Nonclinical Pharmacokinetics and Pharmacodynamics Characterization of Anti-CD79b/CD3 T Cell-Dependent Bispecific Antibody Using a Surrogate Molecule: A Potential Therapeutic Agent for B Cell Malignancies.

The T cell-dependent bispecific (TDB) antibody, anti-CD79b/CD3, targets CD79b and CD3 cell-surface receptors expressed on B cells and T cells, respectively. Since the anti-CD79b arm of this TDB binds only to human CD79b, a surrogate TDB that binds to cynomolgus monkey CD79b (cyCD79b) was used for preclinical characterization. To evaluate the impact of CD3 binding affinity on the TDB pharmacokinetics (PK), we utilized non-tumor-targeting bispecific anti-gD/CD3 antibodies composed of a low/high CD3 affinity arm along with a monospecific anti-gD arm as controls in monkeys and mice. An integrated PKPD model was developed to characterize PK and pharmacodynamics (PD). This study revealed the impact of CD3 binding affinity on anti-cyCD79b/CD3 PK. The surrogate anti-cyCD79b/CD3 TDB was highly effective in killing CD79b-expressing B cells and exhibited nonlinear PK in monkeys, consistent with target-mediated clearance. A dose-dependent decrease in B cell counts in peripheral blood was observed, as expected. Modeling indicated that anti-cyCD79b/CD3 TDB's rapid and target-mediated clearance may be attributed to faster internalization of CD79b, in addition to enhanced CD3 binding. The model yielded unbiased and precise curve fits. These findings highlight the complex interaction between TDBs and their targets and may be applicable to the development of other biotherapeutics.

Novel Anti-LY6G6D/CD3 T-Cell-Dependent Bispecific Antibody for the Treatment of Colorectal Cancer.

New therapeutics and combination regimens have led to marked clinical improvements for the treatment of a subset of colorectal cancer. Immune checkpoint inhibitors have shown clinical efficacy in patients with mismatch-repair-deficient or microsatellite instability-high (MSI-H) metastatic colorectal cancer (mCRC). However, patients with microsatellite-stable (MSS) or low levels of microsatellite instable (MSI-L) colorectal cancer have not benefited from these immune modulators, and the survival outcome remains poor for the majority of patients diagnosed with mCRC. In this article, we describe the discovery of a novel T-cell-dependent bispecific antibody (TDB) targeting tumor-associated antigen LY6G6D, LY6G6D-TDB, for the treatment of colorectal cancer. RNAseq analysis showed that LY6G6D was differentially expressed in colorectal cancer with high prevalence in MSS and MSI-L subsets, whereas LY6G6D expression in normal tissues was limited. IHC confirmed the elevated expression of LY6G6D in primary and metastatic colorectal tumors, whereas minimal or no expression was observed in most normal tissue samples. The optimized LY6G6D-TDB, which targets a membrane-proximal epitope of LY6G6D and binds to CD3 with high affinity, exhibits potent antitumor activity both in vitro and in vivo. In vitro functional assays show that LY6G6D-TDB-mediated T-cell activation and cytotoxicity are conditional and target dependent. In mouse xenograft tumor models, LY6G6D-TDB demonstrates antitumor efficacy as a single agent against established colorectal tumors, and enhanced efficacy can be achieved when LY6G6D-TDB is combined with PD-1 blockade. Our studies provide evidence for the therapeutic potential of LY6G6D-TDB as an effective treatment option for patients with colorectal cancer.

Discovery of Potent and Selective Antibody-Drug Conjugates with Eg5 Inhibitors through Linker and Payload Optimization.

Targeted antimitotic agents are a promising class of anticancer therapies. Herein, we describe the development of a potent and selective antimitotic Eg5 inhibitor based antibody-drug conjugate (ADC). Preliminary studies were performed using proprietary Eg5 inhibitors which were conjugated onto a HER2-targeting antibody using maleimido caproyl valine-citrulline para-amino benzocarbamate, or MC-VC-PABC cleavable linker. However, the resulting ADCs lacked antigen-specificity in vivo, probably from premature release of the payload. Second-generation ADCs were then developed, using noncleavable linkers, and the resulting conjugates (ADC-4 and ADC-10) led to in vivo efficacy in an HER-2 expressing (SK-OV-3ip) mouse xenograft model while ADC-11 led to in vivo efficacy in an anti-c-KIT (NCI-H526) mouse xenograft model in a target-dependent manner.

bisFabs: Tools for rapidly screening hybridoma IgGs for their activities as bispecific antibodies.

Bispecific antibodies are a growing class of therapeutic molecules. Many of the current bispecific formats require DNA engineering to convert the parental monoclonal antibodies into the final bispecific molecules. We describe here a method to generate bispecific molecules from hybridoma IgGs in 3-4 d using chemical conjugation of antigen-binding fragments (Fabs) (bisFabs). Proteolytic digestion conditions for each IgG isotype were analyzed to optimize the yield and quality of the final conjugates. The resulting bisFabs showed no significant amounts of homodimers or aggregates. The predictive value of murine bisFabs was tested by comparing the T-cell redirected cytotoxic activity of a panel of antibodies in either the bisFab or full-length IgG formats. A variety of antigens with different structures and expression levels was used to extend the comparison to a wide range of binding geometries and antigen densities. The activity observed for different murine bisFabs correlated with those observed for the full-length IgG format across multiple different antigen targets, supporting the use of bisFabs as a screening tool. Our method may also be used for the screening of bispecific antibodies with other mechanisms of action, allowing for a more rapid selection of lead therapeutic candidates.

Exosomal levels of miRNA-21 from cerebrospinal fluids associated with poor prognosis and tumor recurrence of glioma patients.

Glioma is a most common type of primary brain tumors. Extracellular vesicles, in the form of exosomes, are known to mediate cell-cell communication by transporting cell-derived proteins and nucleic acids, including various microRNAs (miRNAs). Here we examined the cerebrospinal fluid (CSF) from patients with recurrent glioma for the levels of cancer-related miRNAs, and evaluated the values for prognosis by comparing the measures of CSF-, serum-, and exosome-contained miR-21 levels. Samples from seventy glioma patients following surgery were compared with those from brain trauma patients as a non-tumor control group. Exosomal miR-21 levels in the CSF of glioma patients were found significantly higher than in the controls; whereas no difference was detected in serum-derived exosomal miR-21 expression. The CSF-derived exosomal miR-21 levels correlated with tumor spinal/ventricle metastasis and the recurrence with anatomical site preference. From additional 198 glioma tissue samples, we verified that miR-21 levels associated with tumor grade of diagnosis and negatively correlated with the median values of patient overall survival time. We further used a lentiviral inhibitor to suppress miR-21 expression in U251 cells. The results showed that the levels of miR-21 target genes of PTEN, RECK and PDCD4 were up-regulated at protein levels. Therefore, we concluded that the exosomal miR-21 levels could be demonstrated as a promising indicator for glioma diagnosis and prognosis, particularly with values to predict tumor recurrence or metastasis.

Anti-CD20/CD3 T cell-dependent bispecific antibody for the treatment of B cell malignancies.

Bispecific antibodies and antibody fragments in various formats have been explored as a means to recruit cytolytic T cells to kill tumor cells. Encouraging clinical data have been reported with molecules such as the anti-CD19/CD3 bispecific T cell engager (BiTE) blinatumomab. However, the clinical use of many reported T cell-recruiting bispecific modalities is limited by liabilities including unfavorable pharmacokinetics, potential immunogenicity, and manufacturing challenges. We describe a B cell-targeting anti-CD20/CD3 T cell-dependent bispecific antibody (CD20-TDB), which is a full-length, humanized immunoglobulin G1 molecule with near-native antibody architecture constructed using "knobs-into-holes" technology. CD20-TDB is highly active in killing CD20-expressing B cells, including primary patient leukemia and lymphoma cells both in vitro and in vivo. In cynomolgus monkeys, CD20-TDB potently depletes B cells in peripheral blood and lymphoid tissues at a single dose of 1 mg/kg while demonstrating pharmacokinetic properties similar to those of conventional monoclonal antibodies. CD20-TDB also exhibits activity in vitro and in vivo in the presence of competing CD20-targeting antibodies. These data provide rationale for the clinical testing of CD20-TDB for the treatment of CD20-expressing B cell malignancies.

Gene-based delivery of IFN-beta is efficacious in a murine model of experimental allergic encephalomyelitis.

Experimental allergic encephalomyelitis (EAE) is a model of central nervous system (CNS) inflammation that follows immunization with certain CNS antigens. The course and clinical manifestations of EAE are similar to those of multiple sclerosis (MS) in humans; therefore, EAE has become an accepted animal model to study MS. The purpose of this study was to demonstrate that systemic expression of murine interferon-beta (IFN-beta) (MuIFN-beta), following intramuscular (i.m.) delivery of plasmid DNA encoding MuIFN-beta to the hind limb of mice, is effective in reducing the clinical manifestations of disease in a model of EAE. The results of the study demonstrate that gene-based delivery of MuIFN-beta caused significantly decreased clinical scores compared with delivery of the null vector. A single injection of the MuIFN-beta plasmid was as effective in reducing the severity of the disease as an every other day injection of MuIFN-beta protein.

Transcriptional silencing is associated with extensive methylation of the CMV promoter following adenoviral gene delivery to muscle.

BACKGROUND: Although the transient nature of transgene expression using first-generation adenovirus (Ad) vectors is well known, the exact mechanisms responsible for this phenomenon are uncertain. METHODS: Rats were given intramuscular (i.m.) injections of a first-generation Ad containing the human fibroblast growth factor 4 (hFGF-4) gene driven by the cytomegalovirus (CMV) promoter and enhancer (CMV-PE). The copy number of hFGF-4 mRNA and viral DNA was measured in the same muscles by quantitative RT-PCR and quantitative PCR at times between 1 h and 84 days after virus injection. Quantitative Southern blot analysis for the intact hFGF-4 transcription unit DNA was also performed, and the methylation status of the CMV-PE DNA in the muscle was determined using bisulfite sequencing. RESULTS: The copy number of hFGF-4 mRNA peaked at 6 h then decreased 56-fold by 24 h, and a further 240-fold between days 3 and 28. Although the viral DNA copy number also decreased 23-fold between 6 h and 28 days, the ratio of copies of hFGF-4 mRNA per copy of viral DNA decreased 385-fold over this period. Methylation of the CMV-PE DNA in the muscle at both CpG and non-CpG sites was observed 24 h after virus administration and had increased at day 7. CONCLUSIONS: Decreased transcription associated with extensive methylation of the CMV-PE was the major mechanism responsible for the decrease in transgene mRNA levels. Strategies for preventing transcriptional silencing will be valuable for improving the duration of transgene expression from adenoviral vectors.