Katherine S. Bowdish

CD200 Expression on Tumor Cells Suppresses Antitumor Immunity: New Approaches to Cancer Immunotherapy

Although the immune system is capable of mounting a response against many cancers, that response is insufficient for tumor eradication in most patients due to factors in the tumor microenvironment that defeat tumor immunity. We previously identified the immune-suppressive molecule CD200 as up-regulated on primary B cell chronic lymphocytic leukemia (B-CLL) cells and demonstrated negative immune regulation by B-CLL and other tumor cells overexpressing CD200 in vitro. In this study we developed a novel animal model that incorporates human immune cells and human tumor cells to address the effects of CD200 overexpression on tumor cells in vivo and to assess the effect of targeting Abs in the presence of human immune cells. Although human mononuclear cells prevented tumor growth when tumor cells did not express CD200, tumor-expressed CD200 inhibited the ability of lymphocytes to eradicate tumor cells. Anti-CD200 Ab administration to mice bearing CD200-expressing tumors resulted in nearly complete tumor growth inhibition even in the context of established receptor-ligand interactions. Evaluation of an anti-CD200 Ab with abrogated effector function provided evidence that blocking of the receptor-ligand interaction was sufficient for control of CD200-mediated immune modulation and tumor growth inhibition in this model. Our data indicate that CD200 expression by tumor cells suppresses antitumor responses and suggest that anti-CD200 treatment might be therapeutically beneficial for treating CD200-expressing cancers.

Human antibodies from immunized donors are protective against anthrax toxin in vivo

A panel of Fabs that neutralize anthrax toxin in vitro was selected from libraries generated from human donors vaccinated against anthrax. At least two of these antibodies protect rats from anthrax intoxication in vivo. Fabs 83K7C and 63L1D bind with subnanomolar affinity to protective antigen (PA) 63, and Fab 63L1D neutralizes toxin substoichiometrically, inhibits lethal factor (LF) interaction with PA63 and binds to a conformational epitope formed by PA63.

In vivo targeting of antigens to human dendritic cells through DC-SIGN elicits stimulatory immune responses and inhibits tumor growth in grafted mouse models.

Multiple cancer vaccine trials have been carried out using ex vivo generated autologous dendritic cells (DCs) loaded with tumor antigen before readministration into patients. Though promising, overall immunologic potency and clinical efficacy might be improved with more efficient DC-based therapies that avoid ex vivo manipulations, but are instead based on in vivo targeting of DCs. For initial in vivo proof of concept studies, we evaluated targeting of proteins or peptides to DCs through DC-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN). Because the biology of DC-SIGN is different between mice and humans, we assess human DC-SIGN targeting in the setting of elements of a human immune system in a mouse model. Administration of anti-DC-SIGN antibodies carrying either tetanus toxoid peptides or keyhole limpet hemocyanin (KLH) to Rag2−/−γC−/− mice reconstituted with human immune cells raised stimulatory human T-cell responses to the respective antigen without additional adjuvant requirements. Furthermore, administration of anti-DC-SIGN antibody-KLH conjugate enhanced the adjuvant properties of KLH resulting in inhibition of RAJI (Human Burkitts Lymphoma Cell Line) cell tumor growth in Nonobese Diabetic/Severe Combined Immunodeficient mice transplanted with human immune cells. Thus, mouse models reconstituted with human immune cells seem to be suitable for evaluating DC-targeted vaccines, and furthermore, targeting to DCs in situ via DC-SIGN may provide a promising vaccine platform for inducing strong immune responses against cancer and infectious disease agents.

Blockade of CD200 in the Presence or Absence of Antibody Effector Function: Implications for Anti-CD200 Therapy

CD200 is an immunosuppressive molecule overexpressed in multiple hematologic malignancies such as B cell chronic lymphocytic leukemia, multiple myeloma, and acute myeloid leukemia. We previously demonstrated that up-regulation of CD200 on tumor cells suppresses antitumor immune responses and that antagonistic anti-human CD200 mAbs enabled human PBMC-mediated tumor growth inhibition in xenograft NOD/SCID human (hu)-mouse models. Ab variants with effector function (IgG1 constant region (G1)) or without effector function (IgG2/G4 fusion constant region (G2G4)) exhibited high antitumor activity in a human tumor xenograft model in which CD200 was expressed. In this report, we seek to select the best candidate to move forward into the clinic and begin to decipher the mechanisms of tumor cell killing by comparing anti-CD200-G1 vs anti-CD200-G2G4 in two related animal models. In a CD200-expressing xenograft NOD/SCID hu-mouse model where CD200 ligand/receptor interactions are already established before initiating treatment, we find that anti-CD200-G1 is a less effective Ab compared with anti-CD200-G2G4. Separately, in a model that evaluates the effect of the Abs on the immune cell component of the xenograft NOD/SCID hu-mouse model distinctly from the effects of binding to CD200 on tumor cells, we find that the administration of anti-CD200-G1 Abs completely abolished human PBMC-mediated tumor growth inhibition. Along with supporting in vitro studies, our data indicate that anti-CD200-G1 Abs efficiently mediate Ab-dependent cellular cytotoxicity of activated T cells, critical cells involved in immune-mediated killing. These studies suggest important implications regarding the selection of the constant region in anti-CD200 immunotherapy of cancer patients.

Internalizing Antibodies to the C-Type Lectins, L-SIGN and DC-SIGN, Inhibit Viral Glycoprotein Binding and Deliver Antigen to Human Dendritic Cells for the Induction of T Cell Responses

The C-type lectin L-SIGN is expressed on liver and lymph node endothelial cells, where it serves as a receptor for a variety of carbohydrate ligands, including ICAM-3, Ebola, and HIV. To consider targeting liver/lymph node-specific ICAM-3-grabbing nonintegrin (L-SIGN) for therapeutic purposes in autoimmunity and infectious disease, we isolated and characterized Fabs that bind strongly to L-SIGN, but to a lesser degree or not at all to dendritic cell-specific ICAM-grabbing nonintegrin (DC-SIGN). Six Fabs with distinct relative affinities and epitope specificities were characterized. The Fabs and those selected for conversion to IgG were tested for their ability to block ligand (HIV gp120, Ebola gp, and ICAM-3) binding. Receptor internalization upon Fab binding was evaluated on primary human liver sinusoidal endothelial cells by flow cytometry and confirmed by confocal microscopy. Although all six Fabs internalized, three Fabs that showed the most complete blocking of HIVgp120 and ICAM-3 binding to L-SIGN also internalized most efficiently. Differences among the Fab panel in the ability to efficiently block Ebola gp compared with HIVgp120 suggested distinct binding sites. As a first step to consider the potential of these Abs for Ab-mediated Ag delivery, we evaluated specific peptide delivery to human dendritic cells. A durable human T cell response was induced when a tetanus toxide epitope embedded into a L-SIGN/DC-SIGN-cross-reactive Ab was targeted to dendritic cells. We believe that the isolated Abs may be useful for selective delivery of Ags to DC-SIGN- or L-SIGN-bearing APCs for the modulation of immune responses and for blocking viral infections.

Targeting CUB Domain-Containing Protein 1 with a Monoclonal Antibody Inhibits Metastasis in a Prostate Cancer Model

Through a whole-cell panning approach, we previously identified a panel of antibodies that bound to prostate cancer cell surface antigens. One such antigen, CUB domain-containing protein 1 (CDCP1), was recognized by monoclonal antibody 25A11 and is a single transmembrane molecule highly expressed in several metastatic cancers as well as on CD34(+)CD133(+) myeloid leukemic blast cells. We show CDCP1 expression on prostate cancer cell lines by real-time quantitative PCR (RT-qPCR), flow cytometry, and immunohistochemistry and on prostate cancer patient samples by RT-qPCR and immunohistochemical staining. In cell-based assays, antibody 25A11 inhibited prostate cancer cell migration and invasion in vitro. Further characterization showed that CDCP1 is internalized on antibody binding. When 25A11 was coupled to the cytotoxin saporin either directly or via a secondary antibody, both resulted in prostate cancer cell killing in vitro. In vivo targeting studies with an anti-CDCP1 immunotoxin showed significant inhibition of primary tumor growth as well as metastasis in a mouse xenograft model. These data provide support for continued evaluation of anti-CDCP1 therapy for potential use in cancer in primary and metastatic disease.

A rationally designed agonist antibody fragment that functionally mimics thrombopoietin

By using rational design, antibody fragments (Fabs) that mimic thrombopoietin (TPO) were created. A peptide with cMpl receptor-binding capability was grafted into different complementarity-determining regions of a fully human Fab scaffold. Functional presentation of the peptide was optimized by using phage display and cell-based panning. Select antibodies and fragments containing two grafted peptides were assayed for their ability to stimulate the cMpl receptor in vitro. Several candidates demonstrated agonist activity in an in vitro cMpl receptor signaling reporter assay, including Fab59, which was estimated to be equipotent to TPO. Fab59 additionally was able to effectively stimulate platelet production in normal mice. These rationally designed mimetic Fabs may provide a therapeutic intervention for thrombocytopenia while avoiding the potential generation of neutralizing antibodies to endogenous TPO. Furthermore, this study demonstrates a method by which short-lived linear peptides with binding activity may be converted to more stable and potent agonists capable of activating cell surface receptors.

Antibody Elicited Against the gp41 N-Heptad Repeat (NHR) Coiled-Coil Can Neutralize HIV-1 with Modest Potency but Non-neutralizing Antibodies Also Bind to NHR Mimetics

Following CD4 receptor binding to the HIV-1 envelope spike (Env), the conserved N-heptad repeat (NHR) region of gp41 forms a coiled-coil that is a precursor to the fusion reaction. Although it has been a target of drug and vaccine design, there are few monoclonal antibody (mAb) tools with which to probe the antigenicity and immunogenicity specifically of the NHR coiled-coil. Here, we have rescued HIV-1-neutralizing anti-NHR mAbs from immune phage display libraries that were prepared (i) from b9 rabbits immunized with a previously described mimetic of the NHR coiled-coil, N35(CCG)-N13, and (ii) from an HIV-1 infected individual. We describe a rabbit single-chain Fv fragment (scFv), 8K8, and a human Fab, DN9, which specifically recognize NHR coiled-coils that are unoccupied by peptide corresponding to the C-heptad repeat or CHR region of gp41 (e.g. C34). The epitopes of 8K8 and DN9 were found to partially overlap with that of a previously described anti-NHR mAb, IgG D5; however, 8K8 and DN9 were much more specific than D5 for unoccupied NHR trimers. The mAbs, including a whole IgG 8K8 molecule, neutralized primary HIV-1 of clades B and C in a pseudotyped virus assay with comparable, albeit relatively modest potency. Finally, a human Fab T3 and a rabbit serum (both non-neutralizing) were able to block binding of D5 and 8K8 to a gp41 NHR mimetic, respectively, but not the neutralizing activity of these mAbs. We conclude from these results that NHR coiled-coil analogs of HIV-1 gp41 elicit many Abs during natural infection and through immunization, but that due to limited accessibility to the corresponding region on fusogenic gp41 few can neutralize. Caution is therefore required in targeting the NHR for vaccine design. Nevertheless, the mAb panel may be useful as tools for elucidating access restrictions to the NHR of gp41 and in designing potential improvements to mimetics of receptor-activated Env.

Rationale for anti-CD200 immunotherapy in B-CLL and other hematologic malignancies: new concepts in blocking immune suppression.

Immune evasion in cancer is increasingly recognized as a contributing factor in the failure of a natural host antitumor immune response as well as in the failure of cancer vaccine trials. Immune evasion may be the result of a number of factors, including expansion of regulatory T cells, production of immunosuppressive cytokines, downregulation of HLA class I and tumor-associated antigens and upregulation of immunosuppressive molecules on the surface of tumor cells. CD200, a cell surface ligand that plays a role in regulating the immune system, has been shown to be upregulated on the surface of some hematologic and solid tumor malignancies. This review characterizes the role of CD200 in immune suppression, and describes strategies to target this molecule in the oncology setting, thus directly modulating immune regulation and potentially altering tolerance to tumor antigens.

A step-wise approach significantly enhances protein yield of a rationally-designed agonist antibody fragment in E. coli

Fab59 is a rationally-designed antibody fragment (Fab) that mimics the activity of the cytokine thrombopoietin (TPO). Fab59 activity was initially detected directly from bacterial supernatants in a cell-based assay and was subsequently estimated to be equipotent to TPO using purified material. However, the expression of Fab59 was insufficient to support in vivo characterization of the Fab due to extremely low expression levels from its initial phage display expression vector. To boost expression, a new expression vector was designed and constructed, and Fab59 light chain codons were optimized for bacterial expression. However, from this a new challenge arose, in that the codon-optimized Fab59 was more toxic to Escherichia coli cells than parental Fab59. Co-expression of the bacterial chaperon protein Skp alleviated this toxicity. A two-step purification method was used to isolate monomeric Fab59 from the periplasm. Although Fab59 was prone to form aggregates during the purification process, buffer modification efficiently eliminated this problem. Overall, optimization of Fab59 expression and purification achieved a 100-fold increase in Fab59 production in E. coli relative to the starting yield. The yield of purified monomeric Fab59 from a shake flask reached up to 3.5mg/L, which was sufficient to support testing of the agonist activity of purified monomeric Fab59 in vivo. Even higher yields may be achieved by purification of Fab present in the culture media, as Skp most significantly increased accumulation of Fab59 in that location.