Earl Albone

Targeting endosialin/CD248 through antibody-mediated internalization results in impaired pericyte maturation and dysfunctional tumor microvasculature

Over-expression of endosialin/CD248 (herein referred to as CD248) has been associated with increased tumor microvasculature in various tissue origins which makes it an attractive anti-angiogenic target. In an effort to target CD248, we have generated a human CD248 knock-in mouse line and MORAb-004, the humanized version of the mouse anti-human CD248 antibody Fb5. Here, we report that MORAb-004 treatment significantly impacted syngeneic tumor growth and tumor metastasis in the human CD248 knock-in mice. In comparison with untreated tumors, MORAb-004 treated tumors displayed overall shortened and distorted blood vessels. Immunofluorescent staining of tumor sections revealed drastically more small and dysfunctional vessels in the treated tumors. The CD248 levels on cell surfaces of neovasculature pericytes were significantly reduced due to its internalization. This reduction of CD248 was also accompanied by reduced α-SMA expression, depolarization of pericytes and endothelium, and ultimately dysfunctional microvessels. These results suggest that MORAb-004 reduced CD248 on pericytes, impaired tumor microvasculature maturation and ultimately suppressed tumor development.

Tumor antigen CA125 suppresses antibody-dependent cellular cytotoxicity (ADCC) via direct antibody binding and suppressed Fc-γ receptor engagement

Cancers employ a number of mechanisms to evade host immune responses. Here we report the effects of tumor-shed antigen CA125/MUC16 on suppressing IgG1-mediated antibody-dependent cellular cytotoxicity (ADCC). This evidence stems from prespecified subgroup analysis of a Phase 3 clinical trial testing farletuzumab, a monoclonal antibody to folate receptor alpha, plus standard-of-care carboplatin-taxane chemotherapy in patients with recurrent platinum-sensitive ovarian cancer. Patients with low serum CA125 levels treated with farletuzumab demonstrated improvements in progression free survival (HR 0.49, p = 0.0028) and overall survival (HR 0.44, p = 0.0108) as compared to placebo. Farletuzumab’s pharmacologic activity is mediated in part through ADCC. Here we show that CA125 inhibits ADCC by directly binding to farletuzumab that in turn perturbs Fc-γ receptor engagement on effector cells.Cancers employ a number of mechanisms to evade host immune responses. Here we report the effects of tumor-shed antigen CA125/MUC16 on suppressing IgG1-mediated antibody-dependent cellular cytotoxicity (ADCC). This evidence stems from prespecified subgroup analysis of a Phase 3 clinical trial testing farletuzumab, a monoclonal antibody to folate receptor alpha, plus standard-of-care carboplatin-taxane chemotherapy in patients with recurrent platinum-sensitive ovarian cancer. Patients with low serum CA125 levels treated with farletuzumab demonstrated improvements in progression free survival (HR 0.49, p = 0.0028) and overall survival (HR 0.44, p = 0.0108) as compared to placebo. Farletuzumabs pharmacologic activity is mediated in part through ADCC. Here we show that CA125 inhibits ADCC by directly binding to farletuzumab that in turn perturbs Fc-γ receptor engagement on effector cells.

Correlation of FCGRT genomic structure with serum immunoglobulin, albumin and farletuzumab pharmacokinetics in patients with first relapsed ovarian cancer

Farletuzumab (FAR) is a humanized monoclonal antibody (mAb) that binds to folate receptor alpha. A Ph3 trial in ovarian cancer patients treated with carboplatin/taxane plus FAR or placebo did not meet the primary statistical endpoint. Subgroup analysis demonstrated that subjects with high FAR exposure levels (Cmin>57.6μg/mL) showed statistically significant improvements in PFS and OS. The neonatal Fc receptor (fcgrt) plays a central role in albumin/IgG stasis and mAb pharmacokinetics (PK). Here we evaluated fcgrt sequence and association of its promoter variable number tandem repeats (VNTR) and coding single nucleotide variants (SNV) with albumin/IgG levels and FAR PK in the Ph3 patients. A statistical correlation existed between high FAR Cmin and AUC in patients with the highest quartile of albumin and lowest quartile of IgG1. Analysis of fcgrt identified 5 different VNTRs in the promoter region and 9 SNVs within the coding region, 4 which are novel.

Novel antibody probes for the characterization of endosialin/TEM-1

Endosialin (Tumor Endothelial Marker-1 (TEM-1), CD248) is primarily expressed on pericytes of tumor-associated microvasculature, tumor-associated stromal cells and directly on tumors of mesenchymal origin, including sarcoma and melanoma. While the function of endosialin/TEM-1 is incompletely understood, studies have suggested a role in supporting tumor growth and invasion thus making it an attractive therapeutic target. In an effort to further understand its role in cancer, we previously developed a humanized anti-endosialin/TEM-1 monoclonal antibody (mAb), called ontuxizumab (MORAb-004) for testing in preclinical and clinical studies. We herein report on the generation of an extensive panel of recombinant endosialin/TEM-1 protein extracellular domain (ECD) fragments and novel mAbs against ECD motifs. The domain-specific epitopes were mapped against ECD sub-domains to identify those that can detect distinct structural motifs and can be potentially formatted as probes suitable for diagnostic and functional studies. A number of mAbS were shown to cross-react with the murine and human protein, potentially allowing their use in human animal models and corresponding clinical trials. In addition, pairing of several mAbs supported their use in immunoassays that can detect soluble endosialin/TEM-1 (sEND) in the serum of healthy subjects and cancer patients.

Engineering humanized antibody framework sequences for optimal site-specific conjugation of cytotoxins

ABSTRACT The prevailing techniques used to generate antibody-drug conjugates (ADCs) involve random conjugation of the linker-drug to multiple lysines or cysteines in the antibody. Engineering natural and non-natural amino acids into an antibody has been demonstrated to be an effective strategy to produce homogeneous ADC products with defined drug-to-antibody ratios. We recently reported an efficient residue-specific conjugation technology (RESPECT) where thiol-reactive payloads can be efficiently conjugated to a native unpaired cysteine in position 80 (C80) of rabbit light chains. Deimmunizing the rabbit variable domains through humanization is necessary to reduce the risk of anti-drug antibody responses in patients. However, we found that first-generation humanized RESPECT ADCs showed high levels of aggregation and low conjugation efficiency. We correlated these negative properties to the phenylalanine at position 83 present in most human variable kappa frameworks. When position 83 was substituted with selected amino acids, conjugation was restored and aggregation was reduced to levels similar to the chimeric ADC. This engineering strategy allows for development of second-generation humanized RESPECT ADCs with desirable biopharmaceutical properties.

Generation of therapeutic immunoconjugates via Residue-Specific Conjugation Technology (RESPECT) utilizing a native cysteine in the light chain framework of Oryctolagus cuniculus

ABSTRACT The conjugation of toxins, dyes, peptides, or proteins to monoclonal antibodies is often performed via free thiol groups generated by either partial reduction methods or engineering free cysteine residues into the antibody sequence. Antibodies from the rabbit Oryctolagus cuniculus have an additional intrachain disulfide bond, whereby the light chain variable kappa domain is bridged to the constant kappa region between cysteine residues at positions 80 and 171, respectively. Chimerization of rabbit antibodies with human constant domains allows for the generation of a free thiol group at the light chain position 80 (C80) that can be used for site-specific conjugation. An efficient process for the purification and simultaneous removal of cysteinylation at the C80 site was developed. The unpaired C80 was shown to be efficiently conjugated using several different maleimido-based ligands. REsidue SPEcific Conjugation Technology (RESPECT) antibody-drug conjugates prepared using rabbit-human chimeric anti-human mesothelin rabbit antibodies and maleimido-PEG2-auristatin conjugated to C80 were shown to be highly potent and specific in vitro and effective in vivo in reduction of tumor growth in a highly aggressive mesothelin-expressing xenograft tumor model.

Abstract 644: CA125/MUC16 suppresses antibody-dependent cellular cytotoxicity of IgG1-based therapies via perturbation of antibody Fc-Receptor engagement on immune effector cells

Background: Human cancers employ a number of mechanisms to evade host immune responses. Here we report the effects of the tumor-shed antigen CA125 (MUC16) on suppressing IgG1-mediated antibody-dependent cellular cytotoxicity (ADCC). This evidence stems from prespecified subgroup analysis of an 1100 patient Phase 3 clinical trial testing the investigational agent farletuzumab, a monoclonal antibody (mAb) to folate receptor alpha, plus standard-of-care in patients with recurrent ovarian cancer. In this study, patients with low levels of CA125 [no greater than 3X the upper limit of normal ( Methods: Functional assays employing Jurkat-hCD16 reporter cells as well as primary NK cells were used to measure ADCC activity. CA125 isolates from various patients and cell lines were used in ADCC assays to monitor CA125 effects on immune-effector function. Multiple isogenic tumor cells expressing endogenous CA125 and their CA125-null counterparts were also used to monitor CD16a-mediated ADCC activity. CD16a+ cell binding assays were used to measure the effects of CA125 inhibition on CD16a Fc-Receptor interaction and activation. Recombinant CD16a and humanized mAbs were used in ELISA format to measure molecular binding. Results: Functional assays showed that exogenously added tumor-shed CA125 was able to suppress ADCC mediated by IgG1-type mAbs against several target cell lines. This CA125-mediated ADCC suppression was also observed in cells naturally expressing membrane-bound CA125 but not in isogenic CA125-null cells. Molecular studies suggest that CA125-mediated immunosuppression is caused by the perturbation of antibody interaction with the CD16a Fc-Receptor and that this effect is specific to IgG-type mAbs. Conclusions: Here we demonstrate that the tumor-shed antigen CA125 can elicit immunosuppression by blocking the interaction of tumor-targeting mAbs and CD16a Fc-Receptor on immune effector cells. This effect appears to be through a direct interaction of CA125 with antibody-CD16a proteins. These findings support a biological mechanism for the clinical observation that relapsed ovarian cancer patients with low CA125 blood levels have improved clinical responses to the experimental agent farletuzumab. These findings also provide new insights on the potential biological mechanisms for which tumors produce tumor-shed antigens such as CA125 to suppress host anti-tumor immune responses. Citation Format: J. Bradford Kline, Rina Kennedy, Shawn Fernando, Jennifer McDonough, Earl Albone, Elizabeth B. Somers, Wenquan Wang, Charles Schweizer, Luigi Grasso, Nicholas C. Nicolaides. CA125/MUC16 suppresses antibody-dependent cellular cytotoxicity of IgG1-based therapies via perturbation of antibody Fc-Receptor engagement on immune effector cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 644. doi:10.1158/1538-7445.AM2017-644