Preclinical evidence for the effective use of TL-895, a highly selective and potent second-generation BTK inhibitor, for the treatment of B-cell malignancies.

TL-895 (formerly known as M7583) is a potent, highly selective, adenosine triphosphate (ATP)-competitive, second-generation, irreversible inhibitor of Bruton's tyrosine kinase (BTK). We characterized its biochemical and cellular effects in in vitro and in vivo models. TL-895 was evaluated preclinically for potency against BTK using IC50 concentration-response curves; selectivity using a 270-kinase panel; BTK phosphorylation in Ramos Burkitt's lymphoma cells by ProteinSimple Wes analysis of one study; anti-proliferative effects in primary chronic lymphocytic leukemia (CLL) blasts; cell viability effects in diffuse large B-cell lymphoma (DLBCL) and mantle-cell lymphoma (MCL) cell lines; effects on antibody-dependent cell-mediated cytotoxicity (ADCC) from Daudi cells and chromium-51 release from human tumor cell lines; and efficacy in vivo using four MCL xenograft model and 21 DLBCL patient-derived xenograft (PDX) models (subtypes: 9 ABC, 11 GCB, 1 Unclassified). TL-895 was active against recombinant BTK (average IC50 1.5 nM) and inhibited only three additional kinases with IC50 within tenfold of BTK activity. TL-895 inhibited BTK auto-phosphorylation at the Y223 phosphorylation site (IC50 1-10 nM). TL-895 inhibited the proliferation of primary CLL blasts in vitro and inhibited growth in a subset of activated DLBCL and MCL cell lines. TL-895 inhibited the ADCC mechanism of therapeutic antibodies only at supra-clinical exposure levels. TL-895 significantly inhibited tumor growth in the Mino MCL xenograft model and in 5/21 DLBCL PDX models relative to vehicle controls. These findings demonstrate the potency of TL-895 for BTK and its efficacy in models of B-cell lymphoma despite its refined selectivity.

Identification and characterization of M6903, an antagonistic anti-TIM-3 monoclonal antibody.

T cell immunoglobulin and mucin domain-3 (TIM-3) is an immune checkpoint that regulates normal immune responses but can be exploited by tumor cells to evade immune surveillance. TIM-3 is primarily expressed on immune cells, particularly on dysfunctional and exhausted T cells, and engagement of TIM-3 with its ligands promotes TIM-3-mediated T cell inhibition. Antagonistic ligand-blocking anti-TIM-3 antibodies have the potential to abrogate T cell inhibition, activate antigen-specific T cells, and enhance anti-tumor immunity. Here we describe M6903, a fully human anti-TIM-3 antibody without effector function and with high affinity and selectivity to TIM-3. We demonstrate that M6903 blocks the binding of TIM-3 to three of its ligands, phosphatidylserine (PtdSer), carcinoembryonic antigen cell adhesion-related molecule 1 (CEACAM1), and galectin 9 (Gal-9). These results are supported by an atomic resolution crystal structure and functional assays, which demonstrate that M6903 monotherapy enhanced T cell activation. This activation was further enhanced by the combination of M6903 with bintrafusp alfa, a bifunctional fusion protein that simultaneously blocks the transforming growth factor-ß (TGF-ß) and programmed death ligand 1 (PD-L1) pathways. M6903 and bintrafusp alfa combination therapy also enhanced anti-tumor efficacy in huTIM-3 knock-in mice, relative to either monotherapy. These in vitro and in vivo data, along with favorable pharmacokinetics in marmoset monkeys, suggest that M6903 as a monotherapy warrants further pre-clinical assessment and that M6903 and bintrafusp alfa may be a promising combination therapy in the clinic.

Balancing Selectivity and Efficacy of Bispecific Epidermal Growth Factor Receptor (EGFR) × c-MET Antibodies and Antibody-Drug Conjugates.

Bispecific antibodies (bsAbs) and antibody-drug conjugates (ADCs) have already demonstrated benefits for the treatment of cancer in several clinical studies, showing improved drug selectivity and efficacy. In particular, simultaneous targeting of prominent cancer antigens, such as EGF receptor (EGFR) and c-MET, by bsAbs has raised increasing interest for potentially circumventing receptor cross-talk and c-MET-mediated acquired resistance during anti-EGFR monotherapy. In this study, we combined the selectivity of EGFR × c-MET bsAbs with the potency of cytotoxic agents via bispecific antibody-toxin conjugation. Affinity-attenuated bispecific EGFR × c-MET antibody-drug conjugates demonstrated high in vitro selectivity toward tumor cells overexpressing both antigens and potent anti-tumor efficacy. Due to basal EGFR expression in the skin, ADCs targeting EGFR in general warrant early safety assessments. Reduction in EGFR affinity led to decreased toxicity in keratinocytes. Thus, the combination of bsAb affinity engineering with the concept of toxin conjugation may be a viable route to improve the safety profile of ADCs targeting ubiquitously expressed antigens.

Epitope characterization of an anti-PD-L1 antibody using orthogonal approaches.

The binding of programmed death ligand 1 protein (PD-L1) to its receptor programmed death protein 1 (PD-1) mediates immunoevasion in cancer and chronic viral infections, presenting an important target for therapeutic intervention. Several monoclonal antibodies targeting the PD-L1/PD-1 signaling axis are undergoing clinical trials; however, the epitopes of these antibodies have not been described. We have combined orthogonal approaches to localize and characterize the epitope of a monoclonal antibody directed against PD-L1 at good resolution and with high confidence. Limited proteolysis and mass spectrometry were applied to reveal that the epitope resides in the first immunoglobulin domain of PD-L1. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) was used to identify a conformational epitope comprised of discontinuous strands that fold to form a beta sheet in the native structure. This beta sheet presents an epitope surface that significantly overlaps with the PD-1 binding interface, consistent with a desired PD-1 competitive mechanism of action for the antibody. Surface plasmon resonance screening of mutant PD-L1 variants confirmed that the region identified by HDX-MS is critical for the antibody interaction and further defined specific residues contributing to the binding energy. Taken together, the results are consistent with the observed inhibitory activity of the antibody on PD-L1-mediated immune evasion. This is the first report of an epitope for any antibody targeting PD-L1 and demonstrates the power of combining orthogonal epitope mapping techniques.

Anti-EGFR biparatopic-SEED antibody has enhanced combination-activity in a single molecule.

Certain combinations of non-competitive anti-EGFR antibodies have been reported to produce new effects on cells compared to either antibody used separately. New and enhanced combination-activity includes increased inhibition of signaling, increased receptor internalization and degradation, reduced proliferation of tumor cell lines and induction of complement-dependent cytotoxicity (CDC) effector function. To test requirements and mechanisms to elicit enhanced combination-activity with different EGFR binding domains, we created an anti-EGFR biparatopic antibody. A biparatopic antibody interacts through two different antigen-binding sites to a single antigen. A heterodimeric antibody with one binding domain derived from the C225 antibody and one binding domain derived from the humanized 425 (hu425) antibody was built on the strand-exchange engineered domain (SEED) scaffold. This anti-EGFR biparatopic-SEED antibody was compared to parental antibodies used alone and in combination, and to the corresponding monovalent anti-EGFR-SEED antibodies used alone or in combination. We found that the anti-EGFR biparatopic-SEED had enhanced activity, similar to the combination of the two parental antibodies. Combinations of monovalent anti-EGFR-SEED antibodies did not produce enhanced effectiveness in cellular assays. Our results show that the anti-EGFR biparatopic antibody created using the SEED scaffold has enhanced combination-activity in a single molecule. Furthermore, these data suggest that the potential to cross-link the two different epitopes is an important requirement in the mechanism of enhanced combination-activity.

Therapeutic assessment of SEED: a new engineered antibody platform designed to generate mono- and bispecific antibodies.

The strand-exchange engineered domain (SEED) platform was designed to generate asymmetric and bispecific antibody-like molecules, a capability that expands therapeutic applications of natural antibodies. This new protein engineered platform is based on exchanging structurally related sequences of immunoglobulin within the conserved CH3 domains. Alternating sequences from human IgA and IgG in the SEED CH3 domains generate two asymmetric but complementary domains, designated AG and GA. The SEED design allows efficient generation of AG/GA heterodimers, while disfavoring homodimerization of AG and GA SEED CH3 domains. Using a clinically validated antibody (C225), we tested whether Fab derivatives constructed on the SEED platform retain desirable therapeutic antibody features such as in vitro and in vivo stability, favorable pharmacokinetics, ligand binding and effector functions including antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity. In addition, we tested SEED with combinations of binder domains (scFv, VHH, Fab). Mono- and bivalent Fab-SEED fusions retain full binding affinity, have excellent biochemical and biophysical stability, and retain desirable antibody-like characteristics conferred by Fc domains. Furthermore, SEED is compatible with different combinations of Fab, scFv and VHH domains. Our assessment shows that the new SEED platform expands therapeutic applications of natural antibodies by generating heterodimeric Fc-analog proteins.