Discovery and preclinical development of a therapeutically active nanobody-based chimeric antigen receptor targeting human CD22.

Chimeric antigen receptor (CAR) T cell therapies targeting B cell-restricted antigens CD19, CD20, or CD22 can produce potent clinical responses for some B cell malignancies, but relapse remains common. Camelid single-domain antibodies (sdAbs or nanobodies) are smaller, simpler, and easier to recombine than single-chain variable fragments (scFvs) used in most CARs, but fewer sdAb-CARs have been reported. Thus, we sought to identify a therapeutically active sdAb-CAR targeting human CD22. Immunization of an adult Llama glama with CD22 protein, sdAb-cDNA library construction, and phage panning yielded >20 sdAbs with diverse epitope and binding properties. Expressing CD22-sdAb-CAR in Jurkat cells drove varying CD22-specific reactivity not correlated with antibody affinity. Changing CD28- to CD8-transmembrane design increased CAR persistence and expression in vitro. CD22-sdAb-CAR candidates showed similar CD22-dependent CAR-T expansion in vitro, although only membrane-proximal epitope targeting CD22-sdAb-CARs activated direct cytolytic killing and extended survival in a lymphoma xenograft model. Based on enhanced survival in blinded xenograft studies, a lead CD22sdCAR-T was selected, achieving comparable complete responses to a benchmark short linker m971-scFv CAR-T in high-dose experiments. Finally, immunohistochemistry and flow cytometry confirm tissue and cellular-level specificity of the lead CD22-sdAb. This presents a complete report on preclinical development of a novel CD22sdCAR therapeutic.

A simplified function-first method for the discovery and optimization of bispecific immune engaging antibodies.

Bi-specific T-cell engager antibodies (BiTEs) are synthetic fusion molecules that combine multiple antibody-binding domains to induce active contact between T-cells and antigen expressing cells in the body. Blinatumomab, a CD19-CD3 BiTE is now a widely used therapy for relapsed B-cell malignancies, and similar BiTE therapeutics have shown promise for treating various other forms of cancer. The current process for new BiTE development is time consuming and costly, requiring characterization of the individual antigen binding domains, followed by bi-specific design, protein production, purification, and eventually functional screening. Here, we sought to establish a more cost-efficient approach for generating novel BiTE sequences and assessing bioactivity through a function first approach without purification. We generate a plasmid with a bi-modular structure to allow high-throughput exchange of either binding arm, enabling rapid screening of novel tumour-targeting single chain variable (scFv) domains in combination with the well-characterized OKT3 scFv CD3-targeting domain. We also demonstrate two systems for high throughput functional screening of BiTE proteins based on Jurkat T cells (referred to as BiTE-J). Using BiTE-J we evaluate four EGFRvIII-scFv sequenced in BiTE format, identifying two constructs with superior activity for redirecting T-cells against the EGFRvIII-tumour specific antigen. We also confirm activity in primary T cells, where novel EGFRvIII-BiTEs induced T cell activation and antigen selective tumor killing. We finally demonstrate similar exchange the CD3-interacting element of our bi-modular plasmid. By testing several novel CD3-targeting scFv elements for activity in EGFRvIII-targeted BiTEs, we were able to identify highly active BiTE molecules with desirable functional activity for downstream development. In summary, BiTE-J presents a low cost, high-throughput method for the rapid assessment of novel BiTE molecules without the need for purification and quantification.

Programmable Attenuation of Antigenic Sensitivity for a Nanobody-Based EGFR Chimeric Antigen Receptor Through Hinge Domain Truncation.

Epidermal growth factor family receptor (EGFR) is commonly overexpressed in many solid tumors and an attractive target for chimeric antigen receptor (CAR)-T therapy, but as EGFR is also expressed at lower levels in healthy tissues a therapeutic strategy must balance antigenic responsiveness against the risk of on-target off-tumor toxicity. Herein, we identify several camelid single-domain antibodies (also known as nanobodies) that are effective EGFR targeting moieties for CARs (EGFR-sdCARs) with very strong reactivity to EGFR-high and EGFR-low target cells. As a strategy to attenuate their potent antigenic sensitivity, we performed progressive truncation of the human CD8 hinge commonly used as a spacer domain in many CAR constructs. Single amino acid hinge-domain truncation progressively decreased both EGFR-sdCAR-Jurkat cell binding to EGFR-expressing targets and expression of the CD69 activation marker. Attenuated signaling in hinge-truncated EGFR-sdCAR constructs increased selectivity for antigen-dense EGFR-overexpressing cells over an EGFR-low tumor cell line or healthy donor derived EGFR-positive fibroblasts. We also provide evidence that epitope location is critical for determining hinge-domain requirement for CARs, as hinge truncation similarly decreased antigenic sensitivity of a membrane-proximal epitope targeting HER2-CAR but not a membrane-distal EGFRvIII-specific CAR. Hinge-modified EGFR-sdCAR cells showed clear functional attenuation in Jurkat-CAR-T cells and primary human CAR-T cells from multiple donors in vitro and in vivo. Overall, these results indicate that hinge length tuning provides a programmable strategy for throttling antigenic sensitivity in CARs targeting membrane-proximal epitopes, and could be employed for CAR-optimization and improved tumor selectivity.

Maternal Immune Cell and Cytokine Profiles to Predict Cardiovascular Risk Six Months after Preeclampsia.

Women who develop preeclampsia (PE) are at high risk for cardiovascular disease (CVD). Early identification of women with PE who may benefit the most from early cardiovascular risk screening and interventions remains challenging. Our objective was to assess whether cytokine and immune cell profiles after PE are helpful in distinguishing women at low and high CVD risk at 6-months postpartum. Individuals who developed PE were followed for immune cell phenotyping and plasma cytokine quantification at delivery, at 3-months, and at 6-months postpartum. Lifetime CVD risk was assessed at 6-months postpartum, and the immune cell and cytokine profiles were compared between risk groups at each time point. Among 31 participants, 18 (58.1%) exhibited high CVD-risk profiles at 6-months postpartum. The proportion of circulating NK-cells was significantly lower in high-risk participants at delivery (p = 0.04). At 3-months postpartum, high-risk participants exhibited a lower proportion of FoxP3+ regulatory T-cells (p = 0.01), a greater proportion of CD8+ T cells (p = 0.02) and a lower CD4+:CD8+ ratio (p = 0.02). There were no differences in immune cell populations at 6-months postpartum. There were no differences in plasma cytokines levels between risk groups at any time point. Subtle differences in immune cell profiles may help distinguish individuals at low and high CVD risk in the early postpartum period and warrants further investigation.

A low dose adenovirus vectored vaccine expressing Schistosoma mansoni Cathepsin B protects from intestinal schistosomiasis in mice.

BACKGROUND: Schistosomiasis is an underestimated neglected tropical disease which affects over 236.6 million people worldwide. According to the CDC, the impact of this disease is second to only malaria as the most devastating parasitic infection. Affected individuals manifest chronic pathology due to egg granuloma formation, destroying the liver over time. The only FDA approved drug, praziquantel, does not protect individuals from reinfection, highlighting the need for a prophylactic vaccine. Schistosoma mansoni Cathepsin B (SmCB) is a parasitic gut peptidase necessary for helminth growth and maturation and confers protection as a vaccine target for intestinal schistosomiasis. METHODS: An SmCB expressing human adenovirus serotype 5 (AdSmCB) was constructed and delivered intramuscularly to female C57BL/6 mice in a heterologous prime and boost vaccine with recombinant protein. Vaccine induced immunity was described and subsequent protection from parasite infection was assessed by analysing parasite burden and liver pathology. FINDINGS: Substantially higher humoral and cell-mediated immune responses, consisting of IgG2c, Th1 effectors, and polyfunctional CD4+ T cells, were induced by the heterologous administration of AdSmCB when compared to the other regimens. Though immune responses favoured Th1 immunity, Th2 responses provided by SmCB protein boosts were maintained. This mixed Th1/Th2 immune response resulted in significant protection from S. mansoni infection comparable to other vaccine formulations which are in clinical trials. Schistosomiasis associated liver pathology was also prevented in a murine model. INTERPRETATION: Our study provides missing preclinical data supporting the use of adenoviral vectoring in vaccines for S. mansoni infection. Our vaccination method significantly reduces parasite burden and its associated liver pathology - both of which are critical considerations for this helminth vaccine. FUNDING: This work was supported by the Canadian Institutes of Health Research, R. Howard Webster Foundation, and the Foundation of the McGill University Health Centre.

Self-Cutting and Integrating CRISPR Plasmids Enable Targeted Genomic Integration of Genetic Payloads for Rapid Cell Engineering.

Since observations that CRISPR nucleases function in mammalian cells, many strategies have been devised to adapt them for genetic engineering. Here, we investigated self-cutting and integrating CRISPR-Cas9 plasmids (SCIPs) as easy-to-use gene editing tools that insert themselves at CRISPR-guided locations. SCIPs demonstrated similar expression kinetics and gene disruption efficiency in mouse (EL4) and human (Jurkat) cells, with stable integration in 3-6% of transfected cells. Clonal sequencing analysis indicated that integrants showed bi- or mono-allelic integration of entire CRISPR plasmids in predictable orientations and with limited insertion or deletion formation. Interestingly, including longer homology arms (HAs; 500 bp) in varying orientations only modestly increased knock-in efficiency (by around twofold). Using a SCIP-payload design (SCIPpay) that liberates a promoter-less sequence flanked by HAs thereby requiring perfect homology-directed repair for transgene expression, longer HAs resulted in higher integration efficiency and precision of the payload but did not affect integration of the remaining plasmid sequence. As proofs of concept, we used SCIPpay to insert (1) a gene fragment encoding tdTomato into the CD69 locus of Jurkat cells, thereby creating a cell line that reports T-cell activation, and (2) a chimeric antigen receptor gene into the TRAC locus. Here, we demonstrate that SCIPs function as simple, efficient, and programmable tools useful for generating gene knock-out/knock-in cell lines, and we suggest future utility in knock-in site screening/optimization, unbiased off-target site identification, and multiplexed, iterative, and/or library-scale automated genome engineering.

Adjuvanted Schistosoma mansoni-Cathepsin B With Sulfated Lactosyl Archaeol Archaeosomes or AddaVax™ Provides Protection in a Pre-Clinical Schistosomiasis Model.

Schistosomiasis threatens 800 million people worldwide. Chronic pathology manifests as hepatosplenomegaly, and intestinal schistosomiasis caused by Schistosoma mansoni can lead to liver fibrosis, cirrhosis, and blood in the stool. To assist the only FDA-approved drug, praziquantel, in parasite elimination, the development of a vaccine would be of high value. S. mansoni Cathepsin B (SmCB) is a well-documented vaccine target for intestinal schistosomiasis. Herein, we test the increased efficacy and immunogenicity of SmCB when combined with sulfated lactosyl archaeol (SLA) archaeosomes or AddaVax™ (a squalene based oil-in-water emulsion). Both vaccine formulations resulted in robust humoral and cell mediated immune responses. Impressively, both formulations were able to reduce parasite burden greater than 40% (WHO standard), with AddaVax™ reaching 86.8%. Additionally, SmCB with both adjuvants were able to reduce granuloma size and the amount of larval parasite hatched from feces, which would reduce transmission. Our data support SmCB as a target for S. mansoni vaccination; especially when used in an adjuvanted formulation.

A High-Throughput Method for Characterizing Novel Chimeric Antigen Receptors in Jurkat Cells.

Chimeric antigen receptor (CAR) development involves extensive empirical characterization of antigen-binding domain (ABD)/CAR constructs for clinical suitability. Here, we present a cost-efficient and rapid method for evaluating CARs in human Jurkat T cells. Using a modular CAR plasmid, a highly efficient ABD cloning strategy, plasmid electroporation, short-term co-culture, and flow-cytometric detection of CD69, this assay (referred to as CAR-J) evaluates sensitivity and specificity for ABDs. Assessing 16 novel anti-CD22 single-chain variable fragments derived from mouse monoclonal antibodies, CAR-J stratified constructs by response magnitude to CD22-expressing target cells. We also characterized 5 novel anti-EGFRvIII CARs for preclinical development, identifying candidates with varying tonic and target-specific activation characteristics. When evaluated in primary human T cells, tonic/auto-activating (without target cells) EGFRvIII-CARs induced target-independent proliferation, differentiation toward an effector phenotype, elevated activity against EGFRvIII-negative cells, and progressive loss of target-specific response upon in vitro re-challenge. These EGFRvIII CAR-T cells also showed anti-tumor activity in xenografted mice. In summary, CAR-J represents a straightforward method for high-throughput assessment of CAR constructs as genuine cell-associated antigen receptors that is particularly useful for generating large specificity datasets as well as potential downstream CAR optimization.

Evaluation of recombinant adenovirus vectors and adjuvanted protein as a heterologous prime-boost strategy using HER2 as a model antigen.

Induction of strong antigen-specific cell-mediated and humoral responses are critical to developing a successful therapeutic vaccine. Herein, using HER2 as a model antigen, we aim to evaluate a therapeutic vaccine protocol that elicits anti-tumor antibody and cytotoxic T cells to HER2/neu antigen. Replication-competent (ΔPS AdV) and non-replicating recombinant adenoviral vectors (AdV) expressing a rat HER2/neu (ErbB2) oncogene, were generated and compared for four different doses and over four time points for their ability to induce antigen-specific T and B cell responses in mice. Although ΔPS AdV:Her2 vector was shown to induce more durable antigen-specific CD8+ T cell responses, overall, the AdV:Her2 vector induced broader T and B cell responses. Hence the AdV:Her2 vector was used to evaluate a heterologous prime-boost vaccination regimen using rat HER2 protein encapsulated in archaeosomes composed of a semi-synthetic glycolipid (sulfated S-lactosylarchaeol, SLA; and lactosylarchaeol, LA) (SLA/LA:HER2enc) or admixed with archaeosomes composed of SLA alone (SLA:HER2adm). We first tested AdV:Her2 using a prime-boost approach with SLA/LA:HER2enc, and thereafter evaluated a sub-optimal AdV:Her2 dose in a heterologous prime-boost approach with SLA:HER2adm. A single administration of AdV:Her2 alone induced strong cell-mediated immune responses, whereas SLA/LA:HER2enc alone induced strong antigen-specific IgG titers. In mice primed with a suboptimal dose of AdV:Her2, strong CD8+ T-cell responses were observed after a single dose which were not further augmented by protein boost. AdV:Her2 induced CD4+ specific T-cell responses were augmented by SLA:HER2adm. Homologous vaccination using SLA:HER2adm induced strong antigen-specific antibody responses. However, the overall magnitude of the responses was similar with three doses of SLA:HER2adm or Ad:HER2 prime followed by two doses of SLA:HER2adm. We demonstrate that AdV:Her2 is capable of inducing strong antigen-specific CD8+ T cell responses, even at a low dose, and that these responses can be broadened to include antigen-specific antibody responses by boosting with SLA adjuvanted proteins without compromising CD8 T cell responses elicited by AdV priming.

Chemical Derivatization Strategy for Extending the Identification of MHC Class I Immunopeptides.

Neoantigen-based therapeutic vaccines have a high potential impact on tumor eradication and patient survival. Mass spectrometry (MS)-based immunopeptidomics has the capacity to identify tumor-associated epitopes and pinpoint mutation-bearing major histocompatibility complex (MHC)-binding peptides. This approach presents several challenges, including the identification of low-abundance peptides. In addition, MHC peptides have much lower MS/MS identification rates than tryptic peptides due to their shorter sequence and lack of basic amino acid at C-termini. In this study, we report the development and application of a novel chemical derivatization strategy that combines the analysis of native, dimethylated, and alkylamidated peptides by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to expand the coverage of the MHC peptidome. The results revealed that dimethylation increases hydrophobicity and ionization efficiency of MHC class I peptides, while alkylamidation significantly improves the fragmentation by producing more y-ions during MS/MS fragmentation. Thus, the combination of dimethylation and alkylamidation enabled the identification of peptides that could not be identified from the analysis of their native form. Using this strategy, we identified 3148 unique MHC I peptides from HCT 116 cell lines, compared to only 1388 peptides identified in their native form. Among these, 10 mutation-bearing peptides were identified with high confidence, indicating that this chemical derivatization strategy is a promising approach for neoantigen discovery in clinical applications.

An Archaeosome-Adjuvanted Vaccine and Checkpoint Inhibitor Therapy Combination Significantly Enhances Protection from Murine Melanoma.

Archaeosomes constitute archaeal lipid vesicle vaccine adjuvants that evoke a strong CD8⁺ T cell response to antigenic cargo. Therapeutic treatment of murine B16-ovalbumin (B16-OVA) melanoma with archaeosome-OVA eliminates small subcutaneous solid tumors; however, they eventually resurge despite an increased frequency of circulating and tumor infiltrating OVA-CD8⁺ T cells. Herein, a number of different approaches were evaluated to improve responses, including dose number, interval, and the combination of vaccine with checkpoint inhibitors. Firstly, we found that tumor protection could not be enhanced by repetitive and/or delayed boosting to maximize the CD8⁺ T cell number and/or phenotype. The in vivo cytotoxicity of vaccine-induced OVA-CD8⁺ T cells was impaired in tumor-bearing mice. Additionally, tumor-infiltrating OVA-CD8⁺ T cells had an increased expression of programmed cell death protein-1 (PD-1) compared to other organ compartments, suggesting impaired function. Combination therapy of tumor-bearing mice with the vaccine archaeosome-OVA, and α-CTLA-4 administered concurrently as well as α-PD-1 and an α-PD-L1 antibody administered starting 9 days after tumor challenge given on a Q3Dx4 schedule (days 9, 12, 15 and 18), significantly enhanced survival. Following multi-combination therapy ~70% of mice had rapid tumor recession, with no detectable tumor mass after >80 days in comparison to a median survival of 17-22 days for untreated or experimental groups receiving single therapies. Overall, archaeosomes offer a powerful platform for delivering cancer antigens when used in combination with checkpoint inhibitor immunotherapies.

Immunogenicity of a peptide-based anti-IgE conjugate vaccine in non-human primates.

The anti-human immunoglobulin E (IgE) monoclonal antibody, omalizumab (Xolair®, Genentech, South San Fransisco, CA), is effective in the treatment of poorly controlled moderate to severe allergic asthma and chronic idiopathic urticaria. It acts by specifically binding to the constant domain (Cϵ3) of free human IgE in the blood and interstitial fluid. Although efficacious, use of omalizumab is limited due to restrictions on patient weight and pre-existing IgE levels, and frequent dosing (q2-4 weeks). A vaccine inducing anti-IgE antibodies has the potential for similar clinical benefits with less frequent dosing and relatively lower cost of goods. We developed a vaccine containing two IgE peptide-conjugates targeting the Cϵ3 domain of human IgE. As part of preclinical evaluation of the vaccine to optimize formulation and dose prior to initiating clinical studies, we evaluated the vaccine in non-human primates, and demonstrate the induction of anti-peptide antibodies that can bind to conformationally intact human IgE and are capable, at least in some animals, of substantial lowering circulating IgE levels.

Effects of KLK Peptide on Adjuvanticity of Different ODN Sequences.

Endosomal Toll-like receptors (TLR) such as TLR3, 7, 8 and 9 recognize pathogen associated nucleic acids. While DNA sequence does influence degree of binding to and activation of TLR9, it also appears to influence the ability of the ligand to reach the intracellular endosomal compartment. The KLK (KLKL5KLK) antimicrobial peptide, which is immunostimulatory itself, can translocate into cells without cell membrane permeabilization and thus can be used for endosomal delivery of TLR agonists, as has been shown with the IC31 formulation that contains an oligodeoxynucleotide (ODN) TLR9 agonist. We evaluated the adjuvant activity of KLK combined with CpG or non-CpG (GpC) ODN synthesized with nuclease resistant phosphorothioate (S) or native phosphodiester (O) backbones with ovalbumin (OVA) antigen in mice. As single adjuvants, CpG(S) gave the strongest enhancement of OVA-specific immunity and the addition of KLK provided no benefit and was actually detrimental for some readouts. In contrast, KLK enhanced the adjuvant effects of CpG(O) and to a lesser extent of GpC (S), which on their own had little or no activity. Indeed while CD8 T cells, IFN-γ secretion and humoral response to vaccine antigen were enhanced when CpG(O) was combined with KLK, only IFN-γ secretion was enhanced when GpC (S) was combined to KLK. The synergistic adjuvant effects with KLK/ODN combinations were TLR9-mediated since they did not occur in TLR9 knock-out mice. We hypothesize that a nuclease resistant ODN with CpG motifs has its own mechanism for entering cells to reach the endosome. For ODN without CpG motifs, KLK appears to provide an alternate mechanism for accessing the endosome, where it can activate TLR9, albeit with lower potency than a CpG ODN. For nuclease sensitive (O) backbone ODN, KLK may also provide protection from nucleases in the tissues.

CpG-mediated augmentation of CD8+ T-cell responses in mice is attenuated by a water-in-oil emulsion (Montanide ISA-51) but enhanced by an oil-in-water emulsion (IDRI SE).

Adjuvants are a key component in enhancing immunogenicity of vaccines and play a vital role in facilitating the induction of the correct type of immunity required for each vaccine to be optimally efficacious. Several different adjuvants are found in licensed vaccines, and many others are in pre-clinical or clinical testing. Agonists for TLRs are potent activators of the innate immune system and some, such as CpG (TLR9 agonist), are particularly good for promoting cellular immunity because of the induction of Th1 cytokines. Emulsions that have both delivery and adjuvant properties are classified as water-in-oil (W/O) or oil-in-water (O/W) formulations. The W/O emulsion Montanide ISA-51, often combined with CpG, has been widely tested in cancer vaccine clinical trials. Squalene-based O/W emulsions are in licensed influenza vaccines, and T-cell responses have been assessed pre-clinically. No clinical study has compared the two types of emulsions, and the continued use of W/O with CpG in cancer vaccines may be because the lack of single adjuvant controls has masked the interference issue. These findings may have important implications for the development of vaccines where T-cell immunity is considered essential, such as those for cancer and chronic infections. Using particulate (hepatitis B surface antigen) and soluble protein (ovalbumin) antigen, we show in mice that a W/O emulsion (ISA-51) abrogates CpG-mediated augmentation of CD8(+) T-cell responses, whereas a squalene-based O/W emulsion significantly enhanced them.

Anti-IgE Qb-VLP Conjugate Vaccine Self-Adjuvants through Activation of TLR7.

Qb bacteriophage virus-like particles (Qb-VLP) are utilized as carriers to enhance immune responses to weakly or non-immunogenic antigens such as peptides and haptens. Qb-VLPs are formed through the self-assembly of multiple Qb capsid protein monomers, a process which traps a large amount of bacterial RNA in the core of the VLP. Bacterial RNA is known to activate the innate immune system via TLR 7 and 8 found within the endosomes of certain immune cells and has been shown to contribute to the immunogenicity of Qb-VLP vaccines. Herein, we evaluated an anti-IgE vaccine comprised of two IgE peptides (Y and P) conjugated to Qb-VLP (Qb-Y and Qb-P, respectively) for in vitro stimulation of human PBMCs and in vivo immunogenicity in mice. The in vitro secretion of IFN-α from human PBMCs exposed to Qb-Y is consistent with TLR7 activation. Immunization of mice with the IgE peptide Qb-VLP conjugates induced high titers of anti-IgE antibodies in wild-type mice, but significantly lower titers in TLR7 knockout mice, supporting the self-adjuvanting role of the RNA. Inclusion of alum and alum/CpG as adjuvants partially or completely compensated for the lack of TLR7 activation in TLR7-deficient mice. Our study demonstrates the key role that TLR7 plays in the immunogenicity of the IgE peptide Qb-VLP conjugate vaccine.

CpG ODN and ISCOMATRIX adjuvant: a synergistic adjuvant combination inducing strong T-Cell IFN-γ responses.

For the induction of robust humoral and cellular immune responses, a strong rationale exists to use vaccine-adjuvant combinations possessing both immune modulatory and enhanced delivery capabilities. Herein, we evaluated the combination of 2 different adjuvants, a TLR9 agonist, composed of synthetic oligodeoxynucleotides (ODN) containing immunostimulatory CpG motifs (CpG), and ISCOMATRIX adjuvant (ISCOMATRIX), composed of saponin, phospholipid, and cholesterol, which possesses both immunostimulatory and delivery properties. While both individual adjuvants have been shown effective in numerous preclinical and clinical studies, it is likely that for optimal adjuvant activity a combined adjuvant approach will be necessary. Herein, using three different antigens, namely, hepatitis B surface antigen (HBsAg), ovalbumin (OVA), and influenza A haemagglutinin antigen (HA), we show in mice that some adjuvant effects of CpG and ISCOMATRIX are further enhanced if they are used in combination. In particular, with all three antigens, IFN-γ levels were greatly increased with the CpG/ISCOMATRIX combination. The ability of the CpG/ISCOMATRIX combination to induce antitumor responses when administered with OVA following administration to mice of a highly metastatic OVA-secreting tumor cell line (B16-OVA melanoma) was also demonstrated. Thus the CpG/ISCOMATRIX combination may prove to be a valuable tool in the development of novel or improved vaccines.

Pitfalls of vaccinations with WT1-, Proteinase3- and MUC1-derived peptides in combination with MontanideISA51 and CpG7909.

T cells with specificity for antigens derived from Wilms Tumor gene (WT1), Proteinase3 (Pr3), and mucin1 (MUC1) have been demonstrated to lyse acute myeloid leukemia (AML) blasts and multiple-myeloma (MM) cells, and strategies to enhance or induce such tumor-specific T cells by vaccination are currently being explored in multiple clinical trials. To test safety and immunogenicity of a vaccine composed of WT1-, Pr3-, and MUC1-derived Class I-restricted peptides and the pan HLA-DR T helper cell epitope (PADRE) or MUC1-helper epitopes in combination with CpG7909 and MontanideISA51, four patients with AML and five with MM were repetitively vaccinated. No clinical responses were observed. Neither pre-existing nor naive WT1-/Pr3-/MUC1-specific CD8+ T cells expanded in vivo by vaccination. In contrast, a significant decline in vaccine-specific CD8+ T cells was observed. An increase in PADRE-specific CD4+ T helper cells was observed after vaccination but these appeared unable to produce IL2, and CD4+ T cells with a regulatory phenotype increased. Taken into considerations that multiple clinical trials with identical antigens but different adjuvants induced vaccine-specific T cell responses, our data caution that a vaccination with leukemia-associated antigens can be detrimental when combined with MontanideISA51 and CpG7909. Reflecting the time-consuming efforts of clinical trials and the fact that 1/3 of ongoing peptide vaccination trails use CpG and/or Montanide, our data need to be taken into consideration.

Paclitaxel reduces regulatory T cell numbers and inhibitory function and enhances the anti-tumor effects of the TLR9 agonist PF-3512676 in the mouse.

The anti-tumor properties of Toll-like receptor (TLR) 9 agonist CpG oligodeoxynucleotides (ODN) are enhanced by combinations with several cytotoxic chemotherapy regimens. The mechanisms of this added benefit, however, remain unclear. We now report that, similar to the depletion of regulatory T cells (Treg) using anti-CD25, paclitaxel increased the anti-tumor effect of the TLR9 agonist PF-3512676 in a CD8(+) T cell-dependent fashion. Paclitaxel treatment decreased Treg numbers in a TLR4-independent fashion, and preferentially affected cycling Treg expressing high levels of FoxP3. The paclitaxel-induced reduction in Treg FoxP3 expression was associated with reduced inhibitory function. Adoptively transferred tumor-antigen specific CD8(+) T cells proliferated better in mice treated with paclitaxel and their recruitment in the tumor was increased. However, the systemic frequency of PF-3512676-induced tumor-antigen specific effector CD8(+) T cells decreased with paclitaxel, suggesting opposite effects of paclitaxel on the anti-tumor response. Finally, gene expression profiling and studies of tumor-associated immune cells revealed a complex modulation of the PF-3512676-induced immune response by paclitaxel, including a decrease of IL-10 expression and an increase in IL-17-secreting CD4(+) T cells. Collectively, these data suggest that paclitaxel combined with PF-3512676 may not only promote a better anti-tumor CD8(+) response though increased recruitment in the tumor, possibly through Treg depletion and suppression, but also exerts more complex immune modulatory effects.

Testing of CpG-optimized protein and DNA vaccines against the hepatitis B virus in chimpanzees for immunogenicity and protection from challenge.

Despite the existence for some time of effective prophylactic vaccines, hepatitis B virus (HBV) infection remains an important global concern. Improvements on existing vaccines could be beneficial, especially in situations where it is desirable or necessary to induce protective immunity more rapidly or with fewer doses. We have compared, in chimpanzees, a current HBV vaccine that contains recombinant hepatitis B surface antigen HBsAg) adsorbed to alum, with two novel vaccine strategies that have proven superior to the current vaccine in mice. The first approach was the use of oligodeoxynucleotides containing CpG motifs (CpG ODN) as an adjuvant to Engerix-B, a commercial HBV vaccine. The addition of CpG ODN to Engerix-B greatly improved the kinetics and magnitude of the humoral response, suggesting that CpG ODN might allow induction of protective immunity in humans more quickly and with fewer vaccine doses. All animals receiving either control or CpG-containing subunit vaccines at 0 and 4 weeks attained titers of HBsAg-specific antibody (anti-HBs) considered protective (> or =10 mIU/ml) and were indeed protected from challenge at 8 weeks with 10(3.5) 50% chimp infectious doses (CID(50)) of intravenous HBV. The second approach was a DNA vaccine with a plasmid vector optimized for content of immunostimulatory CpG motifs. Despite the fact that earlier studies had shown four doses of a similar DNA vaccine (except not optimized for CpG content) to induce strong humoral responses in 1 of 2 chimpanzees, in this study two doses of DNA vaccine (at 0 and 4 weeks) did not generate any detectable anti-HBs in either of 2 chimpanzees, although it did protect 1 that rapidly developed anti-HBs during the incubation period, suggesting priming of an antibody response. The poor results may be due to an inadequate number of doses or amount of plasmid DNA in these larger animals, but nevertheless point to the need to improve delivery methods for DNA vaccines for use in larger animals such as primates.

Treatment of intravaginal HSV-2 infection in mice: a comparison of CpG oligodeoxynucleotides and resiquimod (R-848).

The mammalian innate immune system recognizes pathogens via a series of pattern-recognition receptors such as the toll-like receptors (TLR) that interact with pathogen-associated molecular patterns (PAMPs) and lead to the rapid activation of innate immune cells. In this study, we compared the efficacy of CpG ODN (a TLR9 agonist) and resiquimod (R-848; a TLR7/8 agonist) for topical immunoprophylaxis or immunotherapy of vaginal herpes simplex virus type 2 (HSV-2) infection in mice. Efficacy against HSV infection was observed with CpG ODN but less so with R-848, even after repeated administrations. Intravaginal (IVAG) administration of CpG ODN resulted in strong local but relatively weak systemic immune activation, as determined by levels of the chemokines IP-10, MIG and I-TAC in vaginal tissue and plasma, respectively. In contrast, IVAG administration of R-848 resulted in high levels of plasma IP-10, similar to those seen after parenteral administration, but overall, weaker or shorter-lived local immune responses than obtained with CpG ODN. These findings suggest that differences in biodistribution and sites of immune activation between CpG ODN and R-848 after IVAG delivery account for differences in efficacy, and demonstrate the need for local mucosal innate activation for protection against HSV-2.