Conditioning treatment with CD27 Ab enhances expansion and antitumor activity of adoptively transferred T cells in mice.

Cyclophosphamide plus fludarabine (C/F) are currently used to improve the expansion and effectiveness of adoptive cell therapy (ACT). However, these chemotherapeutics cause pan-leukopenia and adverse events, suggesting that safer and more effective conditioning treatments are needed to improve ACT outcomes. Previously, we reported that varlilumab, a CD27-targeting antibody, mediates Treg -preferential T cell depletion, CD8-T cell dominant costimulation, and systemic immune activation in hCD27 transgenic mice and cancer patients. We reasoned that the activities induced by varlilumab may provide an effective conditioning regimen for ACT. Varlilumab pretreatment of hCD27 +/+mCD27 - /- mice resulted in prominent proliferation of transferred T cells isolated from wild-type mice. These studies uncovered a critical role for CD27 signaling for the expansion of transferred T cells, as transfer of T cells from CD27 deficient mice or treatment with a CD70 blocking antibody greatly reduced their proliferation. In this model, varlilumab depletes endogenous hCD27+/+ T cells and blocks their subsequent access to CD70, allowing for more CD70 costimulation available to the mCD27 +/+ transferred T cells. CD27-targeted depletion led to a greater expansion of transferred T cells compared to C/F conditioning and resulted in longer median survival and more cures than C/F conditioning in the E.G7 tumor model receiving OT-I cell therapy. We propose that translation of this work could be achieved through engineering of T cells for ACT to abrogate varlilumab binding but preserve CD70 ligation. Thus, varlilumab could be an option to chemotherapy as a conditioning regimen for ACT.

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.

CD27-Mediated Regulatory T Cell Depletion and Effector T Cell Costimulation Both Contribute to Antitumor Efficacy.

CD27, a member of the TNFR superfamily, is constitutively expressed in most T cells and plays crucial roles in T cell effector functions. The costimulation and antitumor activity of CD27 agonistic Abs have been well documented in mouse models. Clinical testing of a human IgG1 anti-CD27 Ab, varlilumab (clone 1F5), is ongoing in cancer patients. In this study, we set out to further understand CD27 as an immunomodulatory target and to address the mechanism of antitumor efficacy using different IgG isotypes of 1F5 in human CD27-transgenic mice. 1F5mIgG1, the only isotype engaging inhibitory FcγRIIB expressed in B cells, elicited the most potent and broad immune response, but terminal differentiation, exhaustion, and apoptosis in the activated effector T cells were inevitable. Accordingly, this isotype was the most effective in eradicating BCL1 lymphoma but had limited efficacy in s.c. tumors. Conversely, 1F5mIgG2a, which interacts with cells expressing activating FcγRs, led to moderate immune activation, as well as to prominent reduction in the number and suppressive activity of regulatory T cells. These combined mechanisms imparted potent antitumor activity to 1F5mIgG2a, particularly against the s.c. tumors. 1F5hIgG1, varlilumab, showed balanced agonistic activity that was prominent at lower doses and depleting activity that was greater at higher doses. 1F5hIgG1 had good antitumor activity in all tumor models tested. Thus, both agonist and depleting properties contribute to the antitumor efficacy of CD27-targeted immunotherapy, and modulation of these activities in patients may be achieved by varying the dose and regimen.

Development of a Novel Antibody-Drug Conjugate for the Potential Treatment of Ovarian, Lung, and Renal Cell Carcinoma Expressing TIM-1.

T-cell immunoglobulin and mucin domain 1 (TIM-1) is a type I transmembrane protein that was originally described as kidney injury molecule 1 (KIM-1) due to its elevated expression in kidney and urine after renal injury. TIM-1 expression is also upregulated in several human cancers, most notably in renal and ovarian carcinomas, but has very restricted expression in healthy tissues, thus representing a promising target for antibody-mediated therapy. To this end, we have developed a fully human monoclonal IgG1 antibody specific for the extracellular domain of TIM-1. This antibody was shown to bind purified recombinant chimeric TIM-1-Fc protein and TIM-1 expressed on a variety of transformed cell lines, including Caki-1 (human renal clear cell carcinoma), IGROV-1 (human ovarian adenocarcinoma), and A549 (human lung carcinoma). Internalization studies using confocal microscopy revealed the antibody was rapidly internalized by cells in vitro, and internalization was confirmed by quantitative imaging flow cytometry. An antibody-drug conjugate (ADC) was produced with the anti-TIM-1 antibody covalently linked to the potent cytotoxin, monomethyl auristatin E (MMAE), and designated CDX-014. The ADC was shown to exhibit in vitro cytostatic or cytotoxic activity against a variety of TIM-1-expressing cell lines, but not on TIM-1-negative cell lines. Using the Caki-1, IGROV-1, and A549 xenograft mouse models, CDX-014 showed significant antitumor activity in a clinically relevant dose range. Safety evaluation in nonhuman primates has demonstrated a good profile and led to the initiation of clinical studies of CDX-014 in renal cell carcinoma and potentially other TIM-1-expressing tumors. Mol Cancer Ther; 15(12); 2946-54. ©2016 AACR.

Internalization and endosomal degradation of receptor-bound antigens regulate the efficiency of cross presentation by human dendritic cells.

Dendritic cells (DCs) can capture extracellular antigens and load resultant peptides on to MHC class I molecules, a process termed cross presentation. The mechanisms of cross presentation remain incompletely understood, particularly in primary human DCs. One unknown is the extent to which antigen delivery to distinct endocytic compartments determines cross presentation efficiency, possibly by influencing antigen egress to the cytosol. We addressed the problem directly and quantitatively by comparing the cross presentation of identical antigens conjugated with antibodies against different DC receptors that are targeted to early or late endosomes at distinct efficiencies. In human BDCA1+ and monocyte-derived DCs, CD40 and mannose receptor targeted antibody conjugates to early endosomes, whereas DEC205 targeted antigen primarily to late compartments. Surprisingly, the receptor least efficient at internalization, CD40, was the most efficient at cross presentation. This did not reflect DC activation by CD40, but rather its relatively poor uptake or intra-endosomal degradation compared with mannose receptor or DEC205. Thus, although both early and late endosomes appear to support cross presentation in human DCs, internalization efficiency, especially to late compartments, may be a negative predictor of activity when selecting receptors for vaccine development.