The engineered CD80 variant fusion therapeutic davoceticept combines checkpoint antagonism with conditional CD28 costimulation for anti-tumor immunity.

Despite the recent clinical success of T cell checkpoint inhibition targeting the CTLA-4 and PD-1 pathways, many patients either fail to achieve objective responses or they develop resistance to therapy. In some cases, poor responses to checkpoint blockade have been linked to suboptimal CD28 costimulation and the inability to generate and maintain a productive adaptive anti-tumor immune response. To address this, here we utilize directed evolution to engineer a CD80 IgV domain with increased PD-L1 affinity and fuse this to an immunoglobulin Fc domain, creating a therapeutic (ALPN-202, davoceticept) capable of providing CD28 costimulation in a PD-L1-dependent fashion while also antagonizing PD-1 - PD-L1 and CTLA-4-CD80/CD86 interactions. We demonstrate that by combining CD28 costimulation and dual checkpoint inhibition, ALPN-202 enhances T cell activation and anti-tumor efficacy in cell-based assays and mouse tumor models more potently than checkpoint blockade alone and thus has the potential to generate potent, clinically meaningful anti-tumor immunity in humans.

ICOSL+ plasmacytoid dendritic cells as inducer of graft-versus-host disease, responsive to a dual ICOS/CD28 antagonist.

Acute graft-versus-host disease (aGVHD) remains a major complication of allogeneic hematopoietic cell transplantation (HCT). CD146 and CCR5 are proteins that mark activated T helper 17 (Th17) cells. The Th17 cell phenotype is promoted by the interaction of the receptor ICOS on T cells with ICOS ligand (ICOSL) on dendritic cells (DCs). We performed multiparametric flow cytometry in a cohort of 156 HCT recipients and conducted experiments with aGVHD murine models to understand the role of ICOSL+ DCs. We observed an increased frequency of ICOSL+ plasmacytoid DCs, correlating with CD146+CCR5+ T cell frequencies, in the 64 HCT recipients with gastrointestinal aGVHD. In murine models, donor bone marrow cells from ICOSL-deficient mice compared to those from wild-type mice reduced aGVHD-related mortality. Reduced aGVHD resulted from lower intestinal infiltration of pDCs and pathogenic Th17 cells. We transplanted activated human ICOSL+ pDCs along with human peripheral blood mononuclear cells into immunocompromised mice and observed infiltration of intestinal CD146+CCR5+ T cells. We found that prophylactic administration of a dual human ICOS/CD28 antagonist (ALPN-101) prevented aGVHD in this model better than did the clinically approved belatacept (CTLA-4-Fc), which binds CD80 (B7-1) and CD86 (B7-2) and interferes with the CD28 T cell costimulatory pathway. When started at onset of aGVHD signs, ALPN-101 treatment alleviated symptoms of ongoing aGVHD and improved survival while preserving antitumoral cytotoxicity. Our data identified ICOSL+-pDCs as an aGVHD biomarker and suggest that coinhibition of the ICOSL/ICOS and B7/CD28 axes with one biologic drug may represent a therapeutic opportunity to prevent or treat aGVHD.

Partial efficacy of a broadly neutralizing antibody against cell-associated SHIV infection.

Broadly neutralizing antibodies (BnAbs) protect macaques from cell-free simian/human immunodeficiency virus (SHIV) challenge, but their efficacy against cell-associated SHIV is unclear. Virus in cell-associated format is highly infectious, present in transmission-competent bodily fluids, and potentially capable of evading antibody-mediated neutralization. The PGT121 BnAb, which recognizes an epitope consisting of the V3 loop and envelope glycans, mediates antibody-dependent cellular cytotoxicity and neutralization of cell-to-cell HIV-1 transmission. To evaluate whether a BnAb can prevent infection after cell-associated viral challenge, we infused pigtail macaques with PGT121 or an isotype control and challenged animals 1 hour later intravenously with SHIVSF162P3-infected splenocytes. All five controls had high viremia 1 week after challenge. Three of six PGT121-infused animals were completely protected, two of six animals had a 1-week delay in onset of high viremia, and one animal had a 7-week delay in onset of viremia. The infused antibody had decayed on average to 2.0 µg/ml by 1 week after infusion and was well below 1 µg/ml (range, <0.1 to 0.8 µg/ml) by 8 weeks. The animals with a 1-week delay before high viremia had relatively lower plasma concentrations of PGT121. Transfer of 22 million peripheral blood mononuclear cells (PBMCs) stored at weeks 1 to 4 from the animal with the 7-week delayed onset of viremia into uninfected macaques did not initiate infection. Our results show that HIV-1-specific neutralizing antibodies have partial efficacy against cell-associated virus exposure in macaques. We conclude that sustaining high concentrations of bioavailable BnAb is important for protecting against cell-associated virus.

Immunological considerations for developing antibody therapeutics for Influenza A.

Influenza infection can give rise to serious illness leading to complications and hospitalization of patients. The efficacy of current standard of care is very limited and provides little relief for patients hospitalized with serious flu. Human monoclonal antibodies (mAb) against influenza are being developed as new treatment options for this patient population. When developing antibody therapeutics, it is important to consider all possible immunologic effects of the antibodies on viral infection and disease progression including those other than the postulated therapeutic mechanisms. An area of concern is the potential of antibody-dependent enhancement (ADE) of illness. ADE of viral infections has been extensively described for Dengue virus (DENV) but not for influenza. Recently, preliminary results from clinical viral challenge studies of anti-HA-stalk mAbs suggested the possibility of enhanced viral shedding, raising concerns for ADE when utilizing mAbs as therapeutic intervention for influenza although viral shedding was not enhanced in the clinical viral challenge of anti-M2 mAb TCN-032. We herein discuss the known mechanisms of ADE and their relevance to developing mAbs such as anti-HA and anti-M2 for influenza disease.

Efficacy and safety of treatment with an anti-m2e monoclonal antibody in experimental human influenza.

BACKGROUND: The efficacy of TCN-032, a human monoclonal antibody targeting a conserved epitope on M2e, was explored in experimental human influenza. METHODS: Healthy volunteers were inoculated with influenza A/Wisconsin/67/2005 (H3N2) and received a single dose of the study drug, TCN-032, or placebo 24 hours later. Subjects were monitored for symptoms, viral shedding, and safety, including cytokine measurements. Oseltamivir was administered 7 days after inoculation. RESULTS: Although the primary objective of reducing the proportion of subjects developing any grade ≥2 influenza symptom or pyrexia, was not achieved, TCN-032-treated subjects showed 35% reduction (P = .047) in median total symptom area under the curve (days 1-7) and 2.2 log reduction in median viral load area under the curve (days 2-7) by quantitative polymerase chain reaction (P = .09) compared with placebo-treated subjects. TCN-032 was safe and well tolerated with no additional safety signals after administration of oseltamivir. Serum cytokine levels (interferon γ, tumor necrosis factor α, and interleukin 8 and 10) were similar in both groups. Genotypic and phenotypic analyses showed no difference between virus derived from subjects after TCN-032 treatment and parental strain. CONCLUSIONS: These data indicate that TCN-032 may provide immediate immunity and therapeutic benefit in influenza A infection, with no apparent emergence of resistant virus. TCN-032 was safe with no evidence of immune exacerbation based on serum cytokine expression. Clinicaltrials.gov registry number. NCT01719874.

Broadly neutralizing HIV antibodies define a glycan-dependent epitope on the prefusion conformation of gp41 on cleaved envelope trimers.

Broadly neutralizing HIV antibodies are much sought after (a) to guide vaccine design, both as templates and as indicators of the authenticity of vaccine candidates, (b) to assist in structural studies, and (c) to serve as potential therapeutics. However, the number of targets on the viral envelope spike for such antibodies has been limited. Here, we describe a set of human monoclonal antibodies that define what is, to the best of our knowledge, a previously undefined target on HIV Env. The antibodies recognize a glycan-dependent epitope on the prefusion conformation of gp41 and unambiguously distinguish cleaved from uncleaved Env trimers, an important property given increasing evidence that cleavage is required for vaccine candidates that seek to mimic the functional HIV envelope spike. The availability of this set of antibodies expands the number of vaccine targets on HIV and provides reagents to characterize the native envelope spike.

The effects of somatic hypermutation on neutralization and binding in the PGT121 family of broadly neutralizing HIV antibodies.

Broadly neutralizing HIV antibodies (bnAbs) are typically highly somatically mutated, raising doubts as to whether they can be elicited by vaccination. We used 454 sequencing and designed a novel phylogenetic method to model lineage evolution of the bnAbs PGT121-134 and found a positive correlation between the level of somatic hypermutation (SHM) and the development of neutralization breadth and potency. Strikingly, putative intermediates were characterized that show approximately half the mutation level of PGT121-134 but were still capable of neutralizing roughly 40-80% of PGT121-134 sensitive viruses in a 74-virus panel at median titers between 15- and 3-fold higher than PGT121-134. Such antibodies with lower levels of SHM may be more amenable to elicitation through vaccination while still providing noteworthy coverage. Binding characterization indicated a preference of inferred intermediates for native Env binding over monomeric gp120, suggesting that the PGT121-134 lineage may have been selected for binding to native Env at some point during maturation. Analysis of glycan-dependent neutralization for inferred intermediates identified additional adjacent glycans that comprise the epitope and suggests changes in glycan dependency or recognition over the course of affinity maturation for this lineage. Finally, patterns of neutralization of inferred bnAb intermediates suggest hypotheses as to how SHM may lead to potent and broad HIV neutralization and provide important clues for immunogen design.

Highly potent HIV-specific antibody neutralization in vitro translates into effective protection against mucosal SHIV challenge in vivo.

Most animal studies using passive administration of HIV broadly neutralizing monoclonal antibodies (bnMAbs) have associated protection against high-dose mucosal viral challenge with relatively high serum concentrations of antibody. We recently identified several bnMAbs remarkable for their in vitro potency against HIV. Of these bnMAbs, PGT121 is one of the most broad and potent antibodies isolated to date and shows 10- to 100-fold higher neutralizing activity than previously characterized bnMAbs. To evaluate the protective potency of PGT121 in vivo, we performed a protection study in rhesus macaques. Animals were i.v. administered 5 mg/kg, 1 mg/kg, or 0.2 mg/kg PGT121 24 h before being vaginally challenged with a single high dose of chimeric simian-human immunodeficiency virus (SHIV)(SF162P3). Sterilizing immunity was achieved in all animals administered 5 mg/kg and 1 mg/kg and three of five animals administered 0.2 mg/kg PGT121, with corresponding average antibody serum concentrations of 95 µg/mL, 15 µg/mL, and 1.8 µg/mL, respectively. The results suggest that a protective serum concentration for PGT121 is in the single-digit µg/mL for SHIV(SF162P3), showing that PGT121 can mediate sterilizing immunity at serum concentrations that are significantly lower than those observed in previous studies and that may be achievable through vaccination with the development of a suitable immunogen.

Physiological effects of unassembled chaperonin Cct subunits in the yeast Saccharomyces cerevisiae.

Eukaryotic chaperonins, the Cct complexes, are assembled into two rings, each of which is composed of a stoichiometric array of eight different subunits, which are denoted Cct1p-Cct8p. Overexpression of a single CCT gene in Saccharomyces cerevisiae causes an increase of the corresponding Cct subunit, but not of the Cct complex. Nevertheless, overexpression of certain Cct subunits, especially CCT6, suppresses a wide range of abnormal phenotypes, including those caused by the diverse types of conditional mutations tor2-21, lst8-2 and rsp5-9 and those caused by the concomitant overexpression of Sit4p and Sap155p. The examination of 73 altered forms of Cct6p revealed that the cct6-24 mutation, containing GDGTT --> AAAAA replacements of the conserved ATP-binding motif, was unable to suppress any of these traits, although the cct6-24 allele was completely functional for growth. These results provide evidence for functional differences among Cct subunits and for physiological properties of unassembled subunits. We suggest that the suppression is due to the competition of specific Cct subunits for activities that normally modify various cellular components. Furthermore, we also suggest that the Cct subunits can act as suppressors only in certain states, such as when associated with ATP.

Functional interaction of heterogeneous nuclear ribonucleoprotein C with poliovirus RNA synthesis initiation complexes.

We had previously demonstrated that a cellular protein specifically interacts with the 3' end of poliovirus negative-strand RNA. We now report the identity of this protein as heterogeneous nuclear ribonucleoprotein (hnRNP) C1/C2. Formation of an RNP complex with poliovirus RNA was severely impaired by substitution of a lysine, highly conserved among vertebrates, with glutamine in the RNA recognition motif (RRM) of recombinant hnRNP C1, suggesting that the binding is mediated by the RRM in the protein. We have also shown that in a glutathione S-transferase (GST) pull-down assay, GST/hnRNP C1 binds to poliovirus polypeptide 3CD, a precursor to the viral RNA-dependent RNA polymerase, 3D(pol), as well as to P2 and P3, precursors to the nonstructural proteins. Truncation of the auxiliary domain in hnRNP C1 (C1DeltaC) diminished these protein-protein interactions. When GST/hnRNP C1DeltaC was added to in vitro replication reactions, a significant reduction in RNA synthesis was observed in contrast to reactions supplemented with wild-type fusion protein. Indirect functional depletion of hnRNP C from in vitro replication reactions, using poliovirus negative-strand cloverleaf RNA, led to a decrease in RNA synthesis. The addition of GST/hnRNP C1 to the reactions rescued RNA synthesis to near mock-depleted levels. Furthermore, we demonstrated that poliovirus positive-strand and negative-strand RNA present in cytoplasmic extracts prepared from infected HeLa cells coimmunoprecipitated with hnRNP C1/C2. Our findings suggest that hnRNP C1 has a role in positive-strand RNA synthesis in poliovirus-infected cells, possibly at the level of initiation.

A tenascin-C aptamer identified by tumor cell SELEX: systematic evolution of ligands by exponential enrichment.

The targeting of molecular repertoires to complex systems rather than biochemically pure entities is an accessible approach that can identify proteins of biological interest. We have probed antigens presented by a monolayer of tumor cells for their ability to interact with a pool of aptamers. A glioblastoma-derived cell line, U251, was used as the target for systematic evolution of ligands by exponential enrichment by using a single-stranded DNA library. We isolated specifically interacting oligonucleotides, and biochemical strategies were used to identify the protein target for one of the aptamers. Here we characterize the interaction of the DNA aptamer, GBI-10, with tenascin-C, an extracellular protein found in the tumor matrix. Tenascin-C is believed to be involved in both embryogenesis and oncogenesis pathways. Systematic evolution of ligands by exponential enrichment appears to be a successful strategy for the a priori identification of targets of biological interest within complex systems.