Development of ISB 1442, a CD38 and CD47 bispecific biparatopic antibody innate cell modulator for the treatment of multiple myeloma.

Antibody engineering can tailor the design and activities of therapeutic antibodies for better efficiency or other advantageous clinical properties. Here we report the development of ISB 1442, a fully human bispecific antibody designed to re-establish synthetic immunity in CD38+ hematological malignancies. ISB 1442 consists of two anti-CD38 arms targeting two distinct epitopes that preferentially drive binding to tumor cells and enable avidity-induced blocking of proximal CD47 receptors on the same cell while preventing on-target off-tumor binding on healthy cells. The Fc portion of ISB 1442 is engineered to enhance complement dependent cytotoxicity, antibody dependent cell cytotoxicity and antibody dependent cell phagocytosis. ISB 1442 thus represents a CD47-BsAb combining biparatopic targeting of a tumor associated antigen with engineered enhancement of antibody effector function to overcome potential resistance mechanisms that hamper treatment of myeloma with monospecific anti-CD38 antibodies. ISB 1442 is currently in a Phase I clinical trial in relapsed refractory multiple myeloma.

Small molecule and biologic modulators of the immune response to hepatitis C virus.

Hepatitis C virus represents a major global health problem, with approximately 3% of the world population infected. Immune-response modifiers represent the standard of care, given the lack of approved antiviral agents having direct activity against the viral proteins. Although in recent years, improvements in therapy have been attained by combined treatment with pegylated interferon and ribavirin, the discovery and development of next-generation small molecule and biologic agents is ongoing. Several of these newer therapeutics are focused on modulating Toll-like receptors, interferon-alpha signaling, and the pro-inflammatory cytokine balance. A comprehensive account of the lead compounds in development, the bioassays used for optimization of these immune response modifiers and their clinical status is presented.

Analysis of Borrelia burgdorferi gene expression during life cycle phases of the tick vector Ixodes scapularis.

Borrelia burgdorferi exists in nature via an enzootic cycle whereby the organism must adapt to the diverse environmental conditions provided inside the arthropod transmission vector and the mammalian reservoir hosts. B. burgdorferi genes shown to be regulated by temperature, pH and/or cell density during the organism's growth in culture medium were assayed for expression during various stages of the tick feeding cycle by reverse transcription-polymerase chain reaction (RT-PCR). ospA, ospC, flaB, erpA/I/N, erpB/J/O, rev and mlpA, were transcriptionally active following the larval and nymphal stages of feeding as determined by qualitative RT-PCR. During tick resting periods between feedings, ospC, mlpA and rev transcription were undetectable, in contrast to ospA, flaB, erpA/I/N and erpB/J/O. bba64, a gene induced by environmental changes in culture and expressed during mammalian infection, was not detectable during any of the tick life cycle phases. Quantitative PCR to determine B. burgdorferi genome equivalents in these tick samples using DNA co-purified with the RNA allowed an estimation of gene expression relative to the numbers of B. burgdorferi present in the ticks. Although the spirochete totals varied widely between individual tick pools of fed, replete nymphs, the relative expression ratios between individual target genes following a nymphal feed were comparable. Similarly, borrelial gene transcription from the larval feeding and the nymphal feeding were observed and compared. These findings analogize B. burgdorferi gene expression observed by environmental stimuli in vitro with the transcriptional activity occurring during the organism's infectious cycle within the tick.

Leishmania major induces differential expression of costimulatory molecules on mouse epidermal cells.

Levels of expression of costimulatory molecules have been proposed to influence the outcome of antigen-specific T cell priming. We found that Leishmania major selectively modulated the expression of costimulatory molecules on various populations of epidermal cells. B7.2 expression was down-regulated on Thy1.2+ epidermal cells (keratinocytes) from disease-resistant C3H mice, but not from disease-susceptible BALB/c mice. In addition, epidermal cells from BALB/c mice showed a down-regulation of B7.1 expression on NLDC 145+ Langerhans cells. In vitro T cell priming experiments, using syngeneic epidermal cells as antigen-presenting cells (APC), showed that the production of IFN-gamma was inhibited when either B7.1 or B7.2 signaling pathways were blocked. Blockade of B7.2, but not B7.1, significantly inhibited the ability of epidermal cells to induce IL-4 production from CD4+ T cells. In addition, C3H CD4+ T cells, which were unable to secrete detectable levels of IL-4 in cultures with syngeneic APC, were now able to secrete IL-4 following presentation of L. major antigens by congenic BALB/K epidermal cells. Conversely, C3H epidermal cells supported the priming of BALB/K CD4+ T cells for IL-4 production in vitro. Thus, the differential expression of B7 molecules on epidermal cells may not represent the sole factor governing the polarization of L. major-specific CD4+ T cells in vitro.

Major histocompatibility complex class II-independent generation of neutralizing antibodies against T-cell-dependent Borrelia burgdorferi antigens presented by dendritic cells: regulation by NK and gammadelta T cells.

We previously showed that adoptive transfer of Borrelia burgdorferi-pulsed dendritic cells (DCs) into syngeneic mice protects animals from challenge with tick-transmitted spirochetes. Here, we demonstrate that the protective immune response is antibody (Ab) dependent and does not require the presence of major histocompatibility complex (MHC) class II molecules on DCs. Mice sensitized with B. burgdorferi-pulsed MHC class II-deficient (MHC class II(-/-)) DCs mounted a humoral response against protective antigens, including B. burgdorferi outer surface protein A (OspA) and OspC. B-cell help for the generation of neutralizing anti-OspC immunoglobulin G Abs could be provided by gammadelta T cells. In contrast, anti-OspA Ab production required the presence of alphabeta T cells, although this pathway could be independent of MHC class II molecules on antigen-presenting cells. Moreover, depletion of NK cells prior to transfer of antigen-pulsed MHC class II(-/-) DCs resulted in significant increases in the levels of neutralizing Abs induced by DCs. Altogether, these data suggest that the initial interactions between DCs and innate immune cells, such as gammadelta and NK cells, can influence the generation of a protective humoral response against B. burgdorferi antigens.

The immunomodulatory factors of bloodfeeding arthropod saliva.

There are potent immunomodulators in saliva of the bloodfeeding arthropods which transmit many of the world's most serious diseases that may benefit the arthropod by preventing the vertebrate host from becoming sensitized to the saliva. In addition, saliva can enhance transmission of parasites/pathogens by arthropods. As a result, vaccines that target the arthropod (e.g. salivary immunomodulators) should be considered as one component of multisubunit vaccines against arthropod-borne parasites/pathogens. Indeed, since vaccines against the pathogens themselves are often not fully protective, vaccines that target several facets of the life cycle of the pathogen may be the most effective at controlling disease transmission. This review covers known immunomodulatory factors in arthropod vector saliva, focusing mainly on sandflies and ixodid ticks.

Conformational nature of the Borrelia burgdorferi B31 outer surface protein C protective epitope.

Active immunization with Escherichia coli-expressed recombinant outer surface protein C (OspC) of Borrelia burgdorferi has been demonstrated to confer protection against a tick-transmitted infection on laboratory animals. A previous study in this laboratory showed that OspC antibody raised against a denatured immunogen isolated from B. burgdorferi cells failed to provide protective immunity. Therefore, to determine whether the protective epitope of the recombinant antigen was sensitive to denaturation, recombinant OspC preparations were subjected to heat and chemical treatments prior to animal immunization. Following seroconversion to OspC, the animals were challenged with an infectious dose of B. burgdorferi B31 by tick bite. Whereas mice immunized with a soluble, nondenatured form continued to show protection rates close to 100%, mice that had been immunized with denatured antigen were not protected. Furthermore, mice that were immunized with an insoluble (rather than a soluble), nondenatured form of the recombinant OspC showed a protection rate of only 40%. Protective epitope localization experiments showed that either the amino or the carboxy end of the recombinant protein was required to react with a protective OspC-specific monoclonal antibody. The data from these experiments demonstrate that a conformational organization of the protein is essential for the protective capability of the strain B31 OspC immunogen.

An OspC-specific monoclonal antibody passively protects mice from tick-transmitted infection by Borrelia burgdorferi B31.

A murine monoclonal antibody directed against Borrelia burgdorferi B31 outer surface protein C (OspC) antigen was generated by a method whereby borreliae were inoculated into the mouse via the natural transmission mode of tick feeding. Passive immunization with this antibody resulted in protection of C3H/HeJ and outbred mice from a tick-transmitted challenge infection. Immunofluorescence staining of borrelia cells indicated surface exposure of the OspC epitope reactive with the monoclonal antibody.

Phlebotomus papatasi sand fly salivary gland lysate down-regulates a Th1, but up-regulates a Th2, response in mice infected with Leishmania major.

A vertebrate host becomes infected with Leishmania major when the sand fly vector injects parasites into skin along with saliva. Previous studies showed that salivary gland lysate of the New World sand fly Lutzomyia longipalpis markedly enhanced L. major infection in CBA mice. However, L. major is an Old World parasite transmitted in nature by the Old World sand fly Phlebotomus papatasi. Here we examine the ability of P. papatasi salivary gland lysate to enhance infection (lesion size and parasite burden) by L. major. In addition, we examine the effects of salivary gland lysate on the immune response to L. major by monitoring the levels of cytokine mRNA from the lymph nodes draining cutaneous lesions. We found that P. papatasi salivary gland lysate dramatically exacerbated lesion development in disease-resistant CBA mice. This exacerbation of disease correlated with inhibition of the production of Thl cytokines and associated factors (IFN-gamma, IL-12, and inducible nitric oxide synthase), but with enhancement of the Th2 cytokine IL-4, whereas no changes in the levels of IL-10 and TGF-beta were noted. Importantly, salivary gland lysate directly up-regulated expression of IL-4 mRNA in mice in the absence of infection with L. major.

Vector competence of Ixodes scapularis and Ixodes ricinus (Acari: Ixodidae) for three genospecies of Borrelia burgdorferi.

The vector competence of 2 tick species, Ixodes ricinus (L.) and Ixodes scapularis Say, was determined and compared for 3 genospecies of Borrelia burgdorferi. The 3 genospecies of B. burgdorferi used in the following experiments were Borrelia burgdorferi sensu stricto (B-31 and B-31.D1 clone), Borrelia afzelii (strain Pgau. C3), and Borrelia garinii (strain VS286 and VSBP). Spirochetes from all 5 strains were inoculated intradermally into outbred mice; larval ticks of both species were subsequently fed on those mice and replete larvae were assayed for infection by culture in BSK-H media every 7 d for 4 wk. Infection frequencies in I. scapularis exposed to the 5 strains were as follows: B-31 (90%), B-31.D1 (83%), Pgau.C3 (87%), VS286 (10%), and VSBP (5%). The comparable infection frequencies for I. ricinus were B-31 (3%), B-31.D1 (3%), Pgau.C3 (90%), VS286 (5%), and VSBP (3%). Resultant nymphal I. scapularis successfully transmitted B-31, B-31,D1, Pgau.C3, and VS286 to outbred mice. I. ricinus nymphs transmitted Pgau.C3 and VS286. Both species failed to transmit strain VSBP.

A monoclonal antibody generated by antigen inoculation via tick bite is reactive to the Borrelia burgdorferi Rev protein, a member of the 2.9 gene family locus.

Murine monoclonal antibodies directed against proteins of Borrelia burgdorferi B31 (low passage) were generated by the administration of antigen via the bite of borrelia-infected ticks. This strategy was employed as a mechanism to create antibodies against antigens presented by the natural route of tick transmission versus those presented by inoculation with cultured borreliae. One of the resultant antibodies reacted with a 17-kDa antigen from cultured B. burgdorferi, as seen by immunoblot analysis. This antibody was used to screen a B. burgdorferi genomic DNA lambda vector expression library, and an immunoreactive clone was isolated. DNA sequence analysis of this clone, containing a 2.7-kb insert, revealed several open reading frames. These open reading frames were found to be homologs of genes discovered as a multicopy gene family in the 297 strain of B. burgdorferi by Porcella et al. (S. F. Porcella, T. G. Popova, D. R. Akins, M. Li, J. D. Radolf, and M. V. Norgard, J. Bacteriol. 178:3293-3307, 1996). By selectively subcloning genes found in this insert into an Escherichia coli plasmid expression vector, the observation was made that the rev gene product was the protein reactive with the 17-kDa-specific monoclonal antibody. The rev gene product was found to be expressed in low-passage, but not in high-passage, B. burgdorferi B31. Correspondingly, the rev gene was not present in strain B31 genomic DNA from cultures that had been passaged >50 times. Serum samples from Lyme disease patients demonstrated an antibody response against the Rev protein. The generation of an anti-Rev response in Lyme disease patients, and in mice by tick bite inoculation, provides evidence that the Rev protein is expressed and immunogenic during the course of natural transmission and infection.

Effects of Ixodes scapularis and Borrelia burgdorferi on modulation of the host immune response: induction of a TH2 cytokine response in Lyme disease-susceptible (C3H/HeJ) mice but not in disease-resistant (BALB/c) mice.

Previous studies have demonstrated that both Ixodes scapularis saliva and Borrelia burgdorferi antigens modulated lymphokines and monokines in vitro. The studies presented here were designed to delineate the role of I. scapularis and B. burgdorferi in modulation of the host immune response in vivo. Infestation of C3H/HeJ mice with infected I. scapularis resulted in an up regulation of IL-4 as early as 8 days after tick infestation, while the levels of T helper cell type 1 (TH1) cytokines, interleukin-2 (IL-2) and gamma interferon (IFN-gamma), were significantly decreased by days 10 to 12. In contrast, the cytokine profile of BALB/c mice exposed to infected nymphal ticks resulted in only transient alterations in IL-4, IL-2, and IFN-gamma production throughout a 12-day period postinfestation. Although the IL-10 level was elevated in both C3H/HeJ and BALB/c mice infested with infected nymphal ticks, no significant difference in the levels of IL-10 was noted between the mouse strains. Flow-cytometric analysis demonstrated increases in the numbers of splenic B-cell and CD4+ lymphocytes in C3H/HeJ but not BALB/c mice exposed to infected ticks. Cell depletion experiments with C3H/HeJ mice demonstrated that CD4+ cells were the sole producers of IFN-gamma and IL-10 while both CD4+ and CD8+ splenocytes contributed to the production of IL-2 and IL-4. These findings suggest that B and CD4+ splenocytes are activated, increase in number, and produce a polarized TH2 response in C3H/HeJ mice exposed to infected I. scapularis. Given that C3H/HeJ mice are susceptible to Lyme disease and the initial TH2 polarization is not evident in BALB/c mice, effective control of this response may have ramifications for spirochete transmission in vivo.

Borrelia burgdorferi-pulsed dendritic cells induce a protective immune response against tick-transmitted spirochetes.

Borrelia burgdorferi-pulsed dendritic cells and epidermal cells were able to initiate the production of anti-outer surface protein A (OspA) antibody in vitro with normal T and B cells from either BALB/c or C3H/HeJ mice. Inhibition of anti-B. burgdorferi antibody production was observed after 3 days, but not after 2 days, of exposure of the antigen-presenting cells to tumor necrosis factor alpha +/- granulocyte-macrophage colony-stimulating factor. Furthermore, splenic dendritic cells pulsed in vitro with live B. burgdorferi spirochetes and then adoptively transferred into naive syngeneic mice mediated a protective immune response against tick-transmitted spirochetes. This protection appeared not to be due to killing of spirochetes in the feeding ticks, since ticks fed to repletion on B. burgdorferi-pulsed dendritic cell-sensitized mice still harbored live spirochetes. Western blot analysis of the sera collected from dendritic cell-sensitized mice demonstrated that the mice responded to a limited set of B. burgdorferi antigens, including OspA, -B, and -C compared to control groups that either had received unpulsed dendritic cells or were not treated. Finally, mice in the early stage of B. burgdorferi infection were able to develop anti-OspA antibody following injection with B. burgdorferi-pulsed dendritic cells. Our results demonstrate for the first time that adoptive transfer of B. burgdorferi-pulsed dendritic cells induces a protective immune response against tick-transmitted B. burgdorferi and stimulates the production of antibodies specific for a limited set of B. burgdorferi antigens in vivo.

Infiltration of CD4+ CD8+ T cells, and expression of ICAM-1, Ia antigens, IL-1 alpha and TNF-alpha in the skin lesion of BALB/c mice undergoing repeated infestations with nymphal Ixodes ricinus ticks.

The skin cellular immune response of BALB/c mice was examined during three successive infestations with nymphal Ixodes ricinus ticks. An immunohistochemical analysis of skin cryostat sections 72 hr post-tick attachment revealed that CD4+ T cells outnumbered CD8+ T cells in all infestations. The CD4+:CD8+ T-cell ratio was 2.2:1 in the primary infestation, then increased to 3.2:1 and 4.7:1 in the secondary and tertiary infestations. No B lymphocytes (CD45R) were detected in the skin of control and infested mice. A positive staining of intercellular adhesion molecule-1 (ICAM-1) on vascular endothelial cells, dendritic cells and some other mononuclear cells was observed in the dermis. Also, a strong positive staining of Ia antigens on dendritic cells and infiltrated mononuclear cells was noted. The staining pattern was more intense and positive cells increased in number in the skin of re-infested mice compared to the primary infestation. In addition, cells such as epidermal keratinocytes, dermal dendritic cells and infiltrated mononuclear cells positive for the 'pro-inflammatory' cytokines interleukin-1 alpha (IL-1 alpha) and tumour necrosis factor-alpha (TNF-alpha) were localized in the skin of infested mice, as detected at the mRNA level by in situ hybridization and at protein level by immunostaining with antibodies. These results suggest that an antigen was presented to infiltrating T lymphocytes which then became activated. This event may explain the cutaneous delayed-type hypersensitivity previously described in tick-infected BALB/c mice. Importantly, this cutaneous reaction was not sufficient to protect the mouse against tick re-infestation. Furthermore, ICAM-1 could mediate, at least in part, the extravasation of inflammatory cells into the skin of infested mice.

IFN-gamma IL-2, and IL-4 mRNA expression in the skin and draining lymph nodes of BALB/c mice repeatedly infested with nymphal Ixodes ricinus ticks.

The skin and draining lymph nodes of BALB/c mice were examined for IFN-gamma, IL-2, and IL-4 mRNA expression by in situ hybridization in three successive infestations with nymphal Ixodes ricinus ticks. IFN-gamma and IL-2 mRNA positive cells were readily detected in lymph node sections during primary antigenic stimulation (72 hr post-tick attachment), whereas hybridization with IL-4 probe yielded no or only a faint positive signal. No changes in the cytokine pattern were observed in lymph node sections from reinfested mice, with IL-4 mRNA always being expressed to a lesser extent than IFN-gamma and IL-2 mRNA. Seventy-two hours post-tick attachment in primary infestation, some infiltrating cells in the skin were positive for IFN-gamma and IL-4 mRNA, but not for IL-2 mRNA. In skin sections of reinfested mice, mRNA coding for IFN-gamma, IL-2, and IL-4 were detected in infiltrating cells. Cells positive for IL-4 mRNA were lower in number than those positive for IFN-gamma and IL-1 mRNA. A significant decrease in the number of IL-4 mRNA positive cells in the tertiary infestation was noted. All together, these results indicate that I. ricinus nymphal ticks antigens are able to elicit expression of IFN-gamma, IL-2 mRNA and to a lesser extent IL-4 mRNA in both skin and draining lymph nodes. In addition, repeated infestations with ticks led to strong expression of IFN-gamma and IL-2 mRNAs in the skin that may be correlated with previous observations showing the occurrence of cutaneous delayed-type hypersensitivity in tick-infested mice. Notably, the cytokine pattern observed in the skin and draining lymph nodes is not associated with a protective immune response in mice against I. ricinus nymphal ticks infestations.

Absence of acquired resistance to nymphal Ixodes ricinus ticks in BALB/c mice developing cutaneous reactions.

BALB/c mice underwent 3 successive infestations with 15 Ixodes ricinus nymphs. No resistance was acquired as assessed by evaluating tick attachment, duration of blood meal, weights of engorged nymphs, and molting success. However, the hosts developed cutaneous immediate- and delayed-type hypersensitivity reactions when reinfested. Histological examination of tick attachment sites showed that inflammatory cells consisting of neutrophils, eosinophils, and mononuclear cells (lymphocytes and monocytes) infiltrated the skin more intensively during reinfestations. The number of intact mast cells did not vary between successive infestations, whereas the number of degranulated mast cells increased in the early stages of reinfestations. Basophils, which represent 12% of total infiltrating cells, were only observed and quantified in the skin of reinfested mice using transmission electron microscopy (TEM). Degranulating eosinophils were also observed by use of TEM.