Development of CDX-527: a bispecific antibody combining PD-1 blockade and CD27 costimulation for cancer immunotherapy.

CD27 is a costimulatory molecule that provides a complementary target to the PD-1/PD-L1 checkpoint axis on T cells. Combining a CD27 agonist antibody with PD-1/PD-L1 blockade has shown synergistic antitumor activity in preclinical models, which led to clinical studies of the combination in cancer patients. We theorized that coupling CD27 costimulation with PD-1/PD-L1 blockade in a bispecific antibody (BsAb) may provide greater immune activating properties than combining the individual mAbs due to enhanced CD27 activation by cross-linking through PD-L1 and Fc receptors. To test this approach, we developed CDX-527, a tetravalent PD-L1xCD27 IgG1-scFv BsAb. CDX-527 potently inhibits PD-1 signaling and induces CD27-mediated T cell costimulation through PD-L1 cross-linking. In mixed lymphocyte reaction assays, CDX-527 is more potent than the combination of the parental antibodies, suggesting that cross-linking through both Fc receptors and PD-L1 results in enhanced CD27 agonist activity. CDX-527 was shown to mediate effector function against tumor cells overexpressing either CD27 or PD-L1. In human CD27 transgenic mice, we observed that antigen-specific T cell responses to a vaccine are greatly enhanced with a surrogate PD-L1xCD27 BsAb. Furthermore, the BsAb exhibits greater antitumor activity than the combination of the parental antibodies in a syngeneic lymphoma model. A pilot study of CDX-527 in cynomolgus macaques confirmed a mAb-like pharmacokinetic profile without noted toxicities. These studies demonstrate that CDX-527 effectively combines PD-1 blockade and CD27 costimulation into one molecule that is more potent than combination of the parental antibodies providing the rationale to advance this BsAb toward clinical studies in cancer patients.

Development of CDX-1140, an agonist CD40 antibody for cancer immunotherapy.

Limitations of immunotherapy include poorly functioning events early in the immune response cycle, such as efficient antigen presentation and T cell priming. CD40 signaling in dendritic cells leads to upregulation of cell surface costimulatory and MHC molecules and the generation of cytokines, which promotes effective priming of CD8+ effector T cells while minimizing T cell anergy and the generation of regulatory T cells. This naturally occurs through interaction with CD40 ligand (CD40L) expressed on CD4+ T-helper cells. CD40 signaling can also be achieved using specific antibodies, leading to several agonist CD40 antibodies entering clinical development. Our approach to select a CD40 agonist antibody was to define a balanced profile between sufficiently strong immune stimulation and the untoward effects of systemic immune activation. CDX-1140 is a human IgG2 antibody that activates DCs and B cells and drives NFkB stimulation in a CD40-expressing reporter cell line. These activities are Fc-independent and are maintained using an F(ab')2 fragment of the antibody. CDX-1140 binds outside of the CD40L binding site, and addition of recombinant CD40L greatly enhances DC and B activation by CDX-1140, suggesting that CDX-1140 may act synergistically with naturally expressed CD40L. CDX-1140 also has both direct and immune-mediated anti-tumor activity in xenograft models. CDX-1140 does not promote cytokine production in whole blood assays and has good pharmacodynamic and safety profiles in cynomolgus macaques. These data support the potential of CDX-1140 as part of a cancer therapy regimen, and a phase 1 trial has recently commenced.

Toll-like receptor agonists shape the immune responses to a mannose receptor-targeted cancer vaccine.

Previous studies have documented that selective delivery of protein antigens to cells expressing mannose receptor (MR) can lead to enhanced immune responses. We postulated that agents that influenced the MR expression level, and the activation and migration status of MR-expressing antigen presenting cells, would modulate immune responses to MR-targeted vaccines. To address this question, we investigated the effect of clinically used adjuvants in human MR transgenic (hMR-Tg) mice immunized with an MR-targeting cancer vaccine composed of the human anti-MR monoclonal antibody B11 fused with the oncofetal protein, human chorionic gonadotropin beta chain (hCGß), and referred to as B11-hCGß. We found that humoral responses to low doses of B11-hCGß could be enhanced by prior administration of GM-CSF, which upregulated MR expression in vivo. However, co-administration of the Toll-like receptor (TLR) agonists, poly-ICLC and/or CpG with B11-hCGß was required to elicit Th1 immunity, as measured by antigen-specific T-cell production of IFN-γ. The TLR agonists were shown to increase the number of vaccine-containing cells in the draining lymph nodes of immunized hMR-Tg mice. In particular, with B11-hCGß and poly-ICLC, a dramatic increase in vaccine-positive cells was observed in the T-cell areas of the lymph nodes, compared to the vaccine alone or combined with GM-CSF. Importantly, the absence of the TLR agonists during the priming immunization led to antigen-specific tolerance. Therefore, this study provides insight into the mechanisms by which adjuvants can augment immune responses to B11-hCGß and have implications for the rationale design of clinical studies combining MR-targeted vaccination with TLR agonists.

Agonist anti-human CD27 monoclonal antibody induces T cell activation and tumor immunity in human CD27-transgenic mice.

The CD70/CD27 pathway plays a significant role in the control of immunity and tolerance, and previous studies demonstrated that targeting murine CD27 (mCD27) with agonist mAbs can mediate antitumor efficacy. We sought to exploit the potential of this pathway for immunotherapy by developing 1F5, a fully human IgG1 mAb to human CD27 (hCD27) with agonist activity. We developed transgenic mice expressing hCD27 under control of its native promoter for in vivo testing of the Ab. The expression and regulation of hCD27 in hCD27-transgenic (hCD27-Tg) mice were consistent with the understood biology of CD27 in humans. In vitro, 1F5 effectively induced proliferation and cytokine production from hCD27-Tg-derived T cells when combined with TCR stimulation. Administration of 1F5 to hCD27-Tg mice enhanced Ag-specific CD8(+) T cell responses to protein vaccination comparably to an agonist anti-mCD27 mAb. In syngeneic mouse tumor models, 1F5 showed potent antitumor efficacy and induction of protective immunity, which was dependent on CD4(+) and CD8(+) T cells. The requirement of FcR engagement for the agonistic and antitumor activities of 1F5 was demonstrated using an aglycosylated version of the 1F5 mAb. These data with regard to the targeting of hCD27 are consistent with previous reports on targeting mCD27 and provide a rationale for the clinical development of the 1F5 mAb, for which studies in advanced cancer patients have been initiated under the name CDX-1127.

Development of a human monoclonal antibody for potential therapy of CD27-expressing lymphoma and leukemia.

PURPOSE: The TNF receptor superfamily member CD27 is best known for its important role in T-cell immunity but is also recognized as a cell-surface marker on a number of B- and T-cell malignancies. In this article, we describe a novel human monoclonal antibody (mAb) specific for CD27 with properties that suggest a potential utility against malignancies that express CD27. EXPERIMENTAL DESIGN: The fully human mAb 1F5 was generated using human Ig transgenic mice and characterized by analytical and functional assays in vitro. Severe combined immunodeficient (SCID) mice inoculated with human CD27-expressing lymphoma cells were administered 1F5 to investigate direct antitumor effects. A pilot study of 1F5 was conducted in non-human primates to assess toxicity. RESULTS: 1F5 binds with high affinity and specificity to human and macaque CD27 and competes with ligand binding. 1F5 activates T cells only in combination with T-cell receptor stimulation and does not induce proliferation of primary CD27-expressing tumor cells. 1F5 significantly enhanced the survival of SCID mice bearing Raji or Daudi tumors, which may be mediated through direct effector mechanisms such as antibody-dependent cellular cytotoxicity. Importantly, administration of up to 10 mg/kg of 1F5 to cynomolgus monkeys was well tolerated without evidence of significant toxicity or depletion of circulating lymphocytes. CONCLUSIONS: Collectively, the data suggest that the human mAb 1F5, which has recently entered clinical development under the name CDX-1127, may provide direct antitumor activity against CD27-expressing lymphoma or leukemia, independent of its potential to enhance immunity through its agonistic properties.

An extensive phenotypic characterization of the hTNFalpha transgenic mice.

BACKGROUND: Tumor necrosis factor alpha (TNFalpha) is implicated in a wide variety of pathological and physiological processes, including chronic inflammatory conditions, coronary artery disease, diabetes, obesity, and cachexia. Transgenic mice expressing human TNFalpha (hTNFalpha) have previously been described as a model for progressive rheumatoid arthritis. In this report, we describe extensive characterization of an hTNFalpha transgenic mouse line. RESULTS: In addition to arthritis, these hTNFalpha transgenic mice demonstrated major alterations in body composition, metabolic rate, leptin levels, response to a high-fat diet, bone mineral density and content, impaired fertility and male sexual function. Many phenotypes displayed an earlier onset and a higher degree of severity in males, pointing towards a significant degree of sexual dimorphism in response to deregulated expression of TNFalpha. CONCLUSION: These results highlight the potential usefulness of this transgenic model as a resource for studying the progressive effects of constitutively expressed low levels of circulating TNFalpha, a condition mimicking that observed in a number of human pathological conditions.