Mitchell Reff

Targeting the Lymphotoxin-β Receptor with Agonist Antibodies as a Potential Cancer Therapy

The lymphotoxin-beta receptor (LT beta R) is a tumor necrosis factor receptor family member critical for the development and maintenance of various lymphoid microenvironments. Herein, we show that agonistic anti-LT beta R monoclonal antibody (mAb) CBE11 inhibited tumor growth in xenograft models and potentiated tumor responses to chemotherapeutic agents. In a syngeneic colon carcinoma tumor model, treatment of the tumor-bearing mice with an agonistic antibody against murine LT beta R caused increased lymphocyte infiltration and necrosis of the tumor. A pattern of differential gene expression predictive of cellular and xenograft response to LT beta R activation was identified in a panel of colon carcinoma cell lines and when applied to a panel of clinical colorectal tumor samples indicated 35% likelihood a tumor response to CBE11. Consistent with this estimate, CBE11 decreased tumor size and/or improved long-term animal survival with two of six independent orthotopic xenografts prepared from surgical colorectal carcinoma samples. Targeting of LT beta R with agonistic mAbs offers a novel approach to the treatment of colorectal and potentially other types of cancers.

In vitro IgE inhibition in B cells by anti-CD23 monoclonal antibodies is functionally dependent on the immunoglobulin Fc domain.

CD23, the low affinity receptor for IgE (FcvarepsilonRII), is involved in regulation of IgE synthesis by B-lymphocytes. Five monoclonal antibodies to human CD23 were generated from cynomolgus macaques immunized with purified soluble CD23 (sCD23). Four of the five primate antibodies blocked the binding of IgE complexes to CD23 positive cells and also inhibited the production of IgE in vitro by IL-4 induced human peripheral blood mononuclear cells (PBMC). The variable domains of several primate antibodies were utilized to construct chimeric macaque/human (PRIMATIZED((R))) monoclonal antibodies. PRIMATIZED((R)) p5E8G1, containing human gamma 1 constant region, inhibited IgE production in vitro as efficiently as the parent primate antibody, but the human gamma 4 constant version, PRIMATIZED((R)) p5E8G4, was not as effective in IgE inhibition. An F(ab)(2) of p5E8G1 did not inhibit IgE production but did interfere with IgE inhibition by the intact anti-CD23 antibody in a dose dependent fashion. The murine monoclonal antibody MHM6 recognizes human CD23 at a different epitope than primate antibody 5E8, and inhibits IgE production by IL-4 induced PBMC. As with the F(ab)(2) of p5E8G1, the F(ab)(2) of MHM6 also failed to inhibit IgE production. These data imply that the mechanism by which anti-CD23 antibodies inhibit IgE production requires cross-linking of CD23 to an IgG receptor. These data also imply that neither bivalent cross-linking of CD23 alone or inhibition of CD23 binding to its natural ligands is sufficient to inhibit IgE production.

Conserved amino acid networks involved in antibody variable domain interactions

Engineered antibodies are a large and growing class of protein therapeutics comprising both marketed products and many molecules in clinical trials in various disease indications. We investigated naturally conserved networks of amino acids that support antibody VH and VL function, with the goal of generating information to assist in the engineering of robust antibody or antibody‐like therapeutics. We generated a large and diverse sequence alignment of V‐class Ig‐folds, of which VH and VL domains are family members. To identify conserved amino acid networks, covariations between residues at all possible position pairs were quantified as correlation coefficients (ϕ‐values). We provide rosters of the key conserved amino acid pairs in antibody VH and VL domains, for reference and use by the antibody research community. The majority of the most strongly conserved amino acid pairs in VH and VL are at or adjacent to the VH–VL interface suggesting that the ability to heterodimerize is a constraining feature of antibody evolution. For the VH domain, but not the VL domain, residue pairs at the variable‐constant domain interface (VH–CH1 interface) are also strongly conserved. The same network of conserved VH positions involved in interactions with both the VL and CH1 domains is found in camelid VHH domains, which have evolved to lack interactions with VL and CH1 domains in their mature structures; however, the amino acids at these positions are different, reflecting their different function. Overall, the data describe naturally occurring amino acid networks in antibody Fv regions that can be referenced when designing antibodies or antibody‐like fragments with the goal of improving their biophysical properties. Proteins 2009.

Stable IgG-like Bispecific Antibodies Directed toward the Type I Insulin-like Growth Factor Receptor Demonstrate Enhanced Ligand Blockade and Anti-tumor Activity

Bispecific antibodies (BsAbs) target multiple epitopes on the same molecular target or different targets. Although interest in BsAbs has persisted for decades, production of stable and active BsAbs has hindered their clinical evaluation. Here, we describe the production and characterization of tetravalent IgG-like BsAbs that combine the activities of allosteric and competitive inhibitors of the type-I insulin-like growth factor receptor (IGF-1R). The BsAbs, which were engineered for thermal stability, express well, demonstrate favorable biophysical properties, and recognize both epitopes on IGF-1R. Only one BsAb with a unique geometry, denoted BIIB4-5scFv, was capable of engaging all four of its binding arms simultaneously. All the BsAbs (especially BIIB4-5scFv) demonstrated enhanced ligand blocking over the single monoclonal antibodies (mAbs), particularly at high ligand concentrations. The pharmacokinetic profiles of two IgG-like BsAbs were tested in nude mice and shown to be comparable with that of the parental mAbs. The BsAbs, especially BIIB4-5scFv, demonstrated an improved ability to reduce the growth of multiple tumor cell lines and to inhibit ligand-induced IGF-1R signaling in tumor cells over the parental mAbs. BIIB4-5scFv also led to superior tumor growth inhibition over its parental mAbs in vivo. In summary, BsAbs that bridge multiple inhibitory mechanisms against a single target may generally represent a more effective strategy for intervention in oncology or other indications compared with traditional mAb therapy.

Anti-CD23 monoclonal antibody inhibits germline Cε transcription in B cells

Abstract A chimeric macaque/human (PRIMATIZED®) anti-CD23 antibody, p6G5G1, demonstrated a strong inhibitory effect on IL-4- and anti-CD40 antibody-stimulated IgE production by human peripheral blood mononuclear cells (PBMCs). RNA analysis by both reverse transcription-polymerase chain reaction (RT-PCR) and Northern blot showed that p6G5G1 inhibited germline Ce RNA synthesis, but had no effect on CD23 mRNA levels. These data suggest that p6G5G1 may inhibit immunoglobulin class switching to IgE through the inhibition of germline Ce RNA synthesis. Early addition of p6G5G1 after stimulation by IL-4 and anti-CD40 was critical for IgE inhibition. In contrast, later addition of p6G5G1 still showed inhibition of increased levels of surface CD23, which is normally upregulated by stimulation with IL-4 and anti-CD40.

Design, synthesis, and biological evaluation of pyrazolopyrimidine-sulfonamides as potent multiple-mitotic kinase (MMK) inhibitors (part I).

A novel class of pyrazolopyrimidine-sulfonamides was discovered as selective dual inhibitors of aurora kinase A (AKA) and cyclin-dependent kinase 1 (CDK1). These inhibitors were originally designed based on an early lead (compound I). SAR development has led to the discovery of potent inhibitors with single digit nM IC(50)s towards both AKA and CDK1. An exemplary compound 1a has demonstrated good efficacy in an HCT116 colon cancer xenograft model.

Novel Antibody Hinge Regions for Efficient Production of CH2 Domain-deleted Antibodies

HuCC49ΔCH2 is a heavy chain constant domain 2 domain-deleted antibody under development as a radioimmunotherapeutic for treating carcinomas overexpressing the TAG-72 tumor antigen. Mammalian cell culture biosynthesis of HuCC49ΔCH2 produces two isoforms (form A and form B) in an approximate 1:1 ratio, and consequently separation and purification of the desired form A isoform adversely impact process and yield. A protein engineering strategy was used to develop a panel of hinge-engineered HuCC49ΔCH2 antibodies to identify hinge sequences to optimize production of the form A isoform. We found that adding a single proline residue at Kabat position 243, immediately adjacent to the carboxyl end of the core middle hinge CPPC domain, resulted in an increase from 39 to 51% form A isoform relative to the parent HuCC49ΔCH2 antibody. Insertion of the amino acids proline-alanine-proline (PAP) at positions 243-245 enhanced production of the form A isoform to 72%. Insertion of a cysteine-rich 15-amino acid IgG3 hinge motif (CPEPKSCDTPPPCPR) in both of these mutant antibodies resulted in secretion of predominantly form A isoform with little or no detectable form B. Yields exceeding 98% of the form A isoform have been realized. Preliminary peptide mapping and mass spectrometry analysis suggest that at least two, and as many as five, inter-heavy chain disulfide linkages may be present.

Generation, and Characterization of Two Tetravalent CH2 Domain-Deleted CC49 Mabs

of anti-CD3 antibody. Moreover, B7.1/NHS76 fusion protein was found to retain its antigen-binding capacity and good uptake in tumors. To assess their in vivo functions, both fusion proteins were evaluated in murine Colon 26 tumor model. When B7.1/NHS76 (30ug/dose) was administered iv daily3 5 into BALB/c mice bearing 0.5cm sc tumors, a 60% reduction in average tumor size was observed compared to control treated mice at day 19 post-implantation. In contrast, when B7.1/Fc (20ug/dose) was administered in the same protocol, 80% mice underwent complete tumor regression. The therapeutic effects of both fusion proteins were abrogated by depletion of CD8 T cells indicating that this lymphoid subset was instrumental. However, when antisera to CD4 or CD25 Tcell subsets were used to deplete these subsets, the tumors underwent complete regression. Dramatic changes in the infiltration and activation of T-cells could be seen in tumor draining lymph nodes by FACS in the combination therapy groups. In vivo treatment studies using other tumor cell lines and re-challenge studies on previous tumor-free mice are currently in progress. These studies suggest that human B7.1 fusion proteins might be promising antitumor reagents in patients especially when used in combination with methods to delete T-regulatory cells.