Andrea Crocker

Induction of Antigen-Specific Immunity with a Vaccine Targeting NY-ESO-1 to the Dendritic Cell Receptor DEC-205

Dendritic cell targeting safely leads to integrated humoral and cellular immunity when combined with TLR agonists in cancer patients. Start Spreading the News Dendritic cells are the matchmakers of the immune system: They introduce T cells to antigen, providing the right context for the T cell to react. However, tumor alters the nearby microenvironment in such a way as to block immune activation. Dhodapkar et al. attempt to overcome this inhibition by targeting a tumor antigen directly to dendritic cells. The authors tested a vaccine that consisted of a human antibody targeted to the dendritic cell receptor DEC-205 fused with the tumor antigen NY-ESO-1 in a cohort of patients with tumors refractory to other therapies. They also added Toll-like receptor ligands as adjuvants in a dose-escalating study. They found that treatment induced both humoral and cellular immunity in these patients, with no dose-limiting toxicities. What’s more, a subset of patients had either stable disease or disease regression, particularly those who had received immune checkpoint inhibitors. If these data can be reproduced in larger trials, this study suggests that targeting antigen to dendritic cells could be an additional avenue to boost the immune response to cancer. Immune-based therapies for cancer are generating substantial interest because of the success of immune checkpoint inhibitors. This study aimed to enhance anticancer immunity by exploiting the capacity of dendritic cells (DCs) to initiate T cell immunity by efficient uptake and presentation of endocytosed material. Delivery of tumor-associated antigens to DCs using receptor-specific monoclonal antibodies (mAbs) in the presence of DC-activating agents elicits robust antigen-specific immune responses in preclinical models. DEC-205 (CD205), a molecule expressed on DCs, has been extensively studied for its role in antigen processing and presentation. CDX-1401 is a vaccine composed of a human mAb specific for DEC-205 fused to the full-length tumor antigen NY-ESO-1. This phase 1 trial assessed the safety, immunogenicity, and clinical activity of escalating doses of CDX-1401 with the Toll-like receptor (TLR) agonists resiquimod (TLR7/8) and Hiltonol (poly-ICLC, TLR3) in 45 patients with advanced malignancies refractory to available therapies. Treatment induced humoral and cellular immunity to NY-ESO-1 in patients with confirmed NY-ESO-1–expressing tumors across various dose levels and adjuvant combinations. No dose-limiting or grade 3 toxicities were reported. Thirteen patients experienced stabilization of disease, with a median duration of 6.7 months (range, 2.4+ to 13.4 months). Two patients had tumor regression (~20% shrinkage in target lesions). Six of eight patients who received immune-checkpoint inhibitors within 3 months after CDX-1401 administration had objective tumor regression. This first-in-human study of a protein vaccine targeting DCs demonstrates its feasibility, safety, and biological activity and provides rationale for combination immunotherapy strategies including immune checkpoint blockade.

Antigenic Targeting of the Human Mannose Receptor Induces Tumor Immunity

Pattern recognition receptors are preferentially expressed on APCs allowing selective uptake of pathogens for the initiation of antimicrobial immunity. In particular, C-type lectin receptors, including the mannose receptor (MR), facilitate APC-mediated adsorptive endocytosis of microbial glyconjugates. We have investigated the potential of antigenic targeting to the MR as a means to induce Ag-specific humoral and cellular immunity. hMR transgenic (hMR Tg) mice were generated to allow specific targeting with the anti-hMR Ab, B11. We show that hMR targeting induced both humoral and cellular antigenic specific immunity. Immunization of hMR Tg mice with B11 mAbs induced potent humoral responses independent of adjuvant. Injection of hMR Tg mice with mouse anti-hMR Ab clone 19.2 elicited anti-Id-specific humoral immunity while non-Tg mice were unresponsive. B11-OVA fusion proteins (B11-OVA) were efficiently presented to OVA-specific CD4 and CD8 T cells in MR Tg, but not in non-Tg, mice. Effector differentiation of responding T cells in MR Tg mice was significantly enhanced with concomitant immunization with the TLR agonist, CpG. Administration of both CpG and B11-OVA to hMR Tg mice induced OVA-specific tumor immunity while WT mice remained unprotected. These studies support the clinical development of immunotherapeutic approaches in cancer using pattern recognition receptor targeting systems for the selective delivery of tumor Ags to APCs.

Antibody-enhanced cross-presentation of self antigen breaks T cell tolerance.

We have developed a model of autoimmunity to investigate autoantibody-mediated cross-presentation of self antigen. RIP-mOVA mice, expressing OVA in pancreatic beta cells, develop severe autoimmune diabetes when given OT-I cells (OVA-specific CD8(+) T cells) and anti-OVA IgG but not when given T cells alone. Anti-OVA IgG is not directly injurious to the islets but rather enhances cross-presentation of apoptotic islet antigen to the OT-I cells, leading to their differentiation into potent effector cells. Antibody-driven effector T cell activation is dependent on the presence of activating Fc receptors for IgG (FcgammaRs) and cross-priming DCs. As a consequence, diabetes incidence and severity was reduced in mice lacking activating FcgammaRs. An intact complement pathway was also required for disease development, as C3 deficiency was also partially protective. C3-deficient animals exhibited augmented T cell priming overall, indicating a proinflammatory role for complement activation after the T cell priming phase. Thus, we show that autoreactive antibody can potently enhance the activation of effector T cells in response to cross-presented self antigen, thereby contributing to T cell-mediated autoimmunity.

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. Clin Cancer Res; 18(14); 3812–21. ©2012 AACR.

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.

Abstract 5343: Development of novel anti-CD27 human antibodies with therapeutic potential

The costimulatory molecule CD27 is a member of the tumor necrosis factor (TNF) receptor superfamily, and is constitutively expressed on the majority of mature T cells, memory B cells, and a portion of NK cells. The interaction of CD27 with its ligand CD70 plays key roles in T cell activation, proliferation, survival, maturation of effector capacity and memory; in clonal B cell expansion and germinal center formation; and in NK cell cytolytic activity. Previous reports have shown that antigen-specific CD8 + cytolytic T cell immunity can be augmented through CD27 activation in mice. Of particular relevance is the work by M. J. Glennie et al. demonstrating that an agonistic anti-mouse CD27 mAb given without a DC maturation signal has potent anti-tumor activity through boosting of T cell immunity. To explore the therapeutic potential of this target, a panel of fully human antibodies recognizing human CD27 was generated using human Ig transgenic mice immunized with recombinant human CD27. These human anti-CD27 mAbs showed specific and high affinity binding to CD27 and CD27-expressing lymphoma cells. We generated transgenic mice that express human CD27 (hCD27-Tg) to evaluate the anti-human CD27 mAbs in vivo. The expression profile and regulation of the human CD27 transgene driven by its own promoter were similar to that observed with endogenous mouse CD27. Administration of anti-human CD27 mAbs in combination with ovalbumin greatly enhanced antigen specific T cell responses compared to ovalbumin plus human IgG1 isotype control in the hCD27-Tg mice. The SIINFEKL-specific CD8 + T cell proliferation and activation were detected by pentamer staining and IFNγ ELISPOT analysis. These data support the further development of these novel human anti-CD27 antibodies and we are currently investigating their therapeutic activity using various in vivo tumor models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5343.

The mechanism of anti-tumor immunity induced by varlilumab, a CD27 agonist mAb, is model dependent.

The use of agonist antibodies to costimulatory receptors holds significant promise as a complementary approach to the already proven strategy of antibody mediated checkpoint blockade. This is in part due to the multiple mechanisms that have been associated with targeting costimulatory molecules in mouse models such as expansion of antigen-specific effector T cells (Teff), decrease in number or functional activity of regulatory T cells (Treg), and activation of NK cells. Recent reports have suggested that reduction of intratumoral Treg was particularly important for the anti-tumor activity of several immune modulating antibodies. We have developed an agonist anti-human CD27 antibody, varlilumab, and have previously demonstrated its anti-tumor efficacy in several syngeneic tumor models using human CD27 transgenic mice. We set out to understand which mechanism of action of the CD27 antibody is related to its anti-tumor activity by developing mouse isotype variants of the antibody. Varlilumab expressed as a mouse IgG1 isotype (varli-mG1) induces potent costimulation and has no depleting activity, whereas, varlilumab expressed as a mouse IgG2a isotype (varli-mG2a) has moderate costimulatory activity and is capable of depleting cells with high CD27 expression (e.g. Treg). We found that as monotherapy, varli-mG1 was very effective in the BCL1 lymphoma model, and ineffective in the EG.7 thymoma model. In contrast, varli-mG2a was ineffective in the BCL1 model, but had anti-tumor activity in the EG.7 model. Based on correlative flow cytometry studies, we hypothesize that depleting Treg with high CD27 expression is the primary mechanism for anti-CD27 therapy in the EG.7 model, whereas the BCL1 model responds to potent CD27 costimulation. Interestingly, varlilumab (human IgG1 isotype) has a combination of the costimulation and Treg depleting activity and has potent anti-tumor activity in both models. These data suggest that the mechanism of anti-tumor immunity induced by varlilumab is model dependent, and we are currently investigating this in additional tumor models. The results imply that multiple mechanisms are likely to also be relevant in the human setting. The preclinical studies and results from the Phase I trial with varlilumab are consistent with the antibodys capacity to mediate both T cell activation and a reduction in the number of Treg.

Abstract 3519: Appropriate crosslinking is required for co-stimulatory activity of human CD27 antibody in a transgenic mouse model

CD27 is a member of TNFR superfamily. It constitutively expresses on the majority of T cell and a subset of NK cells, playing key roles in T cell activation and survival and in NK cell proliferation and cytotoxicity upon interaction with ligand CD70. Some antibodies to mouse CD27 have been reported that display agonistic and anti-tumor activities while other mAbs had less anti-tumor activity and were depleting. We hypothesized that differences in these antibodies may be due to Fc receptor engagement, as has recently been shown for the adjuvant and anti-tumor activities of agonistic mouse CD40 mAbs, which is also member of TNFR superfamily. We have developed and previously described a human anti-human CD27 antibody (1F5) and a human CD27 transgenic mouse model (hCD27-Tg) to explore the therapeutic potential of targeting CD27. In this study, we examined the effect of modifying the constant regions of the 1F5 mAb on its ability to enhance antigen specific T cell responses. With the original 1F5 hIgG1 as template, a panel of 1F5 variants was made including 1F5 mIgG1, 1F5 mIgG2a, 1F5 mIgG1 D265A and 1F5 hIgG1 N297S using molecular cloning techniques. All of the variants retained equal binding to hCD27 as shown by ELISA and flow cytometry studies. In addition, Biacore analysis confirmed the expected pattern of binding to human and mouse FcαRs. Co-injection of 1F5 or its variants with ovalbumin enhanced antigen-specific CD8 T cell response to different extents, as detected by SIINFEKL-specific IFNα-ELISPOT and ICS. The 1F5 mIgG1 induced the highest number of IFNα-producing CD8 + cells, whereas 1F5 mIgG2a was very weak at enhancing the CD8 T cell response. The hIgG1 version of 1F5 was intermediate in activity. Introduction of the D265A mutation that disrupts FcαRs binding into the mIgG1 eliminated the co-stimulatory function of 1F5. Similarly, the 1F5 hIgG1 N297S also showed reduced activity compared to the original 1F5 hIgG1. The isotype-specific effects on our anti-hCD27 mAb are surprisingly consistent with the findings described for the agonistic anti-mCD40 mAbs, and imply that engagement of the inhibitory Fcα receptors (FcαRIIb) is driving the co-stimulatory activity in this model. Interestingly, the 1F5 hIgG1 triggered a significant T cell response, despite the lack of FcαRIIb binding by Biacore analysis. The effect of these variants on anti-tumor activity in hCD27 transgenic mice is currently being investigated. The 1F5 hIgG1 mAb (CDX-1127) is currently undergoing clinical evaluation in a phase 1 trial of patients with advanced cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3519. doi:1538-7445.AM2012-3519