Harrington, Susan M.

Abstract Description Therapies targeting the PD-1/PD-L1 axis are among the leading treatments of cancer, but only a minority of patients benefit. Many therapies focus on preventing PD-L1 from binding to its receptor, PD-1, but do not address PD-L1’s ability to be recycled to the cell surface and any intrinsic functions it may possess. Our lab has developed an antibody (H1A) that binds to PD-L1 and prevents its association with a stabilizing protein, CMTM6, therefore targeting it for lysosomal degradation. Consequently, H1A limits any PD-L1 intrinsic function while also decreasing interactions with PD-1. Here, we use human-derived immune cells to test H1A’s effect on immune responses through flow cytometry, multiplex screening, and cytotoxicity assays. Additionally, we have developed a humanized PD-1/PD-L1 mouse model that allows us to test H1A’s effect on tumor growth inhibition in an immunocompetent host. Our results show that H1A specifically targets human myeloid cells, resulting in increased co-stimulation, antigen presentation, and cytokine secretion. H1A demonstrated enhanced T cell cytotoxicity in a whole PBMC culture. Finally, H1A resulted in greater tumor growth inhibition and survival in our in vivo tumor models. This work highlights the importance of targeting beyond PD-1/PD-L1 ligation and that myeloid cells are instrumental to enhancing the immune response against cancer. H1A is emerging as a new immunotherapy tool to target PD-L1 and CMTM6 in myeloid cells for treatment of cancers. Funding Sources NIH R01 92389022; NIH T32 AI170478; Mayo Clinic Graduate School of Biomedical Sciences Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Background Immune checkpoint therapies targeting programmed cell death protein 1 (PD-1) and programmed death ligand 1 (PD-L1) have been met with limited clinical responses in the treatment of patients of varying cancer types. Current US Food and Drug Administration (FDA)-approved therapies function to prevent PD-1 binding to PD-L1, thereby preventing T-cell suppression. However, these therapies fail to prevent PD-L1 intrinsic signaling and recycling, which can render these treatments ineffective. Moreover, most research primarily focuses on how these antibodies affect tumor cells and their interactions with T cells. Myeloid cells, which also express PD-L1 and influence T-cell responses, are integral to the efficacy of these therapies. However, strategies that can target these cells to boost antitumor responses have not been effective. Methods Our group has developed a new anti-PD-L1 antibody (H1A), which disrupts PD-L1 recycling and redirects it towards degradation. Due to the loss of available PD-L1 protein, H1A effectively prevents PD-L1 intrinsic signaling. Using in vivo humanized PD-1/PD-L1 mouse tumor models, we evaluated the therapeutic efficacy of H1A against current FDA-approved PD-L1 blocking antibodies. Using human-derived immune cell in vitro assays, we pinpoint the biological consequences of H1A-induced PD-L1 degradation in myeloid cells. Results H1A demonstrated improved tumor control and established immunological memory responses in vivo in the treatment of tumors with moderate immunogenicity that are less responsive to current immunotherapies. This was supported by enhanced activation of myeloid cells (major histocompatibility complex (MHC)-II and CD80) and frequencies of effector T-cell populations found intratumorally. Human myeloid cells treated with H1A were also observed to have increased activation (MHC-II and CD80) and cytokine secretion. Similar to our in vivo findings, human peripheral blood lymphocyte cultures demonstrated increased frequencies of effector T-cell populations and greater tumor cell killing. Conclusions This work highlights the importance of targeting PD-L1 recycling and intrinsic signaling, specifically in myeloid cells, to boost antitumor responses. Our studies suggest that H1A can provide a solution to the lack of responses seen with current therapeutics, while also revealing previously unknown intrinsic functions of PD-L1 in myeloid cells.

458 Background: Immune checkpoint inhibitors (ICI) targeting PD-1/PD-L1 axis has revolutionized the therapeutic landscape of patients with advanced and locally advanced renal cell carcinoma (RCC). However, complete response rates remain low and there is a need for novel therapeutic options for ICI-resistant and refractory RCC. Our group has generated a novel humanized anti-PD-L1 antibody, called H1A, that induces PD-L1 degradation preventing not only its interaction with PD-1 but also inhibiting pro-tumorigenic intracellular signaling. In this study, we compared the antitumor activity of H1A with FDA-approved ICI using patient-derived immune cells and humanized mouse tumor models. Methods: In vivo antitumor activity of H1A and atezolizumab (ATZ) was compared in three humanized PD-1/PD-L1 mouse tumor models with different immunogenicity (E0771:high, MC38:moderate, B16-F10:poor). Peripheral blood mononuclear cells (PBMC) were isolated from 14 RCC patients undergoing with nephrectomy. PBMC were treated with H1A, nivolumab (NIV), atezolizumab (ATZ) and pembrolizumab (PEM) to evaluate their efficacy in inducing tumor cell death in an ex-vivo cytotoxicity assay. Mass cytometry was employed to determine the impact of H1A and atezolizumab on the PBMC profile. Results: MC38 tumors showed moderate response to both H1A and ATZ which was abrogated by CD8 T cell depletion. In E0771 tumors, higher percentage of complete responders was observed with H1A compared to ATZ and E0771 rechallenge revealed memory antitumor immunity with complete rejection of tumors. Patient-derived PBMC treated with H1A showed superior tumor cell killing compared to NIV, PEM and ATZ (p<0.01). While H1A treatment of PBMC induced expansion of effector NK and CD8 T cells (GZMB+ T-bet+), ATZ treatment led to enrichment of Tregs (CD25+,Foxp3+) expressing inhibitory markers PD-1 and LAG-3. Conclusions: H1A demonstrated superior antitumor activity compared to FDA-approved ICI which lays the ground for clinical testing of H1A as a next-generation immune checkpoint inhibitor for the treatment of renal cell carcinoma.

Abstract Though anti-PD-1/PD-L1 immune checkpoint blockade therapy has demonstrated robust anti-tumor immunity in human patients with advanced cancers such as melanoma, only a fraction of patients achieved such durable anti-tumor responses. Among all the non-responders, some patients responded initially but developed resistance to the therapy in the later treatments. This reflects an urgent need to understand the role of PD-1 signals in CD8+ T cells at priming and at effector stages during tumor development for the benefit of immunotherapy. The controversy of rapid tumor progression in PD-1 conditional knockout in regulatory T cells and tumor regression in PD-1 targeted deletion in regulatory T cells indicated germline PD-1 knockout mice might not be sophisticated enough to study the role of PD-1 in CD8+ T cells during tumor development. Thus, we produced a CD8 T cell-specific PD-1 knockout mouse model (CD8 E8ICre-PD-1fl/fl, also called CD8-PD-1 cKO mice) in which PD-1 is deleted in CD8+ T cells in the peripheral tissues. When CD8-PD-1 cKO mice were challenged with B16-OVA mouse melanoma cells, we found a delayed tumor growth and prolonged survival of CD8-PD-1 cKO compared to control mice. However, after 20 days of tumor challenge, the delayed tumor growth rebound in CD8-PD-1 cKO mice. In an OVA antigen immunization model, we found increased effector cells and tissue resident cells in the spleen of CD8-PD-1 cKO mice compared with control mice on day 7 after immunization. Taken together, our results suggest that PD-1 may restrain the expansion of effector T cells that contribute to the early phase of tumor control, but the augmented effector T cell response may cause an attrition of memory T cell pool that will be needed for long term protection. R01CA256927

Abstract The intrinsic and acquired resistance to PD-1/PD-L1 immune checkpoint blockade is an important challenge for patients and clinicians because no reliable tool has been developed to predict individualized response to immunotherapy. In this study, we demonstrate the translational relevance of an ex vivo functional assay that measures the tumor cell killing ability of patient-derived CD8 T and NK cells (referred to as “cytotoxic lymphocytes,” or CLs) isolated from the peripheral blood of patients with renal cell carcinoma. Patient-derived PBMCs were isolated before and after nephrectomy from patients with renal cell carcinoma. We compared the efficacy of U.S. Food and Drug Administration (FDA)-approved PD-1/PD-L1 inhibitors (pembrolizumab, nivolumab, atezolizumab) and a newly developed PD-L1 inhibitor (H1A Ab) in eliciting cytotoxic function. CL activity was improved at 3 mo after radical nephrectomy compared with baseline, and it was associated with higher circulating levels of tumor-reactive effector CD8 T cells (CD11ahighCX3CR1+GZMB+). Treatment of PBMCs with FDA-approved PD-1/PD-L1 inhibitors enhanced tumor cell killing activity of CLs, but a differential response was observed at the individual-patient level. H1A demonstrated superior efficacy in promoting CL activity compared with FDA-approved PD-1/PD-L1 inhibitors. PBMC immunophenotyping by mass cytometry revealed enrichment of effector CD8 T and NK cells in H1A-treated PBMCs and immunosuppressive regulatory T cells in atezolizumab-treated samples. Our study lays the ground for future investigation of the therapeutic value of H1A as a next-generation immune checkpoint inhibitor and the potential of measuring CTL activity in PBMCs as a tool to predict individual response to immune checkpoint inhibitors in patients with advanced renal cell carcinoma.

Abstract Seven different anti–PD-1 and PD-L1 mAbs are now widely used in the United States to treat a variety of cancer types, but no clinical trials have compared them directly. Furthermore, because many of these Abs do not cross-react between mouse and human proteins, no preclinical models exist in which to consider these types of questions. Thus, we produced humanized PD-1 and PD-L1 mice in which the extracellular domains of both mouse PD-1 and PD-L1 were replaced with the corresponding human sequences. Using this new model, we sought to compare the strength of the immune response generated by Food and Drug Administration–approved Abs. To do this, we performed an in vivo T cell priming assay in which anti–PD-1/L1 therapies were given at the time of T cell priming against surrogate tumor Ag (OVA), followed by subsequent B16-OVA tumor challenge. Surprisingly, both control and Ab-treated mice formed an equally robust OVA-specific T cell response at the time of priming. Despite this, anti–PD-1/L1-treated mice exhibited significantly better tumor rejection versus controls, with avelumab generating the best protection. To determine what could be mediating this, we identified the increased production of CX3CR1+PD-1+CD8+ cytotoxic T cells in the avelumab-treated mice, the same phenotype of effector T cells known to increase in clinical responders to PD-1/L1 therapy. Thus, our model permits the direct comparison of Food and Drug Administration–approved anti–PD-1/L1 mAbs and further correlates successful tumor rejection with the level of CX3CR1+PD-1+CD8 + T cells, making this model a critical tool for optimizing and better utilizing anti–PD-1/L1 therapeutics.

Abstract Though anti-PD-1/L1 antibody therapies have generated unprecedented responses in a limited number of patients, the biological and immunological parameters that define their effective use are still largely unknown. The absence of appropriate pre-clinical models has limited our ability to compare the mechanistic differences between the approved PD-1/L1 antibodies in vivo and to determine the context in which each might be most effective. To address this critical need, we have produced humanized PD-1 and PD-L1 mice in which the extracellular domains (antibody binding sites) of mouse PD-1 and PD-L1 are replaced with the sequences of human PD-1 and PD-L1. The PD-1 and PD-L1 in the resulting “HuPD” mice can be recognized by FDA-approved antibodies, while the intracellular domains are kept intact for normal downstream signals. Following characterization of the new mouse model, in our first in vivo treatment studies, HuPD mice were immunized with OVA/Poly I:C in the context of FDA-approved PD-1/L1 antibodies and antigen-specific T cell production was quantified. Nivolumab, Pembrolizumab, and Atezolizumab all increased effector CD8+T cell production against the OVA antigen as expected given their immune stimulating function. Surprisingly, OVA-tetramer staining was unable to detect antigen-specific CD8+T cells in the spleens of immunized mice treated with Avelumab. This may reflect the unique ability of Avelumab to induce ADCC, and indicates that our model is capable of distinguishing key differences among the antibodies in vivo. Ongoing studies will identify how these differences might be exploited to render the most effective results for patients in a variety of tumor types and immune environments.

Abstract The immune response to anti-PD-1 therapy in cancer patients is not completely defined due to lack of reliable biomarkers that can determine T cell function. Bim, a member of the Bcl-2 family, was recently identified as a downstream signaling molecule in the PD-1 pathway. Within CD8 T cells, Bim appears to have a dual function, and can be pro-apoptotic or promote effector activity. Our clinical data has shown that high levels of Bim in CD8 T cells are predictive of response to anti-PD-1 therapy in metastatic melanoma whereas in colorectal cancer, a low level of Bim is associated with treatment response. Thus, we sought to determine what molecular partners might influence the role of Bim. Using co-immunoprecipitation, high resolution microscopy and FRET we identified ANT2 (Adenine Nucleotide Translocase 2), an inner membrane protein of mitochondria, as a protein that interacts with Bim. Accordingly, we found CD8 T cells with low and high mitochondrial potential demonstrated different ANT2/Bim ratios, and a low ANT2/Bim ratio was present in effector T cells with low mitochondrial potential but high cytotoxic activity. Given the pro-survival function of ANT2, our results indicate that ANT2 may balance the pro-apoptotic and effector roles of Bim in mitochondria in order to preserve effector T cells before contraction. Understanding the molecular mechanism of Bim and ANT2 interactions in CD8 T cells may provide a novel marker to predict clinical response to anti-PD-1 therapy.

Abstract We utilized CD11a, an integrin up-regulated on effector and memory CD8+ T cells, as a biomarker to identify, measure, and monitor endogenous tumor-reactive cytotoxic lymphocytes (CTLs) in two mouse tumor models and in melanoma patients. CD11ahigh CD8+ T cells were detected in primary and metastatic tumor sites after tumor cell injection, as well as in spontaneous murine breast cancer tissues. In the peripheral blood of melanoma patients, tumor antigen-specific T cells were detected within the population of CD11ahigh CD8+ T cells that express high levels of PD-1. Infusion of lethally irradiated tumor cells failed to stimulate the expansion of CD11ahigh CD8+ T cells, suggesting that the access of tumor cells to target tissues is required to induce CD11ahigh CD8+ T cell responses. Phenotypic analysis revealed that the CD11ahigh CD8+ T cells detected in our studies were proliferating (Ki67+) activated T cells (CD62L-, CD69+). Although CD11ahigh CD8+ T cells were differentiated effector cells with CTL effector functions, they were typically unable to control tumor growth in our mouse models, possibly due to high expression of PD-1. Our results demonstrate that CD11a can be used as a biomarker to identify and track endogenous tumor-reactive CTLs.

Abstract Seven different anti-PD-1 and PD-L1 monoclonal antibodies are now widely used in the US to treat a variety of cancer types, but there have been no clinical trials comparing them directly. Furthermore, because many of these antibodies do not cross react between mouse and human proteins, no preclinical models exist in which to consider these types of questions. Thus, we produced humanized PD-1 and PD-L1 mice in which the extracellular domains of both mouse PD-1 and PD-L1 were replaced with the corresponding human sequences. Using this new model, we sought to compare the strength of the immune response generated by FDA-approved antibodies. To do this, we performed an in vivo T cell priming assay in which anti-PD-1/L1 therapies were given at the time of T cell priming against surrogate tumor antigen (OVA), followed by subsequent B16-OVA tumor challenge. We found that anti-PD-1/L1-treated mice exhibited significantly better tumor rejection than controls, although both the control and antibody-treated mice generated comparable numbers of OVA-specific T cells at the time of priming. To determine what could mediate this strong antitumor immune response, we identified the increased production of CX3CR1+PD-1+CD8+ cytotoxic T cells in the anti-PD-1/L1-treated mice, the same subset of effector T cells known to increase in clinical responders to PD-1/L1 therapy. Thus, our model permits the direct comparison of FDA-approved anti-PD-1/L1 monoclonal antibodies and further correlates successful tumor rejection with the level of CX3CR1+PD-1+CD8 + T cells, making this model a critical tool for future studies to optimize and better utilize anti-PD-1/L1 therapeutics.