Generation and in vivo validation of an IL-12 fusion protein based on a novel anti-human FAP monoclonal antibody

BACKGROUND In this study, we describe the generation of a fully human monoclonal antibody (named '7NP2') targeting human fibroblast activation protein (FAP), an antigen expressed in the microenvironment of different types of solid neoplasms. METHODS 7NP2 was isolated from a synthetic antibody phage display library and was improved by one round of mutagenesis-based affinity maturation. The tumor recognition properties of the antibody were validated by immunofluorescence procedures performed on cancer biopsies from human patients. A fusion protein consisting of the 7NP2 antibody linked to interleukin (IL)-12 was generated and the anticancer activity of the murine surrogate product (named mIL12-7NP2) was evaluated in mouse models. Furthermore, the safety of the fully human product (named IL12-7NP2) was evaluated in Cynomolgus monkeys. RESULTS Biodistribution analysis in tumor-bearing mice confirmed the ability of the product to selectively localize to solid tumors while sparing healthy organs. Encouraged by these results, therapy studies were conducted in vivo, showing a potent antitumor activity in immunocompetent and immunodeficient mouse models of cancer, both as single agent and in combination with immune checkpoint inhibitors. The fully human product was tolerated when administered to non-human primates. CONCLUSIONS The results obtained in this work provided a rationale for future clinical translation activities using IL12-7NP2

Novel human monoclonal antibodies specific to the alternatively spliced domain D of Tenascin C efficiently target tumors in vivo

Antibody-based delivery of bioactive molecules represents a promising strategy for the improvement of cancer immunotherapy. Here, we describe the generation and characterization of R6N, a novel fully human antibody specific to the alternatively spliced domain D of Tenascin C, which is highly expressed in the stroma of primary tumors and metastasis. The R6N antibody recognized its cognate tumor-associated antigen with identical specificity in mouse and human specimens. Moreover, the antibody was able to selectively localize to solid tumors in vivo as evidenced by immunofluorescence-based biodistribution analysis. Encouraged by these results, we developed a novel fusion protein (termed mIL12-R6N) consisting of the murine interleukin 12 fused to the R6N antibody in homodimeric tandem single-chain variable fragment arrangement. mIL12-R6N exhibited potent antitumor activity in immunodeficient mice bearing SKRC52 renal cell carcinoma, as well as in immunocompetent mice bearing SMA-497 glioma. The experiments presented in this work provide a rationale for possible future applications for the R6N antibody for the treatment of cancer patients

A novel format for recombinant antibody-interleukin-2 fusion proteins exhibits superior tumor-targeting properties in vivo

The targeted delivery of interleukin-2 to the tumor is gaining attention as an avenue to potentiate the action of T and NK cells at the site of disease. We have previously described the fusion of the L19 antibody, specific to the EDB domain of fibronectin, with human interleukin-2, using a non-covalent homodimeric diabody format. Here, we describe four novel formats for the L19-IL2 fusion, featuring different arrangements of antibody and IL2. A comparative quantitative biodistribution analysis in tumor-bearing mice using radioiodinated proteins revealed that the novel format (L19L19-IL2, with the antibody in single-chain diabody format) exhibited the best biodistribution results. In vitro assays on peripheral blood mononuclear cells showed a decrease activation of regulatory T cells when single IL2 domain was used. In vivo, both L19-IL2 and L19L19-IL2 inhibited tumor growth in immunocompetent mouse models of cancer. T-cell analysis revealed similar levels of CD4+ and FoxP3+ cells, with an expansion of the CD8+ T cell in mice treated with L19-IL2 and L19L19-IL2. The percentage of CD4+ regulatory T cells was markedly decreased with L19L19-IL2 combined with a mouse-specific PD-1 blocker. Collectively, these data indicate that the new L19L19-IL2 format exhibits favorable tumor-homing properties and mediates a potent anti-cancer activity in vivo.ISSN:1949-255

Novel human monoclonal antibodies specific to the alternatively spliced domain D of Tenascin C efficiently target tumors in vivo

Antibody-based delivery of bioactive molecules represents a promising strategy for the improvement of cancer immunotherapy. Here, we describe the generation and characterization of R6N, a novel fully human antibody specific to the alternatively spliced domain D of Tenascin C, which is highly expressed in the stroma of primary tumors and metastasis. The R6N antibody recognized its cognate tumor-associated antigen with identical specificity in mouse and human specimens. Moreover, the antibody was able to selectively localize to solid tumors in vivo as evidenced by immunofluorescence-based biodistribution analysis. Encouraged by these results, we developed a novel fusion protein (termed mIL12-R6N) consisting of the murine interleukin 12 fused to the R6N antibody in homodimeric tandem single-chain variable fragment arrangement. mIL12-R6N exhibited potent antitumor activity in immunodeficient mice bearing SKRC52 renal cell carcinoma, as well as in immunocompetent mice bearing SMA-497 glioma. The experiments presented in this work provide a rationale for possible future applications for the R6N antibody for the treatment of cancer patients. (© ©2020 Philochem AG. Published with license by Taylor & Francis Group, LLC.)ISSN:1942-0862ISSN:1942-087

A Novel Antibody–IL15 Fusion Protein Selectively Localizes to Tumors, Synergizes with TNF-based Immunocytokine, and Inhibits Metastasis

IL15 is an immunostimulatory cytokine that holds promises for cancer therapy, but its performance (alone or as partner for fusion proteins) has often been limited by suboptimal accumulation in the tumor and very rapid clearance from circulation. Most recently, the Sushi Domain (SD, the shortest region of IL15 receptor a, capable of binding to IL15) has been fused to IL15-based anticancer products to increase its biological activity. Here, we describe two novel antibody fusion proteins (termed F8-F8-IL15 and F8-F8-SD-IL15), specific to the alternatively spliced EDA domain of fibronectin (a marker of tumor neoangiogenisis, expressed in the majority of solid and hematologic tumors, but absent in normal healthy tissues) and featuring the F8 antibody in single-chain diabody format (with a short linker between VH and VL, thus allowing the domains to pair with the complementary ones of another chain). Unlike previously described fusions of the F8 antibody with human IL15, F8-F8-IL15 and F8-F8-SD-IL15 exhibited a preferential uptake in solid tumors, as evidenced by quantitative biodistribution analysis with radioiodinated protein preparations. Both products were potently active in vivo against mouse metastatic colon carcinomas and in sarcoma lesion in combination with targeted TNF. The results may be of clinical significance, as F8-F8-IL15 and F8-F8-SD-IL15 are fully human proteins, which recognize the cognate tumor-associated antigen with identical affinity in mouse and man.ISSN:1535-7163ISSN:1538-851

Immunogenicity of <i>Escherichia coli</i> Outer Membrane Vesicles: Elucidation of Humoral Responses against OMV-Associated Antigens

Outer membrane vesicles (OMVs) produced by Gram-negative bacteria have emerged as a novel and flexible vaccine platform. OMVs can be decorated with foreign antigens and carry potent immunostimulatory components. Therefore, after their purification from the culture supernatant, they are ready to be formulated for vaccine use. It has been extensively demonstrated that immunization with engineered OMVs can elicit excellent antibody responses against the heterologous antigens. However, the definition of the conditions necessary to reach the optimal antibody titers still needs to be investigated. Here, we defined the protein concentrations required to induce antigen-specific antibodies, and the amount of antigen and OMVs necessary and sufficient to elicit saturating levels of antigen-specific antibodies. Since not all antigens can be expressed in OMVs, we also investigated the effectiveness of vaccines in which OMVs and purified antigens are mixed together without using any procedure for their physical association. Our data show that in most of the cases OMV–antigen mixtures are very effective in eliciting antigen-specific antibodies. This is probably due to the capacity of OMVs to “absorb” antigens, establishing sufficiently stable interactions that allow antigen–OMV co-presentation to the same antigen presenting cell. In those cases when antigen–OMV interaction is not sufficiently stable, the addition of alum to the formulation guarantees the elicitation of high titers of antigen-specific antibodies

Adapting Neutralizing Antibodies to Viral Variants by Structure-Guided Affinity Maturation Using Phage Display Technology

Neutralizing monoclonal antibodies have achieved great efficacy and safety for the treatment of numerous infectious diseases. However, their neutralization potency is often rapidly lost when the target antigen mutates. Instead of isolating new antibodies each time a pathogen variant arises, it can be attractive to adapt existing antibodies, making them active against the new variant. Potential benefits of this approach include reduced development time, cost, and regulatory burden. Here a methodology is described to rapidly evolve neutralizing antibodies of proven activity, improving their function against new pathogen variants without losing efficacy against previous ones. The reported procedure is based on structure-guided affinity maturation using combinatorial mutagenesis and phage display technology. Its use against the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is demonstrated, but it is suitable for any other pathogen. As proof of concept, the method is applied to CoV-X2, a human bispecific antibody that binds with high affinity to the early SARS-CoV-2 variants but lost neutralization potency against Delta. Antibodies emerging from the affinity maturation selection exhibit significantly improved neutralization potency against Delta and no loss of efficacy against the other viral sequences tested. These results illustrate the potential application of structure-guided affinity maturation in facilitating the rapid adaptation of neutralizing antibodies to pathogen variants

Vaccination With a FAT1-Derived B Cell Epitope Combined With Tumor-Specific B and T Cell Epitopes Elicits Additive Protection in Cancer Mouse Models

<p>Human FAT1 is overexpressed on the surface of most colorectal cancers (CRCs) and in particular a 25 amino acid sequence (D8) present in one of the 34 cadherin extracellular repeats carries the epitope recognized by mAb198.3, a monoclonal antibody which partially protects mice from the challenge with human CRC cell lines in xenograft mouse models. Here we present data in immune competent mice demonstrating the potential of the D8-FAT1 epitope as CRC cancer vaccine. We first demonstrated that the mouse homolog of D8-FAT1 (mD8-FAT1) is also expressed on the surface of CT26 and B16F10 murine cell lines. We then engineered bacterial outer membranes vesicles (OMVs) with mD8-FAT1 and we showed that immunization of BALB/c and C57bl6 mice with engineered OMVs elicited anti-mD8-FAT1 antibodies and partially protected mice from the challenge against CT26 and EGFRvIII-B16F10 cell lines, respectively. We also show that when combined with OMVs decorated with the EGFRvIII B cell epitope or with OMVs carrying five tumor-specific CD4+ T cells neoepitopes, mD8-FAT1 OMVs conferred robust protection against tumor challenge in C57bl6 and BALB/c mice, respectively. Considering that FAT1 is overexpressed in both KRAS⁺ and KRAS⁻ CRCs, these data support the development of anti-CRC cancer vaccines in which the D8-FAT1 epitope is used in combination with other CRC-specific antigens, including mutation-derived neoepitopes.</p

Generation and in vivo validation of an IL-12 fusion protein based on a novel anti-human FAP monoclonal antibody

Background In this study, we describe the generation of a fully human monoclonal antibody (named '7NP2') targeting human fibroblast activation protein (FAP), an antigen expressed in the microenvironment of different types of solid neoplasms. Methods 7NP2 was isolated from a synthetic antibody phage display library and was improved by one round of mutagenesis-based affinity maturation. The tumor recognition properties of the antibody were validated by immunofluorescence procedures performed on cancer biopsies from human patients. A fusion protein consisting of the 7NP2 antibody linked to interleukin (IL)-12 was generated and the anticancer activity of the murine surrogate product (named mIL12-7NP2) was evaluated in mouse models. Furthermore, the safety of the fully human product (named IL12-7NP2) was evaluated in Cynomolgus monkeys. Results Biodistribution analysis in tumor-bearing mice confirmed the ability of the product to selectively localize to solid tumors while sparing healthy organs. Encouraged by these results, therapy studies were conducted in vivo, showing a potent antitumor activity in immunocompetent and immunodeficient mouse models of cancer, both as single agent and in combination with immune checkpoint inhibitors. The fully human product was tolerated when administered to non-human primates. Conclusions The results obtained in this work provided a rationale for future clinical translation activities using IL12-7NP2.ISSN:2051-142