The Bispecific Tumor Antigen-Conditional 4-1BB x 5T4 Agonist, ALG.APV-527, Mediates Strong T-Cell Activation and Potent Antitumor Activity in Preclinical Studies.

4-1BB (CD137) is an activation-induced costimulatory receptor that regulates immune responses of activated CD8 T and natural killer cells, by enhancing proliferation, survival, cytolytic activity, and IFNγ production. The ability to induce potent antitumor activity by stimulating 4-1BB on tumor-specific cytotoxic T cells makes 4-1BB an attractive target for designing novel immuno-oncology therapeutics. To minimize systemic immune toxicities and enhance activity at the tumor site, we have developed a novel bispecific antibody that stimulates 4-1BB function when co-engaged with the tumor-associated antigen 5T4. ALG.APV-527 was built on the basis of the ADAPTIR bispecific platform with optimized binding domains to 4-1BB and 5T4 originating from the ALLIGATOR-GOLD human single-chain variable fragment library. The epitope of ALG.APV-527 was determined to be located at domain 1 and 2 on 4-1BB using X-ray crystallography. As shown in reporter and primary cell assays in vitro, ALG.APV-527 triggers dose-dependent 4-1BB activity mediated only by 5T4 crosslinking. In vivo, ALG.APV-527 demonstrates robust antitumor responses, by inhibiting growth of established tumors expressing human 5T4 followed by a long-lasting memory immune response. ALG.APV-527 has an antibody-like half-life in cynomolgus macaques and was well tolerated at 50.5 mg/kg. ALG.APV-527 is uniquely designed for 5T4-conditional 4-1BB-mediated antitumor activity with potential to minimize systemic immune activation and hepatotoxicity while providing efficacious tumor-specific responses in a range of 5T4-expressing tumor indications as shown by robust activity in preclinical in vitro and in vivo models. On the basis of the combined preclinical dataset, ALG.APV-527 has potential as a promising anticancer therapeutic for the treatment of 5T4-expressing tumors.

Sequential immunization with a subtype B HIV-1 envelope quasispecies partially mimics the in vivo development of neutralizing antibodies.

A major goal of human immunodeficiency virus type 1 (HIV-1) vaccine efforts is the design of Envelope (Env)-based immunogens effective at eliciting heterologous or broad neutralizing antibodies (NAbs). We hypothesized that programming the B-cell response could be achieved by sequentially exposing the host to a collection of env variants representing the viral quasispecies members isolated from an individual that developed broad NAbs over time. This ordered vaccine approach (sequential) was compared to exposure to a cocktail of env clones (mixture) and to a single env variant (clonal). The three strategies induced comparable levels of the autologous and heterologous neutralization of tier 1 pseudoviruses. Sequential and mixture exposure to quasispecies led to epitope targeting similar to that observed in the simian-human immunodeficiency virus (SHIV)-infected animal from which the env variants were cloned, while clonal and sequential exposure led to greater antibody maturation than the mixture. Therefore, the sequential vaccine approach best replicated the features of the NAb response observed in that animal. This study is the first to explore the use of a collection of HIV-1 env quasispecies variants as immunogens and to present evidence that it is possible to educate the B-cell response by sequential exposure to native HIV-1 quasispecies env variants derived from an individual with a broadened NAb response.

Evidence for globally shared, cross-reacting polymorphic epitopes in the pregnancy-associated malaria vaccine candidate VAR2CSA.

Pregnancy-associated malaria (PAM) is characterized by the placental sequestration of Plasmodium falciparum-infected erythrocytes (IEs) with the ability to bind to chondroitin sulfate A (CSA). VAR2CSA is a leading candidate for a pregnancy malaria vaccine, but its large size ( approximately 350 kDa) and extensive polymorphism may pose a challenge to vaccine development. In this study, rabbits were immunized with individual VAR2CSA Duffy binding-like (DBL) domains expressed in Pichia pastoris or var2csa plasmid DNA and sera were screened on different CSA-binding parasite lines. Rabbit antibodies to three recombinant proteins (DBL1, DBL3, and DBL6) and four plasmid DNAs (DBL1, DBL3, DBL5, and DBL6) reacted with homologous FCR3-CSA IEs. By comparison, antibodies to the DBL4 domain were unable to react with native VAR2CSA protein unless it was first partially proteolyzed with trypsin or chymotrypsin. To investigate the antigenic relationship of geographically diverse CSA-binding isolates, rabbit immune sera were screened on four heterologous CSA-binding lines from different continental origins. Antibodies did not target conserved epitopes exposed in all VAR2CSA alleles; however, antisera to several DBL domains cross-reacted on parasite isolates that had polymorphic loops in common with the homologous immunogen. This study demonstrates that VAR2CSA contains common polymorphic epitopes that are shared between geographically diverse CSA-binding lines.

Mutations in envelope gp120 can impact proteolytic processing of the gp160 precursor and thereby affect neutralization sensitivity of human immunodeficiency virus type 1 pseudoviruses.

The design of an efficient human immunodeficiency virus (HIV) immunogen able to generate broad neutralizing antibodies (NAbs) remains an elusive goal. As more data emerge, it is becoming apparent that one important aspect of such an immunogen will be the proper representation of the envelope protein (Env) as it exists on native virions. Important questions that are yet to be fully addressed include what factors dictate Env processing, how different Env forms are represented on the virion, and ultimately how these issues influence the development and efficacy of NAbs. Recent data have begun to illuminate the extent to which changes in gp41 can impact the overall structure and neutralizing sensitivity of Env. Here, we present evidence to suggest that minor mutations in gp120 can significantly impact Env processing. We analyzed the gp120 sequences of 20 env variants that evolved in multiple macaques over 8 months of infection with simian/human immunodeficiency virus 89.6P. Variant gp120 sequences were subcloned into gp160 expression plasmids with identical cleavage motifs and gp41 sequences. Cells cotransfected with these plasmids and delta env genomes were able to produce competent virus. The resulting pseudoviruses incorporated high levels of Env onto virions that exhibited a range of degrees of virion-associated Env cleavage (15 to 40%). Higher levels of cleavage correlated with increased infectivity and increased resistance to macaque plasma, HIV immunoglobulin, soluble CD4, and human monoclonal antibodies 4E10, 2F5, and b12. Based on these data, we discuss a model whereby changes in gp120 of 89.6P impact Env processing and thereby mediate escape from a range of neutralizing agents.

Evidence for persistent, occult infection in neonatal macaques following perinatal transmission of simian-human immunodeficiency virus SF162P3.

To model human immunodeficiency virus (HIV) perinatal transmission, we studied infection of simian-human immunodeficiency virus (SHIV) SF162P3 in 10 pregnant Macaca nemestrina females and their offspring. Four of nine infants born to and suckled by these dams had evidence of infection, a transmission rate of 44.4% (95% confidence interval, 13.7% to 78.8%). We quantified transplacentally acquired and de novo Env-specific immunoglobulin G (IgG), IgM, and neutralizing antibodies in newborns. Transmission of escape variants was confirmed. In utero infection (n = 1) resulted in high viremia, depletion of peripheral CD4+ T cells, and rapid evolution of env in blood and tissues. Peripartum or postpartum SHIV infection (n = 3) resulted in postacute viral control that was undetectable by very sensitive multiplex PCR, despite increasing antibodies. Seropositive infants with highly controlled viremia had homogeneous peripheral blood env sequences, and their tissues had <3 copies per million cells. A high incidence of seropositive virus-low or -negative SHIV infection in infant macaques has implications for HIV type 1 perinatal transmission and detection.