Multifaceted Bioanalytical Methods for the Comprehensive Pharmacokinetic and Catabolic Assessment of MEDI3726, an Anti-Prostate-Specific Membrane Antigen Pyrrolobenzodiazepine Antibody-Drug Conjugate.

Complex biotherapeutic modalities, such as antibody-drug conjugates (ADC), present significant challenges for the comprehensive bioanalytical characterization of their pharmacokinetics (PK) and catabolism in both preclinical and clinical settings. Thus, the bioanalytical strategy for ADCs must be designed to address the specific structural elements of the protein scaffold, linker, and warhead. A typical bioanalytical strategy for ADCs involves quantification of the Total ADC, Total IgG, and Free Warhead concentrations. Herein, we present bioanalytical characterization of the PK and catabolism of a novel ADC. MEDI3726 targets prostate-specific membrane antigen (PMSA) and is comprised of a humanized IgG1 antibody site-specifically conjugated to tesirine (SG3249). The MEDI3726 protein scaffold lacks interchain disulfide bonds and has an average drug to antibody ratio (DAR) of 2. Based on the structural characteristics of MEDI3726, an array of 4 bioanalytical assays detecting 6 different surrogate analyte classes representing at least 14 unique species was developed, validated, and employed in support of a first-in-human clinical trial (NCT02991911). MEDI3726 requires the combination of heavy-light chain structure and conjugated warhead to selectively deliver the warhead to the target cells. Therefore, both heavy-light chain dissociation and the deconjugation of the warhead will affect the activity of MEDI3726. The concentration-time profiles of subjects dosed with MEDI3726 revealed catabolism of the protein scaffold manifested by the more rapid clearance of the Active ADC, while exhibiting minimal deconjugation of the pyrrolobenzodiazepine (PBD) warhead (SG3199).

Chk1 inhibition in p53-deficient cell lines drives rapid chromosome fragmentation followed by caspase-independent cell death.

Activation of Checkpoint kinase 1 (Chk1) following DNA damage mediates cell cycle arrest to prevent cells with damaged DNA from entering mitosis. Here we provide a high-resolution analysis of cells as they undergo S- and G2-checkpoint bypass in response to Chk1 inhibition with the selective Chk1 inhibitor GNE-783. Within 4-8 h of Chk1 inhibition following gemcitabine induced DNA damage, cells with both sub-4N and 4N DNA content prematurely enter mitosis. Coincident with premature transition into mitosis, levels of DNA damage dramatically increase and chromosomes condense and attempt to align along the metaphase plate. Despite an attempt to congress at the metaphase plate, chromosomes rapidly fragment and lose connection to the spindle microtubules. Gemcitabine mediated DNA damage promotes the formation of Rad51 foci; however, while Chk1 inhibition does not disrupt Rad51 foci that are formed in response to gemcitabine, these foci are lost as cells progress into mitosis. Premature entry into mitosis requires the Aurora, Cdk1/2 and Plk1 kinases and even though caspase-2 and -3 are activated upon mitotic exit, they are not required for cell death. Interestingly, p53, but not p21, deficiency enables checkpoint bypass and chemo-potentiation. Finally, we uncover a differential role for the Wee-1 checkpoint kinase in response to DNA damage, as Wee-1, but not Chk1, plays a more prominent role in the maintenance of S- and G2-checkpoints in p53 proficient cells.

Subcutaneous versus intravenous administration of rituximab: pharmacokinetics, CD20 target coverage and B-cell depletion in cynomolgus monkeys.

The CD20-specific monoclonal antibody rituximab (MabThera(®), Rituxan(®)) is widely used as the backbone of treatment for patients with hematologic disorders. Intravenous administration of rituximab is associated with infusion times of 4-6 hours, and can be associated with infusion-related reactions. Subcutaneous administration of rituximab may reduce this and facilitate administration without infusion-related reactions. We sought to determine the feasibility of achieving equivalent efficacy (measured by endogenous B-cell depletion) and long-term durability of CD20 target coverage for subcutaneously administered rituximab compared with intravenous dosing. In these preclinical studies, male cynomolgus monkeys were treated with either intravenous rituximab or novel subcutaneous formulation of rituximab containing human recombinant DNA-derived hyaluronidase enzyme. Peripheral blood samples were analyzed for serum rituximab concentrations, peripheral B-cell depletion, and CD20 target coverage, including subset analysis according to CD21+ status. Distal lymph node B-cell depletion and CD20 target coverage were also measured. Initial peak serum concentrations of rituximab were significantly higher following intravenous administration than subcutaneous. However, the mean serum rituximab trough concentrations were comparable at 2 and 7 days post-first dose and 9 and 14 days post-second dose. Efficacy of B-cell depletion in both peripheral blood and distal lymph nodes was comparable for both methods. In lymph nodes, 9 days after the second dose with subcutaneous and intravenous rituximab, B-cell levels were decreased by 57% and 42% respectively. Similarly, levels of peripheral blood B cells were depleted by >94% for both subcutaneous and intravenous dosing at all time points. Long-term recovery of free unbound surface CD20 levels was similar, and the duration of B-cell depletion was equally sustained over 2 months for both methods. These results demonstrate that, despite initial peak serum drug level differences, subcutaneous rituximab has similar durability, pharmacodynamics, and efficacy compared with intravenous rituximab.

Suppression of phosphatidylinositol 3,4,5-trisphosphate production is a key determinant of B cell anergy.

Anergy is a critical physiologic mechanism to sensor self-reactive B cells. However, a biochemical understanding of how anergy is achieved and maintained is lacking. Herein, we investigated the role of the phosphoinositide 3-kinase (PI3K) lipid product PI(3,4,5)P(3) in B cell anergy. We found reduced generation of PI(3,4,5)P(3) in anergic B cells, which was attributable to reduced phosphorylation of the PI3K membrane adaptor CD19, as well as increased expression of the inositol phosphatase PTEN. Sustained production of PI(3,4,5)P(3) in B cells, achieved through conditional deletion of Pten, resulted in failed tolerance induction and abundant autoantibody production. In contrast to wild-type immature B cells, B cell receptor engagement of PTEN-deficient immature B cells resulted in activation and proliferation, indicating a central defect in early B cell responsiveness. These findings establish repression of the PI3K signaling pathway as a necessary condition to avert the generation, activation, and persistence of self-reactive B cells.

A critical role for complement C3d and the B cell coreceptor (CD19/CD21) complex in the initiation of inflammatory arthritis.

Complement C3 cleavage products mediate the recognition and clearance of toxic or infectious agents. In addition, binding of the C3d fragment to Ag promotes B lymphocyte activation through coengagment of the BCR and complement receptor 2 (CD21). Signal augmentation is thought to be achieved through enhanced recruitment and activation of CD21-associated CD19. In this study we show, using the DBA/1 collagen-induced arthritis (CIA) model, that conjugation of C3d to heterologous type II collagen is sufficient to cause disease in the absence of the mycobacterial components of CFA. Transient depletion of C3 during the inductive phase of CIA delays and lessens the severity of disease, and DBA/1 mice deficient for coreceptor components CD19 or CD21 are not susceptible to CIA. Adoptive transfer experiments revealed that CD21 expression on either B cells or follicular dendritic cells is sufficient to acquire disease susceptibility. Although CD19(-/-) and CD21(-/-) mice produce primary Ab responses to heterologous and autologous type II collagen, they are impaired in the ability to activate T cells, form germinal centers, and produce secondary autoantibody responses. These findings indicate that binding of C3d to self-Ags can promote autoimmunity through enhanced Ag retention and presentation by follicular dendritic cells and B cells, respectively.

CD19 function in central and peripheral B-cell development.

Although the B-cell antigen receptor (BCR) factors most prominently in the maintenance and differentiation of mature B cells, it is now appreciated that co-receptor molecules can positively or negatively modulate signals through the BCR. Co-receptors are functionally defined as modifiers of BCR engagement and signal transduction, and are distinct from other accessory molecules that act independently to regulate B-cell growth. The co-receptor CD19 functions to augment signals by the pre-BCR/BCR and in doing so can modulate B-cell fate decisions at multiple stages of development. In mature B cells, CD19 also associates with complement receptor 2 (CR2/CD21) and is pivotal for transducing signals induced by co-recognition of complement C3d-fixed antigens by the BCR and CD21. In this article, we focus on recent progress in the understanding of CD19 function through the characterization of mouse models that relate in vivo function to biochemical properties of CD19.