Innate immune signals mediated by Toll-like receptors (TLRs) have been thought to contribute considerably to the antibody-enhancing effects of vaccine adjuvants. However, we report here that mice deficient in the critical signaling components for TLR mount robust antibody responses to T cell-dependent antigen given in four typical adjuvants: alum, Freund's complete adjuvant, Freund's incomplete adjuvant, and monophosphoryl-lipid A/trehalose dicorynomycolate adjuvant. We conclude that TLR signaling does not account for the action of classical adjuvants and does not fully explain the action of a strong adjuvant containing a TLR ligand. This may have important implications in the use and development of vaccine adjuvants.

Aging is associated with a gradual loss of naïve T cells and a reciprocal increase in the proportion of memory T cells. While reduced thymic output is important, age-dependent changes in factors supporting naïve T cells homeostasis may also be involved. Indeed, we noted a dramatic decrease in the ability of aged mice to support survival and homeostatic proliferation of naïve T cells. The defect was not due to a reduction in IL-7 expression, but from a combination of changes in the secondary lymphoid environment that impaired naïve T cell entry and access to key survival factors. We observed an age-related shift in the expression of homing chemokines and structural deterioration of the stromal network in T cell zones. Treatment with IL-7/mAb complexes can restore naïve T cell homeostatic proliferation in aged mice. Our data suggests that homeostatic mechanisms that support the naïve T cell pool deteriorate with age.

Clostridium difficile is the cause of emerging nosocomial infections that result in abundant morbidity and mortality worldwide. Thus, the development of a vaccine to kill the bacteria to prevent this disease is highly desirable. Several recently identified bacterial surface glycans, such as PS-I and PS-II, are promising vaccine candidates to preclude C. difficile infection. To circumvent difficulties with the generation of natural PS-I due to its low expression levels in bacterial cultures, improved chemical synthesis protocols for the pentasaccharide repeating unit of PS-I and oligosaccharide substructures were utilized to produce large quantities of well-defined PS-I related glycans. The analysis of stool and serum samples obtained from C. difficile patients using glycan microarrays of synthetic oligosaccharide epitopes revealed humoral immune responses to the PS-I related glycan epitopes. Two different vaccine candidates were evaluated in the mouse model. A synthetic PS-I repeating unit CRM197 conjugate was immunogenic in mice and induced immunoglobulin class switching as well as affinity maturation. Microarray screening employing PS-I repeating unit substructures revealed the disaccharide Rha-(1→3)-Glc as a minimal epitope. A CRM197-Rha-(1→3)-Glc disaccharide conjugate was able to elicit antibodies recognizing the C. difficile PS-I pentasaccharide. We herein demonstrate that glycan microarrays exposing defined oligosaccharide epitopes help to determine the minimal immunogenic epitopes of complex oligosaccharide antigens. The synthetic PS-I pentasaccharide repeating unit as well as the Rha-(1→3)-Glc disaccharide are promising novel vaccine candidates against C. difficile that are currently in preclinical evaluation.

Interleukin-7 (IL-7) availability determines the size and proliferative state of the resting T cell pool. However, the mechanisms that regulate steady-state IL-7 amounts are unclear. Using experimental lymphopenic mouse models and IL-7-induced homeostatic proliferation to measure IL-7 availability in vivo, we found that radioresistant cells were the source of IL-7 for both CD4 and CD8 T cells. Hematopoietic lineage cells, although irrelevant as a source of IL-7, were primarily responsible for limiting IL-7 availability via their expression of IL-7R. Unexpectedly, innate lymphoid cells were found to have a potent influence on IL-7 amounts in the primary and secondary lymphoid tissues. These results demonstrate that IL-7 homeostasis is achieved through consumption by multiple subsets of innate and adaptive immune cells.

Synthetic cell-surface glycans are promising vaccine candidates against Clostridium difficile. The complexity of large, highly antigenic and immunogenic glycans is a synthetic challenge. Less complex antigens providing similar immune responses are desirable for vaccine development. Based on molecular-level glycan–antibody interaction analyses, we here demonstrate that the C. difficile surface polysaccharide-I (PS-I) can be resembled by multivalent display of minimal disaccharide epitopes on a synthetic scaffold that does not participate in binding. We show that antibody avidity as a measure of antigenicity increases by about five orders of magnitude when disaccharides are compared with constructs containing five disaccharides. The synthetic, pentavalent vaccine candidate containing a peptide T-cell epitope elicits weak but highly specific antibody responses to larger PS-I glycans in mice. This study highlights the potential of multivalently displaying small oligosaccharides to achieve antigenicity characteristic of larger glycans. The approach may result in more cost-efficient carbohydrate vaccines with reduced synthetic effort.

Interactions between glycans and glycan binding proteins are essential for numerous processes in all kingdoms of life. Glycan microarrays are an excellent tool to examine protein–glycan interactions. Here, we present a microbe-focused glycan microarray platform based on oligosaccharides obtained by chemical synthesis. Glycans were generated by combining different carbohydrate synthesis approaches including automated glycan assembly, solution-phase synthesis, and chemoenzymatic methods. The current library of more than 300 glycans is as diverse as the mammalian glycan array from the Consortium for Functional Glycomics and, due to its microbial focus, highly complementary. This glycan platform is essential for the characterization of various classes of glycan binding proteins. Applications of this glycan array platform are highlighted by the characterization of innate immune receptors and bacterial virulence factors as well as the analysis of human humoral immunity to pathogenic glycans.

P-BCMA-101 is a novel chimeric antigen receptor (CAR)-T cell therapeutic targeting BCMA, which is highly expressed on MM cells. It is designed to increase efficacy while minimizing toxicity through reduced immunogenicity, lack of tonic signaling, a safety switch, and a product comprised predominantly of early memory T cells that are effectively all CAR-positive. Rather than using a traditional antibody-based binder, P-BCMA-101 utilizes an anti-BCMA Centyrin™ fused to a CD3ζ/4-1BB signaling domain. Centyrins are fully human and have high binding affinities, but are smaller, more stable and potentially less immunogenic. P-BCMA-101 is produced using the piggyBac™ (PB) DNA Modification System instead of a viral vector, and requires only plasmid DNA and mRNA. This eliminates the need for virus, is less costly, and produces a purified population of CAR+ cells with a preponderance of the favorable stem cell memory T phenotype (TSCM). The higher cargo capacity permits the incorporation of other genes, a safety switch that allows for rapid depletion of product in vivo if indicated by adverse events, and a selection gene that allows for enrichment of CAR+ cells. These features are predicted to result in a greater therapeutic index. Efficacy of P-BCMA-101 in NSG mice bearing aggressive human MM.1S and p53 -/- MM.1S MM was reported (Hermanson, AACR 2016). Whereas control animals died early, tumor burden was reduced to the limit of detection after P-BCMA-101 treatment, and recurrences were spontaneously re-controlled without re-administration of product. A Phase 1, 3+3 dose escalation trial is being conducted in patients with r/r MM (≥ 3 prior lines, including a proteasome inhibitor and an IMiD, or double refractory) to assess the safety and efficacy of P-BCMA-101 (NCT03288493). No pre-specified level of BCMA expression was required. Patients are apheresed to harvest T cells, P-BCMA-101 is then manufactured and administered to patients as a single intravenous (IV) dose after a standard 3-day cyclophosphamide (300 mg/m2/day) / fludarabine (30 mg/m2/day) conditioning regimen. As of 31Jul18, 12 patients have been treated with 48, 50, 55, 118, 122, 124, 143, 155, 164, 238, 324 and 430 x 106 P-BCMA-101 CAR-T cells in 3 weight-based cohorts. Patients were heavily pre-treated (3-9 prior therapies), 100% had failed IMiDs, proteasome inhibitors and daratumumab, and 64% had high-risk cytogenetics. Nine patients have yet reached their first 2-week response assessment. All patients have shown some improvement in myeloma assessments on study, yet only 1 patient (8%) has developed any cytokine release syndrome (CRS) (limited Grade 2). Of 3 patients in the first cohort 1 attained a PR and 1 with non-secretory disease near CR of her plasmacytomas on PET/CT. Of the subsequent 6 patients, 3 patients have thus far reached a PR, 1 a VGPR, and 1 a sCR. Thus of the yet evaluable patients treated above Cohort 1, the overall response rate (ORR) is 83% (5/6), in spite of only one experiencing CRS. This CRS was scored as Grade 2, based on short-lived fever and hypotension managed with IV fluids and antibiotics, with minimal CRS marker elevations. Likewise, CRS markers were minimally elevated in other patients. The maximal IL-6 level in any patient was 86 pg/mL, which is orders of magnitude lower than levels generally reported in patients experiencing meaningful CRS after treatment with CAR-T products. No patients required treatment with tocilizumab or safety switch activation. There have been no patient deaths, and no neurotoxicity, DLTs or unexpected/off-target toxicities related to treatment. Generally, infusions were well-tolerated, with cytopenias, including transfusion requiring cytopenias and febrile neutropenia, being the most common Grade 3+ adverse events. Consistent with the hypothesis of the early memory phenotype conveying durability, circulating P-BCMA-101 cells were detected in the blood by flow and PCR, peaking at 2-3 weeks, and remaining detectable at the last timepoint tested in all patients (3 patients thus far assessed at 3 months). In conclusion, current clinical trial data in patients with r/r MM support preclinical findings that the novel design of P-BCMA-101 can produce significant efficacy, comparing favorably with other anti-BCMA CAR-T products at similar doses, with notably less CRS and no neurotoxicity, consistent with the hypothesis of an improved therapeutic index. Funding by Poseida Therapeutics and CIRM. Disclosures Gregory: Poseida Therapeutics, Inc.: Research Funding. Cohen:Seattle Genetics: Consultancy; Kite Pharma: Consultancy; Oncopeptides: Consultancy; Poseida Therapeutics, Inc.: Research Funding; GlaxoSmithKline: Consultancy, Research Funding; Bristol Meyers Squibb: Consultancy, Research Funding; Celgene: Consultancy; Janssen: Consultancy; Novartis: Research Funding. Costello:Celgene: Consultancy; Poseida Therapeutics, Inc.: Research Funding; Takeda: Consultancy. Ali:Celgene Inc: Research Funding; Aduro Biotech: Consultancy, Research Funding; Amgen Inc: Consultancy; Juno: Consultancy; Takeda Oncology: Consultancy; Poseida Therapeutics: Research Funding. Berdeja:Genentech: Research Funding; Bluebird: Research Funding; Glenmark: Research Funding; Celgene: Research Funding; Takeda: Research Funding; Teva: Research Funding; Janssen: Research Funding; Sanofi: Research Funding; Novartis: Research Funding; Poseida Therapeutics, Inc.: Research Funding; Bristol-Myers Squibb: Research Funding; Amgen: Research Funding. Ostertag:Poseida Therapeutics, Inc.: Employment, Equity Ownership. Martin:Poseida Therapeutics, Inc.: Employment, Equity Ownership. Shedlock:Poseida Therapeutics, Inc.: Employment, Equity Ownership. Resler:Poseida Therapeutics, Inc.: Employment, Equity Ownership. Spear:Poseida Therapeutics, Inc.: Employment, Equity Ownership. Orlowski:Millenium Pharmaceuticals: Consultancy, Research Funding; Poseida: Research Funding; BioTheryX, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Consultancy; Genentech: Consultancy; Janssen Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Patel:Poseida Therapeutics, Inc.: Research Funding; Takeda: Research Funding; Abbvie: Research Funding; Celgene: Research Funding.

Glycoconjugate vaccines based on isolated capsular polysaccharide (CPS) save millions of lives annually by preventing invasive pneumococcal disease caused by Some components of the glycoconjugate vaccine Prevnar13 that contains CPS antigens from 13 serotypes undergo modifications or degradation during isolation and conjugation, resulting in production problems and lower efficacy. We illustrate how stable, synthetic oligosaccharide analogs of labile CPS induce a specific protective immune response against native CPS using serotype 5 (ST-5), a problematic CPS component of Prevnar13. The rare aminosugar l-PneuNAc and a branched l-FucNAc present in the natural repeating unit (RU) are essential for antibody recognition and avidity. The epitope responsible for specificity differs from the part of the antigen that is stabilized by chemical modification. Glycoconjugates containing stable, monovalent synthetic oligosaccharide analogs of ST-5 CPS RU induced long-term memory and protective immune responses in rabbits superior to those elicited by the ST-5 CPS component in multivalent Prevnar13.