Pharmacodynamic and Antitumor Activity of BI 836880, a Dual Vascular Endothelial Growth Factor and Angiopoietin 2 Inhibitor, Alone and Combined with Programmed Cell Death Protein-1 Inhibition.

Vascular endothelial growth factor (VEGF) and angiopoietin (ANG)-2 have complementary roles in angiogenesis and promote an immunosuppressive tumor microenvironment. It is anticipated that the combination of VEGF and ANG2 blockade could provide superior activity to the blockade of either pathway alone and that the addition of VEGF/ANG2 inhibition to an anti-programmed cell death protein-1 (PD-1) antibody could change the tumor microenvironment to support T-cell-mediated tumor cytotoxicity. Here, we describe the pharmacologic and antitumor activity of BI 836880, a humanized bispecific nanobody comprising two single-variable domains blocking VEGF and ANG2, and an additional module for half-life extension in vivo. BI 836880 demonstrated high affinity and selectivity for human VEGF-A and ANG2, resulting in inhibition of the downstream signaling of VEGF/ANG2 and a decrease in endothelial cell proliferation and survival. In vivo, BI 836880 exhibited significant antitumor activity in all patient-derived xenograft models tested, showing significantly greater tumor growth inhibition (TGI) than bevacizumab (VEGF inhibition) and AMG386 (ANG1/2 inhibition) in a range of models. In a Lewis lung carcinoma syngeneic tumor model, the combination of PD-1 inhibition with VEGF inhibition showed superior efficacy versus the blockade of either pathway alone. TGI was further increased with the addition of ANG2 inhibition to VEGF/PD-1 blockade. VEGF/ANG2 inhibition had a strong antiangiogenic effect. Our data suggest that the blockade of VEGF and ANG2 with BI 836880 may offer improved antitumor activity versus the blockade of either pathway alone and that combining VEGF/ANG2 inhibition with PD-1 blockade can further enhance antitumor effects. SIGNIFICANCE STATEMENT: Vascular endothelial growth factor (VEGF) and angiopoietin (ANG)-2 play key roles in angiogenesis and have an immunosuppressive effect in the tumor microenvironment. This study shows that BI 836880, a bispecific nanobody targeting VEGF and ANG2, demonstrates substantial antitumor activity in preclinical models. Combining VEGF/ANG2 inhibition with the blockade of the PD-1 pathway can further improve antitumor activity.

High Throughput Combinatorial Formatting of PcrV Nanobodies for Efficient Potency Improvement.

Improving potencies through concomitant blockage of multiple epitopes and avid binding by fusing multiple (different) monovalent Nanobody building blocks via linker sequences into one multivalent polypeptide chain is an elegant alternative to affinity maturation. We explored a large and random formatting library of bivalent (combinations of two identical) and biparatopic (combinations of two different) Nanobodies for functional blockade of Pseudomonas aeruginosa PcrV. PcrV is an essential part of the P. aeruginosa type III secretion system (T3SS), and its oligomeric nature allows for multiple complex binding and blocking options. The library screening yielded a large number of promising biparatopic lead candidates, revealing significant (and non-trivial) preferences in terms of Nanobody building block and epitope bin combinations and orientations. Excellent potencies were confirmed upon further characterization in two different P. aeruginosa T3SS-mediated cytotoxicity assays. Three biparatopic Nanobodies were evaluated in a lethal mouse P. aeruginosa challenge pneumonia model, conferring 100% survival upon prophylactic administration and reducing lung P. aeruginosa burden by up to 2 logs. At very low doses, they protected the mice from P. aeruginosa infection-related changes in lung histology, myeloperoxidase production, and lung weight. Importantly, the most potent Nanobody still conferred protection after therapeutic administration up to 24 h post-infection. The concept of screening such formatting libraries for potency improvement is applicable to other targets and biological therapeutic platforms.

Trivalency of a Nanobody Specific for the Human Respiratory Syncytial Virus Fusion Glycoprotein Drastically Enhances Virus Neutralization and Impacts Escape Mutant Selection.

ALX-0171 is a trivalent Nanobody derived from monovalent Nb017 that binds to antigenic site II of the human respiratory syncytial virus (hRSV) fusion (F) glycoprotein. ALX-0171 is about 6,000 to 10,000 times more potent than Nb017 in neutralization tests with strains of hRSV antigenic groups A and B. To explore the effect of this enhanced neutralization on escape mutant selection, viruses resistant to either ALX-0171 or Nb017 were isolated after serial passage of the hRSV Long strain in the presence of suboptimal concentrations of the respective Nanobodies. Resistant viruses emerged notably faster with Nb017 than with ALX-0171 and in both cases contained amino acid changes in antigenic site II of hRSV F. Detailed binding and neutralization analyses of these escape mutants as well as previously described mutants resistant to certain monoclonal antibodies (MAbs) offered a comprehensive description of site II mutations which are relevant for neutralization by MAbs and Nanobodies. Notably, ALX-0171 showed a sizeable neutralization potency with most escape mutants, even with some of those selected with the Nanobody, and these findings make ALX-0171 an attractive antiviral for treatment of hRSV infections.

Generation and Characterization of ALX-0171, a Potent Novel Therapeutic Nanobody for the Treatment of Respiratory Syncytial Virus Infection.

Respiratory syncytial virus (RSV) is an important causative agent of lower respiratory tract infections in infants and elderly individuals. Its fusion (F) protein is critical for virus infection. It is targeted by several investigational antivirals and by palivizumab, a humanized monoclonal antibody used prophylactically in infants considered at high risk of severe RSV disease. ALX-0171 is a trimeric Nanobody that binds the antigenic site II of RSV F protein with subnanomolar affinity. ALX-0171 demonstrated in vitro neutralization superior to that of palivizumab against prototypic RSV subtype A and B strains. Moreover, ALX-0171 completely blocked replication to below the limit of detection for 87% of the viruses tested, whereas palivizumab did so for 18% of the viruses tested at a fixed concentration. Importantly, ALX-0171 was highly effective in reducing both nasal and lung RSV titers when delivered prophylactically or therapeutically directly to the lungs of cotton rats. ALX-0171 represents a potent novel antiviral compound with significant potential to treat RSV-mediated disease.

High efficacy of huCD20-targeted AcTaferon in humanized patient derived xenograft models of aggressive B cell lymphoma.

Type I interferon (IFN) is a potent antitumoral drug, with an important history in the treatment of hematologic malignancies. However, its pleiotropic nature leads to severe dose-limiting toxicities that blunt its therapeutic potential. To achieve selective targeting of specific immune or tumor cells, AcTakines (Activity-on-Target Cytokines), i.e., immunocytokines utilizing attenuated cytokines, and clinically optimized A-Kines™ were developed. In syngeneic murine models, the CD20-targeted murine IFNα2-based AcTaferons (AFNs) have demonstrated clear antitumoral effects, with excellent tolerability. The current study explores the antitumoral potential of the humanized huCD20-Fc-AFN in 5 different humanized patient derived xenograft (PDX) models of huCD20+ aggressive B non-Hodgkin lymphomas (B-NHLs). The huCD20-Fc-AFN consists of a huCD20-specific single-domain antibody (VHH) linked through a heterodimeric 'knob-in-hole' human IgG1 Fc molecule to an attenuated huIFNα2 sequence. An in vitro targeting efficacy of up to 1.000-fold could be obtained, without detectable in vivo toxicities, except for selective (on-target) and reversible B cell depletion. Treatment with huCD20-Fc-AFN significantly increased the median overall survival (mOS) in both non-humanized (mOS 31 to 45 days; HR = 0.26; p = 0.001), and humanized NSG/NOG mice (mOS 34 to 80 days; HR = 0.37; p < 0.0001). In humanized mice, there was a trend for increased survival when compared to equimolar rituximab (mOS 49 to 80 days; HR = 0.73; p = 0.09). The antitumoral effects of huCD20-Fc-AFN were partly due to direct effects of type I IFN on the tumor cells, but additional effects via the human immune system are essential to obtain long-term remissions. To conclude, huCD20-Fc-AFN could provide a novel therapeutic strategy for huCD20-expressing aggressive B-NHLs.

Knocking Out CD70 Rescues CD70-Specific NanoCAR T Cells from Antigen-Induced Exhaustion.

CD70 is an attractive target for chimeric antigen receptor (CAR) T-cell therapy for the treatment of both solid and liquid malignancies. However, the functionality of CD70-specific CAR T cells is modest. We optimized a CD70-specific VHH-based CAR (nanoCAR). We evaluated the nanoCARs in clinically relevant models in vitro, using co-cultures of CD70-specific nanoCAR T cells with malignant rhabdoid tumor organoids, and in vivo, using a diffuse large B-cell lymphoma patient-derived xenograft (PDX) model. Although the nanoCAR T cells were highly efficient in organoid co-cultures, they showed only modest efficacy in the PDX model. We determined that fratricide was not causing this loss in efficacy but rather CD70 interaction in cis with the nanoCAR-induced exhaustion. Knocking out CD70 in nanoCAR T cells using CRISPR/Cas9 resulted in dramatically enhanced functionality in the diffuse large B-cell lymphoma PDX model. Through single-cell transcriptomics, we obtained evidence that CD70 knockout CD70-specific nanoCAR T cells were protected from antigen-induced exhaustion. In addition, we demonstrated that wild-type CD70-specific nanoCAR T cells already exhibited signs of exhaustion shortly after production. Their gene signature strongly overlapped with gene signatures of exhausted CAR T cells. Conversely, the gene signature of knockout CD70-specific nanoCAR T cells overlapped with the gene signature of CAR T-cell infusion products leading to complete responses in chronic lymphatic leukemia patients. Our data show that CARs targeting endogenous T-cell antigens negatively affect CAR T-cell functionality by inducing an exhausted state, which can be overcome by knocking out the specific target.

Clearance of genotype 1b hepatitis C virus in chimpanzees in the presence of vaccine-induced E1-neutralizing antibodies.

Accumulating evidence indicates that neutralizing antibodies play an important role in protection from chronic hepatitis C virus (HCV) infection. Efforts to elicit such responses by immunization with intact heterodimeric E1E2 envelope proteins have met with limited success. To determine whether antigenic sites, which are not exposed by the combined E1E2 heterodimer structure, are capable of eliciting neutralizing antibody responses, we expressed and purified each as separate recombinant proteins E1 and E2, from which the immunodominant hypervariable region (HVR-1) was deleted. Immunization of chimpanzees with either E1 or E2 alone induced antigen-specific T-helper cytokines of similar magnitude. Unexpectedly, the capacity to neutralize HCV was observed in E1 but not in animals immunized with E2 devoid of HVR-1. Furthermore, in vivo only E1-vaccinated animals exposed to the heterologous HCV-1b inoculum cleared HCV infection.

Neutralizing host responses in hepatitis C virus infection target viral entry at postbinding steps and membrane fusion.

BACKGROUND & AIMS: Hepatitis C virus (HCV) is a leading cause of chronic hepatitis worldwide. Viral attachment and entry, representing the first steps of virus-host cell interactions, are major targets of adaptive host cell defenses. The mechanisms of antibody-mediated neutralization by host neutralizing responses in HCV infection are only poorly understood. Retroviral HCV pseudotypes (HCVpp) and recombinant cell culture-derived HCV (HCVcc) have been successfully used to study viral entry and antibody-mediated neutralization. METHODS: In this study, we used these model systems to investigate the mechanism of antibody-mediated neutralization by monoclonal antienvelope antibodies and polyclonal anti-HCV immunoglobulins purified from HCV-infected patients. RESULTS: Using a panel of monoclonal antienvelope antibodies, we identified an epitope within the E1 glycoprotein targeted by human neutralizing antibodies during postbinding events. Interestingly, we observed that host neutralizing responses in the majority of HCV-infected individuals include antibodies targeting HCV entry after binding of the virus to the target cell membrane. Using a kinetic assay based on HCVpp and HCVcc entry, we demonstrate that purified antiviral immunoglobulins derived from individual HCV-infected patients appear to inhibit HCV infection at an entry step closely linked to CD81 and scavenger receptor BI (SR-BI). CONCLUSIONS: Our results indicate that host neutralizing responses in HCV-infected patients target viral entry after HCV binding most likely related to HCV-CD81, and HCV-SR-BI interactions, as well as membrane fusion. These findings have implications not only for the understanding of the pathogenesis of HCV infection but also for the design of novel immunotherapeutic and preventive strategies.

Isolation and characterization of broadly neutralizing human monoclonal antibodies to the e1 glycoprotein of hepatitis C virus.

The relative importance of humoral and cellular immunity in the prevention or clearance of hepatitis C virus (HCV) infection is poorly understood. However, there is considerable evidence that neutralizing antibodies are involved in disease control. Here we describe the detailed analysis of human monoclonal antibodies (MAbs) directed against HCV glycoprotein E1, which may have the potential to control HCV infection. We have identified two MAbs that can strongly neutralize HCV-pseudotyped particles (HCVpp) bearing the envelope glycoproteins of genotypes 1a, 1b, 4a, 5a, and 6a and less strongly neutralize HCVpp bearing the envelope glycoproteins of genotype 2a. Genotype 3a was not neutralized. The epitopes for both MAbs were mapped to the region encompassing amino acids 313 to 327. In addition, robust neutralization was also observed against cell culture-adapted viruses of genotypes 1a and 2a. Results from this study suggest that these MAbs may have the potential to prevent HCV infection.

Rational design of a multiepitope vaccine encoding T-lymphocyte epitopes for treatment of chronic hepatitis B virus infections.

Protein sequences from multiple hepatitis B virus (HBV) isolates were analyzed for the presence of amino acid motifs characteristic of cytotoxic T-lymphocyte (CTL) and helper T-lymphocyte (HTL) epitopes with the goal of identifying conserved epitopes suitable for use in a therapeutic vaccine. Specifically, sequences bearing HLA-A1, -A2, -A3, -A24, -B7, and -DR supertype binding motifs were identified, synthesized as peptides, and tested for binding to soluble HLA. The immunogenicity of peptides that bound with moderate to high affinity subsequently was assessed using HLA transgenic mice (CTL) and HLA cross-reacting H-2(bxd) (BALB/c x C57BL/6J) mice (HTL). Through this process, 30 CTL and 16 HTL epitopes were selected as a set that would be the most useful for vaccine design, based on epitope conservation among HBV sequences and HLA-based predicted population coverage in diverse ethnic groups. A plasmid DNA-based vaccine encoding the epitopes as a single gene product, with each epitope separated by spacer residues to enhance appropriate epitope processing, was designed. Immunogenicity testing in mice demonstrated the induction of multiple CTL and HTL responses. Furthermore, as a complementary approach, mass spectrometry allowed the identification of correctly processed and major histocompatibility complex-presented epitopes from human cells transfected with the DNA plasmid. A heterologous prime-boost immunization with the plasmid DNA and a recombinant MVA gave further enhancement of the immune responses. Thus, a multiepitope therapeutic vaccine candidate capable of stimulating those cellular immune responses thought to be essential for controlling and clearing HBV infection was successfully designed and evaluated in vitro and in HLA transgenic mice.