Scott Koenig

Development of a Humanized Monoclonal Antibody (MEDI-493) with Potent In Vitro and In Vivo Activity against Respiratory Syncytial Virus

Neutralizing polyclonal antibody to respiratory syncytial virus (RSV) has been shown to be an effective prophylactic agent when administered intravenously in high-risk infants. This study describes the generation of a humanized monoclonal antibody, MEDI-493, that recognizes a conserved neutralizing epitope on the F glycoprotein of RSV. The affinity of MEDI-493 was found to be equal to or slightly better than an isotype-matched chimeric derivative of the parent antibody. In plaque reduction, microneutralization, and fusion-inhibition assays, MEDI-493 was significantly more potent than the polyclonal preparation. Broad neutralization of a panel of 57 clinical isolates of the RSV A and B subtypes was demonstrated. Pretreatment of cotton rats with MEDI-493 resulted in 99% reduction of lung RSV titers at a dose of 2.5 mg/kg, corresponding to a serum concentration of 25-30 microg/mL. Further, MEDI-493 did not induce increased RSV infection or pathology in either a primary or a secondary challenge.

Use of a Whole Genome Approach To Identify Vaccine Molecules Affording Protection against Streptococcus pneumoniae Infection

ABSTRACT Microbial targets for protective humoral immunity are typically surface-localized proteins and contain common sequence motifs related to their secretion or surface binding. Exploiting the whole genome sequence of the human bacterial pathogen Streptococcus pneumoniae, we identified 130 open reading frames encoding proteins with secretion motifs or similarity to predicted virulence factors. Mice were immunized with 108 of these proteins, and 6 conferred protection against disseminated S. pneumoniaeinfection. Flow cytometry confirmed the surface localization of several of these targets. Each of the six protective antigens showed broad strain distribution and immunogenicity during human infection. Our results validate the use of a genomic approach for the identification of novel microbial targets that elicit a protective immune response. These new antigens may play a role in the development of improved vaccines against S. pneumoniae.

Fc Optimization of Therapeutic Antibodies Enhances Their Ability to Kill Tumor Cells In vitro and Controls Tumor Expansion In vivo via Low-Affinity Activating Fcγ Receptors

Monoclonal antibodies (mAb) are widely used in the treatment of non-Hodgkins lymphoma and autoimmune diseases. Although the mechanism of action in vivo is not always known, the therapeutic activity of several approved mAbs depends on the binding of the Fcgamma regions to low-affinity Fcgamma receptors (FcgammaR) expressed on effector cells. We did functional genetic screens to identify IgG1 Fc domains with improved binding to the low-affinity activating Fc receptor CD16A (FcgammaRIIIA) and reduced binding to the low-affinity inhibitory Fc receptor, CD32B (FcgammaRIIB). Identification of new amino acid residues important for FcgammaR binding guided the construction of an Fc domain that showed a dramatically enhanced CD16A binding and greater than a 100-fold improvement in antibody-dependent cell-mediated cytotoxicity. In a xenograft murine model of B-cell malignancy, the greatest enhancement of an Fc-optimized anti-human B-cell mAb was accounted for by improved binding to FcgammaRIV, a unique mouse activating FcgammaR that is expressed by monocytes and macrophages but not natural killer (NK) cells, consistent with experimental and clinical data suggesting that mononuclear phagocytes, effector cells expressing both activating and inhibitory FcgammaR, are critical mediators of B-cell depletion in vivo. By using mice transgenic for human CD16A, enhanced survival was observed due to expression of CD16A-158(phe) on monocytes and macrophages as well as on NK cells in these mice. The design of new generations of improved antibodies for immunotherapy should aim at Fc optimization to increase the engagement of activating FcgammaR present on the surface of tumor-infiltrating effector cell populations.

Immunopathogenic Mechanisms in Human Immunodeficiency Virus (HIV) Infection

Infection with HIV can result in a complex array of immunopathogenic effects. HIV infection involves both a direct quantitative depletion of T4 lymphocytes as well as an indirect qualitative effect on the function of several types of immune effector cells. The combination of T4-cell destruction and functional abnormalities contributes to the broad scope of immunologic aberrations and opportunistic diseases seen in HIV-infected individuals. In addition, HIV infection of monocyte/macrophages may play an important role as a reservoir or sanctuary of infection in the host and contribute to the characteristically long incubation period between HIV infection and disease. The activation of HIV from latent or chronically infected cells in vitro by mitogens, antigens, heterologous viruses, and cytokines represents a potential mechanism whereby HIV infection in individuals progresses from an asymptomatic carrier state to clinical AIDS. The release of virus from activated cells can lead to the spread of the virus to other target cells and result in both a qualitative or quantitative defect in immunocompetent cells and subsequent immunosuppression. It is also clear that HIV infection can result in the modulation of expression of certain cellular genes, thereby potentially compounding immunoregulatory abnormalities. Further knowledge of the complex relation between HIV and its target cells will be essential to our understanding of the myriad of potential pathogenic mechanisms of HIV infection and may lead to ways of interrupting the progression of HIV-induced disease.

Selective dysregulation of the FcγIIB receptor on memory B cells in SLE

The inappropriate expansion and activation of autoreactive memory B cells and plasmablasts contributes to loss of self-tolerance in systemic lupus erythematosus (SLE). Defects in the inhibitory Fc receptor, FcγRIIB, have been shown to contribute to B cell activation and autoimmunity in several mouse models of SLE. In this paper, we demonstrate that expression of FcγRIIB is routinely up-regulated on memory B cells in the peripheral blood of healthy controls, whereas up-regulation of FcγRIIB is considerably decreased in memory B cells of SLE patients. This directly correlates with decreased FcγRIIB-mediated suppression of B cell receptor–induced calcium (Ca2+) response in those B cells. We also found substantial overrepresentation of African-American patients among those who failed to up-regulate FcγRIIB. These results suggest that the inhibitory receptor, FcγRIIB, may be impaired at a critical checkpoint in SLE in the regulation of memory B cells; thus, FcγRIIB represents a novel target for therapeutic interventions in this disease.

Identification and Characterization of a Novel Family of Pneumococcal Proteins That Are Protective against Sepsis

ABSTRACT Four pneumococcal genes (phtA, phtB, phtD, andphtE) encoding a novel family of homologous proteins (32 to 87% identity) were identified from the Streptococcus pneumoniae genomic sequence. These open reading frames were selected as potential vaccine candidates based upon their possession of hydrophobic leader sequences which presumably target these proteins to the bacterial cell surface. Analysis of the deduced amino acid sequences of these gene products revealed the presence of a histidine triad motif (HxxHxH), termed Pht (pneumococcal histidine triad) that is conserved and repeated several times in each of the four proteins. The four pht genes (phtA, phtB, phtD, and a truncated version of phtE) were expressed inEscherichia coli. A flow cytometry-based assay confirmed that PhtA, PhtB, PhtD and, to a lesser extent, PhtE were detectable on the surface of intact bacteria. Recombinant PhtA, PhtB, and PhtD elicited protection against certain pneumococcal capsular types in a mouse model of systemic disease. These novel pneumococcal antigens may serve as effective vaccines against the most prevalent pneumococcal serotypes.

Application of dual affinity retargeting molecules to achieve optimal redirected T-cell killing of B-cell lymphoma.

We describe the application of a novel, bispecific antibody platform termed dual affinity retargeting (DART) to eradicate B-cell lymphoma through coengagement of the B cell-specific antigen CD19 and the TCR/CD3 complex on effector T cells. Comparison with a single-chain, bispecific antibody bearing identical CD19 and CD3 antibody Fv sequences revealed DART molecules to be more potent in directing B-cell lysis. The enhanced activity with the CD19xCD3 DART molecules was observed on all CD19-expressing target B cells evaluated using resting and prestimulated human PBMCs or purified effector T-cell populations. Characterization of a CD19xTCR bispecific DART molecule revealed equivalent potency with the CD19xCD3 DART molecule, demonstrating flexibility of the DART structure to support T-cell/B-cell associations for redirected T cell-killing applications. The enhanced level of killing mediated by DART molecules was not accompanied by any increase in nonspecific T-cell activation or lysis of CD19(-) cells. Cell-association studies indicated that the DART architecture is well suited for maintaining cell-to-cell contact, apparently contributing to the high level of target cell killing. Finally, the ability of the CD19xTCR DART to inhibit B-cell lymphoma in NOD/SCID mice when coadministered with human PBMCs supports further evaluation of DART molecules for the treatment of B-cell malignancies.

Monoclonal antibodies capable of discriminating the human inhibitory Fcγ-receptor IIB (CD32B) from the activating Fcγ-receptor IIA (CD32A): biochemical, biological and functional characterization

Human CD32B (FcγRIIB), the low‐affinity inhibitory Fcγ receptor (FcγR), is highly homologous in its extracellular domain to CD32A (FcγRIIA), an activating FcγR. Available monoclonal antibodies (mAb) against the extracellular region of CD32B recognize both receptors. Through immunization of mice transgenic for human CD32A, we generated a set of antibodies specific for the extracellular region of CD32B with no cross‐reactivity with CD32A, as determined by enzyme‐linked immunosorbent assay and surface plasmon resonance with recombinant CD32A and CD32B, and by fluorescence‐activated cell sorting analysis of CD32 transfectants. A high‐affinity mAb, 2B6, was used to explore the expression of CD32B by human peripheral blood leucocytes. While all B lymphocytes expressed CD32B, only a fraction of monocytes and almost no polymorphonuclear cells stained with 2B6. Likewise, natural killer cells, which express CD32C, a third CD32 variant, did not react with 2B6. Immune complexes co‐engage the inhibitory receptor with activating Fcγ receptors, a mechanism that limits cell responses. 2B6 competed for immune complex binding to CD32B as a monomeric Fab, suggesting that it directly recognizes the Fc‐binding region of the receptor. Furthermore, when co‐ligated with an activating receptor, 2B6 triggered CD32B‐mediated inhibitory signalling, resulting in diminished release of inflammatory mediators by FcεRI in an in vitro allergy model or decreased proliferation of human B cells induced by B‐cell receptor stimulation. These antibodies form the basis for the development of investigational tools and therapeutics with multiple potential applications, ranging from adjuvants in FcγR‐mediated responses to the treatment of allergy and autoimmunity.

Effector Cell Recruitment with Novel Fv-based Dual-affinity Re-targeting Protein Leads to Potent Tumor Cytolysis and in Vivo B-cell Depletion

Bispecific antibodies capable of redirecting the lytic potential of immune effector cells to kill tumor targets have long been recognized as a potentially potent biological therapeutic intervention. Unfortunately, efforts to produce such molecules have been limited owing to inefficient production and poor stability properties. Here, we describe a novel Fv-derived strategy based on a covalently linked bispecific diabody structure that we term dual-affinity re-targeting (DART). As a model system, we linked an Fv specific for human CD16 (FcgammaRIII) on effector cells to an Fv specific for mouse or human CD32B (FcgammaRIIB), a normal B-cell and tumor target antigen. DART proteins were produced at high levels in mammalian cells, retained the binding activity of the respective parental Fv domains as well as bispecific binding, and showed extended storage and serum stability. Functionally, the DART molecules demonstrated extremely potent, dose-dependent cytotoxicity in retargeting human PBMC against B-lymphoma cell lines as well as in mediating autologous B-cell depletion in culture. In vivo studies in mice demonstrated effective B-cell depletion that was dependent on the transgenic expression of both CD16A on the effector cells and CD32B on the B-cell targets. Furthermore, DART proteins showed potent in vivo protective activity in a human Burkitts lymphoma cell xenograft model. Thus, DART represents a biologically potent format that provides a versatile platform for generating bispecific antibody fragments for redirected killing and, with the selection of appropriate binding partners, applications outside of tumor cell cytotoxicity.

Immunology of Human Immunodeficiency Virus Infection and the Acquired Immunodeficiency Syndrome: An Update

Recent advances in the understanding of the pathogenesis of infection with human immunodeficiency virus (HIV) stems from the demonstration that the membrane glycoprotein, CD4, is the cellular receptor for HIV. This glycoprotein is found mainly on the surface of a major subpopulation of T lymphocytes and also on macrophages, natural killer cells, some B lymphocytes, and neuronal cells. Cells infected with HIV may be destroyed or have their normal function impaired. Host immune responses to HIV are poor and are not sustained. Neutralizing antibody often is not produced, or HIV may escape from normal immunosuppressive mechanisms through the process of rapid antigenic variation. Factors and markers that may be important in the outcome or that may predict progression of HIV infection are genetic (Gc type), environmental (nutritional status or intercurrent sexually transmitted diseases sustained by the host), and immunologic (rate of decline in number and impairment of function of CD4 lymphocytes and of decline in antibody titers to HIV core protein, p24). A recombinant vaccine will probably be developed for testing in future clinical trials.