque system despite evolutionary divergence in the Fc-FcR systems of the two species. Thirdly, I examined the downstream effects of antibody-mediated HIV-1 virion phagocytosis, and demonstrated that virions phagocytosed by antibody-dependent mechanisms likely do not cause enhanced infection, and also do not elicit additional inflammatory cytokines including IL-1β, IL-6, and TNFα. Thus, my work contributes to the understanding of antibody Fc-mediated phagocytosis function, with implications for HIV-1 vaccine and passive immunotherapy strategies, and broader relevance also for other infectious diseases as well as antibody-based cancer immunotherapy.

Many human monoclonal antibodies that neutralize multiple clades of HIV-1 are polyreactive and bind avidly to mammalian autoantigens. Indeed, the generation of neutralizing antibodies to the 2F5 and 4E10 epitopes of HIV-1 gp41 in man may be proscribed by immune tolerance since mice expressing the VH and VL of 2F5 have an arrested B-cell development characteristic of central tolerance. This developmental blockade implies the presence of tolerizing autoantigens that mitigate effective humoral responses. I hypothesize that discreet human antigens are mimicked by the membrane-proximal external region (MPER) of HIV-1 gp41, and that such mimicry is a wide-spread strategy for HIV-1 to evade immune attacks to its vulnerable neutralizing epitopes.

In the first part of the study, I propose to identify autoantigens mimicked by the 2F5 and 4E10 epitopes. I used immunoprecipitation coupled with mass spectrometry as well as protein arrays to identify the self-antigens recognized by 2F5 and 4E10. The binding of antigens was confirmed using serological assays and targeted mutagenesis was used to map the binding epitope. We identified human kynureninase (KYNU) and splicing factor 3b subunit 3 (SF3B3) as the primary conserved, vertebrate self-antigens recognized by the 2F5 and 4E10 antibodies, respectively. 2F5 binds the H4 domain of KYNU which contains the complete 2F5 linear epitope (ELDKWA). 4E10 recognizes a conformational epitope of SF3B3 that is strongly dependent on hydrophobic interactions. Opossums carry a rare KYNU H4 domain that abolishes 2F5 binding, but retain all SF3B3 4E10 epitopes. Immunization of opossums with HIV-1 gp140 induced extraordinary titers of serum antibody to the 2F5 ELDKWA epitope but little or nothing to the 4E10 determinant.

Our identification of structural motif shared by vertebrates and HIV-1 provides direct evidence that immunological tolerance can impair humoral responses to HIV-1. In the second part of the project, I propose to study the mechanisms of immune tolerance to B cells expressing the 2F5 antibodies. To determine the B cell repertoire before and after tolerance checkpoints, I used the Nojima-Kitamura single B-cell culture that supports differentiation into IgG-secreting plasma cells, even autoreactive cells that are normally subject to tolerization in vivo. I found that the pre-tolerance compartment (small pre-B) from 2F5 KI mice are cells that express the 2F5 V(D)J rearrangements and bind HIV-1 gp41, KYNU, and cardiolipin. Mature, post-tolerance B cells from 2F5 KI mice, however, are purged of gp41- and KYNU-reactivity, but retain cardiolipin-binding, and sequence analysis revealed extensive light-chain editing. The anergic B cells in the post-tolerance compartment are enriched with self-reactivity to KYNU and maintain binding to HIV-1 gp41. Our results demonstrate that tolerance of the 2F5 epitope is driven by specific reactivity to KYNU, but not general cross-reactivity to cardiolipin. In addition, that the peripheral anergic B cells retain self-reactivity and binding to HIV-1 gp41 suggests a potential target for activation by immunizations.

Lastly, we sought to determine whether the host mimicry by 4E10 and 2F5 epitopes is also present in other HIV-1 epitopes, including additional conserved neutralizing epitopes and more importantly, non-neutralizing epitopes. We used protein microarrays to assess autoreactivity of HIV-1 broadly neutralizing antibodies (bnAbs) and non-neutralizing antibodies (nnAbs) and found that as a class, bnAbs are more polyreactive and autoreactive than nnAbs. The poly- and autoreactive property is therefore not a result of chronic inflammation, but rather uniquely associated with neutralization, consistent with the role of heteroligation for HIV-1 neutralizing activity. In addition, mutation frequencies of bNAbs and nnAbs per se do not correlate with poly- and autoreactivity. Our results demonstrate that HIV-1 bnAbs are significantly more polyreactive and self-reactive than non-neutralizers, which may subject them to immunological tolerance control in vivo. Infrequent poly- or autoreactivity among nnAbs implies that their dominance in humoral responses is due to the absence of negative control by immune tolerance.

The results of this study indicate that mimicry of host antigens by HIV-1 is an effective mechanism to camouflage vulnerable neutralizing epitopes of HIV-1 and evade host immune responses. As a result, protective HIV-1 bnAbs are rare and often poly- or autoreactive, constituting a major hurdle that must be overcome to effectively elicit protective responses by an HIV-1 vaccine.

A successful HIV-1 vaccine is likely to be comprised of a polyclonal response with multiple antiviral functions that generate both broadly neutralizing antibodies (bNAbs) that neutralize multiple strains of HIV and/ or functional non-neutralizing antibodies that participate in numerous antiviral mechanisms. It is suggested that poly-/autoreactivity is a feature of bNabs but the interplay of autoimmunity and bNab development is unknown. Furthermore the non-neutralizing antibody specificities and functions are unknown for generating a potentially protective anti-HIV response. Understanding the development of rare neutralizing antibodies and the specificity and effector functions of non-neutralizing antibodies is critical for generating a preventative HIV vaccine. This thesis focuses on 1) the development of broadly neutralizing antibodies; specifically those that are present in both the face of autoimmunity when both self-antigen and HIV-1 is a determinant in disease outcome and 2) the mechanisms of non-neutralizing antibodies in mediating protection against HIV-1.

In the first part of this thesis, I chart the evolution of an HIV-1 antibody lineage that has reactivity to self antigen. One HIV-1 broadly neutralizing antibody targeting the membrane proximal external region, 2F5, shares sequence identity with the human self-antigen kynureninase, (KYNU). We previously reported that an HIV-1 chronically infected patient had both evidence of antibodies targeting self-proteins and circulating 2F5-like antibodies in serum. Whether development of 2F5-like antibodies to a self-antigen can lead to a broadly neutralizing antibody response or whether reactivity to self-antigen is a requirement for the neutralization breadth of 2F5-like antibodies has been a question in the HIV vaccine community. Evolution of 2F5 mAb-like antibody neutralization breadth and autoreactivity has not been previously demonstrated due to the scarcity of this specificity of antibody found circulating in HIV-1 infected individuals. In this study, I found that an unmutated common ancestor derived from a isolated mAb from this patient did not bind KYNU but more mature antibodies in the constructed antibody lineage developed reactivity to KYNU and other self-antigens due to an accumulation of antibody VH mutations. At the same time, the accumulation of mutations in the m66 lineage VH resulted in decreased 2F5 mAb binding affinity and virus neutralization potency, consistent with host mechanisms that limit antibody autoreactivity by placing constraints on affinity maturation. This work highlights the complex maturation pathways involving autoantigen reactivity in the development of broadly neutralizing antibodies.

The second part of the study centers on non-neutralizing antibody functions in defense against HIV-1. Elucidation of antibody effector functions responsible for protective immunity against HIV-1 acquisition is a major goal for the HIV-1 vaccine field. Immunoglobulin A (IgA) is an important part of the host defense against pathogens; however, little is known about the role of vaccine-elicited IgA and their capacity to mediate antiviral functions. To identify the antiviral functions of HIV-1-specific IgA elicited by vaccination, we cloned HIV-1 envelope specific IgA monoclonal antibodies by memory B cell cultures from peripheral blood mononuclear cells from a vaccinee from the only partially efficacious HIV vaccine regimen to date, RV144. We produced two IgA clonal cell lines (HG129 and HG130) producing native, non-recombinant IgA monoclonal antibodies (mAbs). HG129 is crossreactive and binds the variable loop (V3) of the HIV envelope; HG130 is a conformational gp120 antibody that interacts with the CD4 binding site. HG129 and HG130 mAbs mediated phagocytosis of HIV infectious virions and HIV antigen-coated beads respectively, and HG129 blocked HIV-1 Env glycoprotein binding to galactosylceramide, an alternative HIV-1 receptor. These findings elucidate potential antiviral functions of vaccine-elicited HIV-1 envelope specific IgA that may act to block HIV-1 acquisition at the portal of entry by preventing HIV-1 binding to galactosylceramide and mediating antibody Fc receptor mediated virion phagocytosis. Furthermore, these findings highlight the complex and diverse interactions of vaccine-elicited IgA with pathogens that depend on IgA fine specificity and form (e.g. multimeric, monomeric) in the systemic circulation and mucosal compartments.

The results of this work demonstrate that whether an antibody is broadly neutralizing or non-broadly neutralizing, it is the fine specificity to the HIV-1 envelope that is crucial for its functional activity. The functional activity is dependent on antibody specificity and isotype due to engagement of effector cells at mucosal sites. The production of potentially protective antibody envelope specificities, antibody isotypes, and functions should be considered in the immunogen design for the development of safe and effective vaccine approaches.