Therapeutic Targeting of LIF Overcomes Macrophage-mediated Immunosuppression of the Local Tumor Microenvironment.

PURPOSE: Leukemia inhibitory factor (LIF) is a multifunctional cytokine with numerous reported roles in cancer and is thought to drive tumor development and progression. Characterization of LIF and clinical-stage LIF inhibitors would increase our understanding of LIF as a therapeutic target. EXPERIMENTAL DESIGN: We first tested the association of LIF expression with transcript signatures representing multiple processes regulating tumor development and progression. Next, we developed MSC-1, a high-affinity therapeutic antibody that potently inhibits LIF signaling and tested it in immune competent animal models of cancer. RESULTS: LIF was associated with signatures of tumor-associated macrophages (TAM) across 7,769 tumor samples spanning 22 solid tumor indications. In human tumors, LIF receptor was highly expressed within the macrophage compartment and LIF treatment drove macrophages to acquire immunosuppressive capacity. MSC-1 potently inhibited LIF signaling by binding an epitope that overlaps with the gp130 receptor binding site on LIF. MSC-1 showed monotherapy efficacy in vivo and drove TAMs to acquire antitumor and proinflammatory function in syngeneic colon cancer mouse models. Combining MSC-1 with anti-PD1 leads to strong antitumor response and a long-term tumor-free survival in a significant proportion of treated mice. CONCLUSIONS: Overall, our findings highlight LIF as a therapeutic target for cancer immunotherapy.

In situ antibody phage display yields optimal inhibitors of integrin α11/ß1.

Integrins are transmembrane multi-conformation receptors that mediate interactions with the extracellular matrix. In cancer, integrins influence metastasis, proliferation, and survival. Collagen-binding integrin-α11/ß1, a marker of aggressive tumors that is involved in stroma-tumor crosstalk, may be an attractive target for anti-cancer therapeutic antibodies. We performed selections with phage-displayed synthetic antibody libraries for binding to either purified integrin-α11/ß1 or in situ on live cells. The in-situ strategy yielded many diverse antibodies, and strikingly, most of these antibodies did not recognize purified integrin-α11/ß1. Conversely, none of the antibodies selected for binding to purified integrin-α11/ß1 were able to efficiently recognize native cell-surface antigen. Most importantly, only the in-situ selection yielded functional antibodies that were able to compete with collagen-I for binding to cell-surface integrin-α11/ß1, and thus inhibited cell adhesion. In-depth characterization of a subset of in situ-derived clones as full-length immunoglobulins revealed high affinity cellular binding and inhibitory activities in the single-digit nanomolar range. Moreover, the antibodies showed high selectivity for integrin-α11/ß1 with minimal cross-reactivity for close homologs. Taken together, our findings highlight the advantages of in-situ selections for generation of anti-integrin antibodies optimized for recognition and inhibition of native cell-surface proteins, and our work establishes general methods that could be extended to many other membrane proteins.

Homology modeling and structure-based design improve hydrophobic interaction chromatography behavior of integrin binding antibodies.

Monoclonal antibody (mAb) candidates from high-throughput screening or binding affinity optimization often contain mutations leading to liabilities for further development of the antibody, such as aggregation-prone regions and lack of solubility. In this work, we optimized a candidate integrin α11-binding mAb for developability using molecular modeling, rational design, and hydrophobic interaction chromatography (HIC). A homology model of the parental mAb Fv region was built, and this revealed hydrophobic patches on the surface of the complementarity-determining region loops. A series of 97 variants of the residues primarily responsible for the hydrophobic patches were expressed and their HIC retention times (RT) were measured. As intended, many of the computationally designed variants reduced the HIC RT compared to the parental mAb, and mutating residues that contributed most to hydrophobic patches had the greatest effect on HIC RT. A retrospective analysis was then performed where 3-dimentional protein property descriptors were evaluated for their ability to predict HIC RT using the current series of mAbs. The same descriptors were used to train a simple multi-parameter protein quantitative structure-property relationship model on this data, producing an improved correlation. We also extended this analysis to recently published HIC data for 137 clinical mAb candidates as well as 31 adnectin variants, and found that the surface area of hydrophobic patches averaged over a molecular dynamics sample consistently correlated to the experimental data across a diverse set of biotherapeutics.

Fully human antibodies against the Protease-Activated Receptor-2 (PAR-2) with anti-inflammatory activity.

PAR-2 belongs to a family of G-protein coupled Protease-Activated Receptors (PAR) which are activated by specific proteolytic cleavage in the extracellular N-terminal region. PAR-2 is activated by proteases such as trypsin, tryptase, proteinase 3, factor VIIa, factor Xa and is thought to be a mediator of inflammation and tissue injury, where elevated levels of proteases are found. Utilizing the HuCAL GOLD® phage display library we generated fully human antibodies specifically blocking the protease cleavage site in the N-terminal domain. In vitro affinity optimization resulted in antibodies with up to 1000-fold improved affinities relative to the original parental antibodies with dissociation constants as low as 100 pM. Corresponding increases in potency were observed in a mechanistic protease cleavage assay. The antibodies effectively inhibited PAR-2 mediated intracellular calcium release and cytokine secretion in various cell types stimulated with trypsin. In addition, the antibodies demonstrated potent inhibition of trypsin induced relaxation of isolated rat aortic rings ex vivo. In a short term mouse model of inflammation, the trans vivo DTH model, anti-PAR-2 antibodies showed inhibition of the inflammatory swelling response. In summary, potent inhibitors of PAR-2 were generated which allow further assessment of the role of this receptor in inflammation and evaluation of their potential as therapeutic agents.

The determination and correlation of molecular and cellular equilibrium Kd and kinetic k(off) values for small molecule allosteric antagonists of LFA-1.

Molecular K(d) and k(off) parameters are often used to define the molecular potency of drugs. These constants, however, are determined on purified target proteins, and their relationship to in vivo binding phenomena is poorly understood. Herein, we report two novel assays to determine the off-rates of allosteric antagonists from lymphocyte function-associated antigen 1 (LFA-1). The SPR assay involves using the non-blocking mAb TS2/4 to immobilize full-length LFA-1 on a hydrophilic chip surface, and the soluble, native ligand sICAM-1 to probe the fraction of free LFA-1. To determine the fraction of free LFA-1 on cell surfaces, a flow cytometry assay was developed utilizing the fluorophore-labeled Fab R3.1. The R3.1 antibody has been previously demonstrated to block the ability of both ICAM-1 and antagonists to bind to purified and cell-surface LFA-1. The molecular and ex vivo cellular parameters were determined for a set of nine structurally-related LFA-1 allosteric antagonists. The relationships between the parameters determined in the ELISA (K(d)), SPR (k(off)), and flow cytometry (k(off)) assays were shown to be linear with slopes approximately equal to 1, and a correlation analysis showed that the three assay datasets were equivalent at the alpha=0.05 level. These results were unexpected, as the ELISA and SPR assays involve high affinity LFA-1, and the flow cytometry assays involve cell surface LFA-1 in whole-blood, in which a distribution of affinity states would be expected. Nevertheless, the results presented herein show that the K(d) and k(off)'s determined in molecular assays can be used as predictors of LFA-1 receptor occupancy in ex vivo assays.

Small molecule LFA-1 antagonists compete with an anti-LFA-1 monoclonal antibody for binding to the CD11a I domain: development of a flow-cytometry-based receptor occupancy assay.

The beta(2) integrin LFA-1 (CD11a/CD18) is a leukocyte-specific adhesion molecule that mediates leukocyte extravasation, antigen presentation, and T-cell-mediated cytolysis through its interaction with its counter-receptors, ICAM-1, ICAM-2, and ICAM-3. We have recently described a small molecule antagonist of LFA-1 (BIRT 377) that inhibits LFA-1/ICAM-1 molecular interactions, LFA-1-dependent adhesion assays, antigen-induced proliferation of T-cells, and superantigen-induced production of IL-2 in vivo in mice. We have also recently described a unique monoclonal antibody, R3.1, which competes with BIRT 377 and its analogs for binding to both purified full-length LFA-1 and the purified recombinant I domain module. In this manuscript, we extend these studies to cell-based systems and utilize this unique reagent for the development of a receptor occupancy assay. Exploiting these observations, we have designed and validated an assay that allows us to measure receptor occupancy in vitro on monkey and human peripheral blood leukocytes and ex vivo in whole blood from monkeys dosed with small molecule LFA-1 antagonists. Further refinement of these reagents has led to the development of a Fab-based assay that allows rapid and reproducible analysis of whole blood samples. These optimized reagents allow for quantification of the number of receptors expressed on the cell surface and a more accurate quantitation of receptor occupancy.