Costimulatory Receptor Research Articles

A costimulatory chimeric antigen receptor targeting TROP2 enhances the cytotoxicity of NK cells expressing a T cell receptor reactive to human papillomavirus type 16 E7

Immune cells modified to express a tumor-reactive T cell receptor (TCR) have shown limited efficacy as stand-alone therapy against solid tumors. Genital and oropharyngeal carcinomas induced by human papillomavirus (HPV) type 16 express constitutively its E6 and E7 oncoproteins, which makes them convenient targets for adoptive cell immunotherapy. However, viral antigen presentation by tumor cells is low and limits the anti-tumor efficacy of CD8+ T cells. To enhance the functionality of immune effector cells, we have devised a strategy combining a costimulatory chimeric antigen receptor (CAR) with a TCR. We used a clinically tested TCR specific to E7 (E7-TCR) of HPV16 and a newly constructed CAR targeting the trophoblast cell surface antigen 2 (TROP2), which carried the intracellular costimulatory domains CD28 and 4-1BB, but was devoid of the CD3ζ domain. Flow cytometry analyses showed a notable upregulation of activation markers and of cytolytic molecule release by NK-92 cells genetically engineered to express CD3, CD8 and both E7-TCR and TROP2-CAR, after co-incubation with HPV16+ cervical cancer cells. Furthermore, the E7-TCR/TROP2-CAR NK-92 cells demonstrated enhanced antigen-specific activation and augmented cytotoxicity against tumor cells compared with NK-92 cells expressing the E7-TCR alone. A costimulatory TROP2-CAR can synergistically cooperate with the E7-TCR in NK cells thereby enhancing their signaling strength and antigen-specific cytotoxicity. This approach might improve the outcome of adoptive cell immunotherapies for HPV16+ cancer patients that are currently under investigation.

Beyond CTLA-4 and PD-1 Inhibition: Novel Immune Checkpoint Molecules for Melanoma Treatment.

More than ten years after the approval of ipilimumab, immune checkpoint inhibitors (ICIs) against PD-1 and CTLA-4 have been established as the most effective treatment for locally advanced or metastatic melanoma, achieving durable responses either as monotherapies or in combinatorial regimens. However, a considerable proportion of patients do not respond or experience early relapse, due to multiple parameters that contribute to melanoma resistance. The expression of other immune checkpoints beyond the PD-1 and CTLA-4 molecules remains a major mechanism of immune evasion. The recent approval of anti-LAG-3 ICI, relatlimab, in combination with nivolumab for metastatic disease, has capitalized on the extensive research in the field and has highlighted the potential for further improvement of melanoma prognosis by synergistically blocking additional immune targets with new ICI-doublets, antibody-drug conjugates, or other novel modalities. Herein, we provide a comprehensive overview of presently published immune checkpoint molecules, including LAG-3, TIGIT, TIM-3, VISTA, IDO1/IDO2/TDO, CD27/CD70, CD39/73, HVEM/BTLA/CD160 and B7-H3. Beginning from their immunomodulatory properties as co-inhibitory or co-stimulatory receptors, we present all therapeutic modalities targeting these molecules that have been tested in melanoma treatment either in preclinical or clinical settings. Better understanding of the checkpoint-mediated crosstalk between melanoma and immune effector cells is essential for generating more effective strategies with augmented immune response.

TB granuloma: CD30 co-stimulation for CD4+ T cell co-operation.

Tuberculosis granuloma T cells express an array of mediators including the CD30 co-stimulatory receptor and its ligand, CD153. CD4 T effector cells require signals through CD30, potentially provided co-operatively by other T cells, to completely differentiate and protect against disease (Foreman et al., 2023. J. Exp. Med.https://doi.org/10.1084/jem.20222090).

Cis-B7:CD28 interactions at invaginated synaptic membranes provide CD28 co-stimulation and promote CD8+ T cell function and anti-tumor immunity

B7 ligands (CD80 and CD86), expressed by professional antigen-presenting cells (APCs), activate the main co-stimulatory receptor CD28 on Tcells in trans. However, in peripheral tissues, APCs expressing B7 ligands are relatively scarce. This raises the questions of whether and how CD28 co-stimulation occurs in peripheral tissues. Here, we report that CD8+ Tcells displayed B7 ligands that interacted with CD28 in cis at membrane invaginations of the immunological synapse as a result of membrane remodeling driven by phosphoinositide-3-kinase (PI3K) and sorting-nexin-9 (SNX9). cis-B7:CD28 interactions triggered CD28 signaling through protein kinase C theta (PKCθ) and promoted CD8+ Tcell survival, migration, and cytokine production. In mouse tumor models, loss of Tcell-intrinsic cis-B7:CD28 interactions decreased intratumoral Tcells and accelerated tumor growth. Thus, B7 ligands on CD8+ Tcells can evoke cell-autonomous CD28 co-stimulation in cis in peripheral tissues, suggesting cis-signaling as a general mechanism for boosting Tcell functionality.

B7-H3 specific CAR-T cells exhibit potent activity against prostate cancer

B7-H3 is an attractive target for immunotherapy because of its high expression across multiple solid tumors, including prostate cancer, and restricted expression in normal tissues. Among various types of tumor immunotherapy, chimeric antigen receptor T (CAR-T) cell therapy has shown remarkable success in hematological tumors. However, the potency of CAR-T cell therapy in solid tumors is still limited. Here, we examined the expression of B7-H3 in prostate cancer tissues and cells and developed a second-generation CAR that specifically targets B7-H3 and CD28 as costimulatory receptor to explore its tumoricidal potential against prostate cancer in vitro and in vivo. The high expression of B7-H3 was detected on both the surface of PC3, DU145 and LNCaP cells and prostate cancer tissues. B7-H3 CAR-T cells efficiently controlled the growth of prostate cancer in an antigen-dependent manner in vitro and in vivo. Moreover, tumor cells could induce the proliferation of CAR-T cells and the release of high levels of cytokines of IFN-γ and TNF-α in vitro. Results demonstrated that B7-H3 is a potential target for prostate cancer therapy that supports the clinical development of B7-H3 specific CAR-T cells for prostate cancer.

Mechanisms of action of monoclonal antibodies in oncology integrated in IMGT/mAb-DB.

Cancer cells activate different immune checkpoint (IC) pathways in order to evade immunosurveillance. Immunotherapies involving ICs either block or stimulate these pathways and enhance the efficiency of the immune system to recognize and attack cancer cells. In this way, the development of monoclonal antibodies (mAbs) targeting ICs has significant success in cancer treatment. Recently, a systematic description of the mechanisms of action (MOA) of the mAbs has been introduced in IMGT/mAb-DB, the IMGT® database dedicated to mAbs for therapeutic applications. The characterization of these antibodies provides a comprehensive understanding of how mAbs work in cancer. In depth biocuration taking advantage of the abundant literature data as well as amino acid sequence analyses from mAbs managed in IMGT/2Dstructure-DB, the IMGT® protein database, allowed to define a standardized and consistent description of the MOA of mAbs targeting immune checkpoints in cancer therapy. A fine description and a standardized graphical representation of the MOA of selected mAbs are integrated within IMGT/mAb-DB highlighting two main mechanisms in cancer immunotherapy, either Blocking or Agonist. In both cases, the mAbs enhance cytotoxic T lymphocyte (CTL)-mediated anti-tumor immune response (Immunostimulant effect) against tumor cells. On the one hand, mAbs targeting co-inhibitory receptors may have a functional Fc region to increase anti-tumor activity by effector properties that deplete Treg cells (Fc-effector function effect) or may have limited FcγR binding to prevent Teff cells depletion and reduce adverse events. On the other hand, agonist mAbs targeting co-stimulatory receptors may bind to FcγRs, resulting in antibody crosslinking (FcγR crosslinking effect) and substantial agonism. In IMGT/mAb-DB, mAbs for cancer therapy are characterized by their chains, domains and sequence and by several therapeutic metadata, including their MOA. MOAs were recently included as a search criterion to query the database. IMGT® is continuing standardized work to describe the MOA of mAbs targeting additional immune checkpoints and novel molecules in cancer therapy, as well as expanding this study to other clinical domains.

4-1BB costimulation following mRNA vaccination improves CD8 T cell responses

Abstract mRNA vaccines have received full FDA approval to prevent COVID-19, and are being explored for multiple infectious diseases as well as cancer. While they have shown high efficacy at preventing severe disease caused by SARS-CoV-2, waning immunity has motivated the development of improved mRNA vaccine regimens. 4-1BB (also known as CD137) is a costimulatory receptor that has been shown to be important for T cell responses following viral infections and cancer. We asked whether triggering 4-1BB costimulation with agonistic antibodies could improve immune responses elicited by mRNA vaccines in C57BL/6 mice. Here, we show that triggering 4-1BB costimulation at the time of mRNA vaccination impairs CD8 T cell responses, whereas triggering 4-1BB costimulation after day 4 of mRNA vaccination improves CD8 T cell immune responses. These data demonstrate time-dependent effects of 4-1BB costimulation on cellular responses elicited by mRNA vaccines, and suggest that delayed provision of 4-1BB costimulation could offer immunologic benefits following mRNA vaccination. This work was supported by a DP2 New Innovator Award to P.P.M. This work was supported by a DP2 New Innovator Award to P.P.M.

Discovery of TIGIT inhibitors by phage display

Abstract TIGIT is an emerging immune checkpoint receptor expressed higher than PD-1 in some tumors especially anti-PD1 resistant tumors. By competing with the co-stimulatory receptor CD226/DNAM-1 for binding to CD155/PVR, TIGIT transduces inhibitory signals and suppresses the immune response. Therefore, we sought to discover TIGIT inhibitors for improving tumor immunotherapy. Through directional cloning, bacterial transformation and phage amplification techniques, we generated a novel genetically encoded, phage-displayed bicyclic peptide library comprised of more than a million peptide sequences. Depending on the two combinations of different disulfide bonds, each peptide backbone is predicted to be folded into either endo-bicyclic or exo-bicyclic structure. We conducted three rounds of phage display panning against recombinant TIGIT IgV domain that enriched high-affinity peptides and identified promising TIGIT binding peptides. Phage ELISA showed binding of phage-presented peptides to recombinant TIGIT IgV and competitive ELISA evaluated the ability of the peptides to disrupt interactions between TIGIT and PVR. This work validated the efficiency of the phage-displayed bicyclic peptide library for discovery of ligands targeting protein receptors and led to identification of novel TIGIT inhibitors. AAI Careers in Immunology Fellowship

Abstract PR03: ADCLEC.syn1 CAR T cells detect combinatorial target signatures to safely prevent antigen-low AML escape

Abstract CAR therapy is efficacious in relapsed/refractory CD19-positive malignancies including B-ALL. In AML, conventional 2nd-generation CAR therapies are being evaluated clinically. Some of the potential obstacles are antigen-low leukemia escape and on-target toxicities due to widespread CAR target expression in normal hematopoietic cells. We hypothesized that novel targets and fine-tuned combinatorial CAR approaches may enable distinctive target elimination based on differential antigen co-expression patterns in AML vs normal cells, thereby maintaining anti-leukemic efficacy while limiting toxicity on normal cells. Here, we present our target phenotypic profiling of primary AML (n>30 r/r AML patients) and normal hematopoietic cells, and link it with CAR efficacy and changes in normal hematopoiesis. Using spectral flow cytometry on bone marrow and peripheral blood from AML patients and healthy controls, we quantified differential target co-expression of several AML targets, including CD33, CD123, CLEC12A and the novel target ADGRE2. We estimated the numbers of surface molecules per cell and created two-dimensional maps of combinatorial target densities in AML vs normal. In this analysis, the combination of ADGRE2+CLEC12A offered the best therapeutic window. We developed a combinatorial CAR+CCR platform, ADCLEC.syn1, consisting of an ADGRE2-targeting 28z1XX-CAR and a CLEC12A-targeting chimeric costimulatory receptor (CCR) providing additional 4-1BB costimulation. This CAR+CCR configuration triggers killing of cells with high CAR target density alone, whereas cells with low CAR target density are only killed if the CCR target is co-expressed (IF-BETTER gate). Both in vitro and in vivo, we used MOLM13 AML cell line variants matching a variety of different ADGRE2 and CLEC12A target density combinations found in AML or normal hematopoietic cells. The observed target density thresholds for ADCLEC.syn1-mediated cell lysis demonstrated potential for sparing vital normal hematopoietic cells. In addition, we evaluated ADCLEC.syn1 in several molecularly distinct AML PDX models with clinically representative target phenotypes, achieving long-term AML remissions in all studied PDX models. In a humanized mouse model co-engrafted with MOLM13 AML cells, ADCLEC.syn1 T cells rapidly induced complete and durable AML remissions in all mice, with minimal toxicity of normal hematopoietic cells compared to control CARs. Overall, we profiled antigen densities of several CAR target combinations in AML and normal hematopoietic cells, which informed the development of the ADCLEC.syn1 platform. Pre-clinical in vivo models based on AML with clinically representative target densities demonstrated high anti-leukemic efficacy of ADCLEC.syn1 and established killing thresholds with potential for sparing normal hematopoietic cells. A first-in-human phase 1 clinical trial evaluating ADCLEC.syn1 T cells is anticipated to be initiated at MSKCC in 2023. Citation Format: Sascha Haubner, Jorge Mansilla-Soto, Sarah Nataraj, Friederike Kogel, Qing Chang, Elisa De Stanchina, Kathryn Fraser, Jae H. Park, Xiuyan Wang, Isabelle Rivière, Michel Sadelain. ADCLEC.syn1 CAR T cells detect combinatorial target signatures to safely prevent antigen-low AML escape [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr PR03.

Delivering co-stimulatory tumor necrosis factor receptor agonism for cancer immunotherapy: past, current and future perspectives.

The tumor necrosis factor superfamily (TNFSF) and their receptors (TNFRSF) are important regulators of the immune system, mediating proliferation, survival, differentiation, and function of immune cells. As a result, their targeting for immunotherapy is attractive, although to date, under-exploited. In this review we discuss the importance of co-stimulatory members of the TNFRSF in optimal immune response generation, the rationale behind targeting these receptors for immunotherapy, the success of targeting them in pre-clinical studies and the challenges in translating this success into the clinic. The efficacy and limitations of the currently available agents are discussed alongside the development of next generation immunostimulatory agents designed to overcome current issues, and capitalize on this receptor class to deliver potent, durable and safe drugs for patients.

Expression and function of CD51 on CD8 T cells as an immunomodulatory target

T cell responses are regulated by co-stimulatory and inhibitory receptors along with T cell receptor- and cytokine-mediated signals. CD51 is a transmembrane glycoprotein of the integrin family that plays a role in cell adhesion, migration, tumorigenesis, and other cellular functions. In this study, we aimed to investigate the expression and function of CD51 on CD8 T cells. Upon in vitro T cell activation, CD51 expression was delayed but subsequently was upregulated in CD8 T cells upon cell division. Furthermore, CD51 was highly expressed in exhausted CD8 T cells in chronic LCMV infection, B16F10 melanoma, and CT26 colon carcinoma, and its expression level increased as cells became more differentiated. Using CRISPR-mediated knockdown, we found that the absence of CD51 led to a lower number of virus-specific CD8 T cells upon chronic lymphocytic choriomeningitis virus (LCMV) infection, although their granzyme B expression and cytokine production were maintained. Blocking CD51 also inhibited the in vitro proliferation of CD8 T cells. These results suggest that CD51 plays an important role in the early expansion of CD8 T cells and may have potential as an immunomodulatory target.

Abstract CT253: Phase 1/2 study of the safety, pharmacokinetics, and preliminary clinical activity of BT7480 in patients with nectin-4 associated advanced malignancies

Abstract Background: Bicycles are a novel class of synthetic molecules formed by using a central scaffold to constrain short linear peptides into a stabilized bi-cyclic structure, which can then be readily conjugated to other molecules. BT7480 is a trimeric first-in-class Bicycle tumor-targeted immune cell agonist (Bicycle TICA) comprised of two unique bicyclic peptides: one that binds to Nectin-4 and two identical Bicycles that bind and agonize CD137. The resulting multi-specific molecule stimulates an immune response in the presence of both Nectin-4-expressing tumor cells as well as CD137-expressing immune cells. Nectin-4, a cell adhesion molecule overexpressed on the surface of tumor cells and CD137, a costimulatory receptor expressed on immune cells, are co-expressed in a variety of solid tumors including lung, head and neck, breast, ovarian, and bladder. Co-ligation of Nectin-4 and CD137 by BT7480 is hypothesized to induce oligomerization and activation of CD137, resulting in a tumor-localized costimulatory signal. A favorable preclinical profile supported the initiation of this first-in-human study to investigate the safety and efficacy of BT7480 in patients with solid tumors associated with Nectin-4 expression (NCT05163041). Methods: This is a phase 1/2, open-label, multicenter study to assess safety, clinical activity, and pharmacokinetics (PK) and pharmacodynamics (PD) of BT7480. Patients must have an advanced malignancy associated with Nectin-4 expression that is not eligible for standard therapy. Additional key criteria include ≥18 years of age, ECOG of 0 or 1, adequate organ function, and no prior therapy with a CD137-targeted agent. Patients are treated at one of the escalating doses (3+3 design) by weekly IV infusion in a 4-week cycle. Primary objectives are to assess safety (phase 1) and clinical activity per RECIST v1.1 (phase 2). Secondary objectives include assessment of PK parameters, measurement of anti-drug antibodies, and evaluation of CD137 target engagement in blood. Key exploratory objectives include evaluating pharmacogenomics, ctDNA, and biomarkers in blood and tumors associated with pharmacological activity, including signals of immune activations and target expression. PK and PD analyses will be performed to support dose escalation decisions and to further the understanding of safety and clinical activity signals as a result of treatment with BT7480. This study is actively recruiting. Citation Format: Thomas R. Evans, Afshin Dowlati, Juanita S. Lopez, Mohammed M. Milhem, Jordi Rodón Ahnert, Alexander Spira, Richard D. Carvajal, Matthew Zibelman, Sebastien J. Hazard, Amy Dickson, Lei Xu, Heather Cohen, Justin Bader, Kristen Hurov, Rajiv Sharma, Dominic Smethurst, Kyriakos P. Papadopoulos. Phase 1/2 study of the safety, pharmacokinetics, and preliminary clinical activity of BT7480 in patients with nectin-4 associated advanced malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT253.

Abstract CT154: Combination of cinrebafusp alfa with ramucirumab and paclitaxel is well tolerated and elicits encouraging clinical activity in patients with HER2-positive gastric/gastroesophageal junction (GEJ) adenocarcinoma

Abstract Introduction: Cinrebafusp alfa is a first-in-class bispecific antibody-Anticalin® fusion protein that targets HER2 and the costimulatory receptor 4-1BB, leading to enhanced activation of T cells in the tumor, while avoiding liver toxicity. In a Phase 1 monotherapy study, cinrebafusp alfa was well tolerated and showed single agent activity in patients with HER2-positive malignancies. Based on pharmacokinetics (PK), pharmacodynamics and clinical efficacy data, a loading dose of 18mg/kg Q2W in cycle 1 followed by 8mg/kg Q2W in subsequent cycles as maintenance dose was chosen for the Phase 2 study. Methods: This Phase 2 study enrolled patients with metastatic gastric/gastroesophageal junction cancer and confirmed HER2-high status (IHC 3+ or IHC 2+ with HER2/neu gene amplification) who had received one or two prior lines of treatment. Patients received cinrebafusp alfa in combination with ramucirumab and paclitaxel. Primary endpoint was objective response rate (ORR), and key secondary endpoints included safety profile, PK, and immunogenicity. Summary of data: 5 patients were enrolled before enrollment ceased for reasons unrelated to safety or efficacy profile. As of the cut-off date (19-Dec-2022), 5 out of 5 patients achieved a partial response (PR) as best overall response with tumor lesion shrinkage ranging between 35% and 66%. Two of the PRs have been confirmed, one PR is unconfirmed, and 2 patients with currently unconfirmed PRs are continuing to receive treatment. In total, 2 of 5 patients discontinued treatment due to disease progression after 140 and 113 days (patient discontinued cinrebafusp alfa after 42 days but continued on ramucirumab and paclitaxel); 3 patients remain on treatment. Median duration of response was 3.8 months at time of data cut-off. The most frequent treatment emergent adverse events (TEAEs) included Grade 1/2 fatigue (4 patients) and Grade 1-3 diarrhea (4 patients). Infusion related reactions (Grade 2/3) were seen in 1 patient, leading eventually to discontinuation of cinrebafusp alfa. With regards to prior treatment history, all patients received trastuzumab and chemotherapy. 2 patients also received trastuzumab deruxtecan while 4 patients previously received anti-PD1 therapy. Conclusions: The combination of cinrebafusp alfa with ramucirumab and paclitaxel was safe and tolerated in the 5 patients treated. Preliminary activity of cinrebafusp alfa in combination with ramucirumab and paclitaxel demonstrated a high response rate indicating that the combination can elicit clinical responses in patients who have progressed on trastuzumab deruxtecan or checkpoint inhibitor regimens. Citation Format: Geoffrey Ku, Jeeyun Lee, Kayti Aviano, Tim Demuth, Laura-Carolin Hasenkamp, Shane A. Olwill. Combination of cinrebafusp alfa with ramucirumab and paclitaxel is well tolerated and elicits encouraging clinical activity in patients with HER2-positive gastric/gastroesophageal junction (GEJ) adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT154.

Xenorecognition and costimulation of porcine endothelium-derived extracellular vesicles in initiating human porcine-specific T cell immune responses

Porcine vascular endothelial cells (PECs) form a mechanistic centerpiece of xenograft rejection. Here, we determined that resting PECs release swine leukocyte antigen class I (SLA-I) but not swine leukocyte antigen class-II DR (SLA-DR) expressing extracellular vesicles (EVs) and investigated whether these EVs proficiently initiate xenoreactive T cell responses via direct xenorecognition and costimulation. Human T cells acquired SLA-I+ EVs with or without direct contact to PECs, and these EVs colocalized with T cell receptors. Although interferon gamma-activated PECs released SLA-DR+ EVs, the binding of SLA-DR+ EVs to T cells was sparse. Human T cells demonstrated low levels of proliferation without direct contact to PECs, but marked T cell proliferation was induced following exposure to EVs. EV-induced proliferation proceeded independent of monocytes/macrophages, suggesting that EVs delivered both a T cell receptor signal and costimulation. Costimulation blockade targeting B7, CD40L, or CD11a significantly reduced T cell proliferation to PEC-derived EVs. These findings indicate that endothelial-derived EVs can directly initiate T cell-mediated immune responses, and suggest that inhibiting the release of SLA-I EVs from organ xenografts has the potential to modify the xenograft rejection. We propose a secondary-direct pathway for T cell activation via xenoantigen recognition/costimulation by endothelial-derived EVs.

Abstract 4057: T cells co-expressing a highly potent NY-ESO-1-specific TCR and a chimeric PD1-41BB co-stimulatory switch receptor show a favorable polyfunctional profile for the treatment of solid tumors

Abstract T cell receptor (TCR)-based immunotherapies have shown great potential for the safe and efficacious treatment of patients suffering from various solid malignancies. However, novel strategies are needed to overcome the immunosuppressive tumor microenvironment (TME) and to enhance the efficacy of TCR-T cell (TCR-T) therapies in solid tumors. NY-ESO-1 is a cancer/testis antigen that is highly expressed in various solid tumor indications while its expression in healthy tissues is restricted to male germ cells. Our HLA-A2-restricted, NY-ESO-1-specific TCR used here was selected from a non-tolerized T cell repertoire of a healthy donor using Medigene’s proprietary Allo-HLA TCR priming technology and high-throughput TCR isolation and characterization processes. To prevent inhibition of TCR-T cell function via the PD1-PDL1 axis within the TME, the NY-ESO-1-TCR was co-expressed with a chimeric PD1-41BB co-stimulatory switch receptor, consisting of the extracellular domain of PD1 and the intracellular domain of the 41BB co-stimulatory receptor. Thereby, an inhibitory signal from the PD1-PD-L1 axis is turned into a co-stimulatory one, which results in a dual mechanistic benefit, and superior TCR-T cells with respect to tumor cell killing, cell proliferation and poly-cytokine secretion. Co-expression of PD1-41BB on NY-ESO-1 TCR-T cells led to a 3-fold increase in IFN-γ release and a higher proliferation index (~1.5 fold) (after antigen encounter. Under conditions of repeated antigen exposure in vitro using HLA-A2/NY-ESO-1/PDL1-positive tumor spheroids, TCR-T cells co-expressing PD1-41BB showed enhanced functional activities compared to TCR-T cells carrying the TCR alone. In vitro single-cell secretome analyses demonstrated up to a 5-fold increase in polyfunctional T cells and a higher polyfunctional strength index (PSI) for NY-ESO-1 TCR-T cells expressing PD1-41BB compared to “naked” NY-ESO-1 TCR-T cells lacking PD1-41BB. Typical Th1 cytokines/proteins like IFN-γ, TNF-α and Granzyme B mainly contributed to the superior PSI of NY-ESO-1-TCR-T cells co-expressing PD1-41BB. Importantly, our data indicate that transgenic PD1-41BB has the capacity to provide a co-stimulatory signal despite the presence of endogenous PD1 which is upregulated in stimulated “naked“ NY-ESO-1 TCR-T cells. In summary, we find that co-expression of a chimeric PD1-41BB co-stimulatory switch receptor significantly enhances in vitro NY-ESO-1 TCR-T cell proliferation, functionality and anti-tumor activity. Analyses of single-cell T cell cytokine polyfunctionality helped us to unravel the mode of action of NY-ESO-1 TCR-T cells co-expressing PD1-41BB and point to their complex display of effector, stimulatory and chemoattractant cytokines as a potential driver for superior recognition of tumor cells. Citation Format: Andrea Coluccio, Stefanie Tippmer, Petra Prinz, Maja Buerdek, Kathrin Mutze, Barbara Loesch, Kathrin Davari, Giulia Longinotti, Dolores J. Schendel. T cells co-expressing a highly potent NY-ESO-1-specific TCR and a chimeric PD1-41BB co-stimulatory switch receptor show a favorable polyfunctional profile for the treatment of solid tumors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4057.

Abstract 3274: Novel cell-based bioassays for mono- and multi-specific antibodies targeting PD-1/PD-L1 for combination immunotherapy

Abstract Programmed cell death protein 1 (PD-1) is an immune checkpoint (IC) receptor that negatively regulates T cell function. Blocking the interaction of PD-1 with its ligands PD-L1 or PD-L2 unleashes the immune system and enhances anti-tumor responses. Immunotherapy using PD-1 or PD-L1 blocking antibodies has led to a paradigm shift in cancer drug discovery, due to its durable effect against a wide variety of cancers. However, despite its tremendous clinical success in cancer treatment, a significant fraction of patients and tumor types remain unresponsive to this therapy. This has led to the development of combination therapies linking PD-1 checkpoint inhibitors with inhibitors of several other IC receptors. A major challenge in immunotherapy drug development is the lack of quantitative and reproducible functional assays. Existing methods rely heavily on primary immune cells are highly variable and labor-intensive. Here, we report the development of a suite of cell line-based reporter bioassays for monoclonal antibodies targeting human PD-1/PD-L1, or bispecific antibodies targeting PD-1/PD-L1 and a co-stimulatory receptor (e.g., 4-1BB, OX40, ICOS) or an immune inhibitory receptor (e.g., CTLA-4, LAG-3, TIGIT). A mouse PD-1/PD-L1 assay and a human PD-1/PD-L2 assay are also developed and can be used in parallel with human PD-1/PD-L1 assay to enable preclinical studies and assess assay specificity. Each of these assays consist of two engineered cell lines: a T effector cell line that express a luciferase reporter driven by specific promoter/response elements responding to the intracellular signals mediated by the T cell receptor (TCR), with modulation from PD-1 and the second immune receptor, and an engineered artificial antigen presenting cell (aAPC). Importantly, these combination bioassays demonstrate the synergetic effect of PD-1 blockade with blockade of a second IC inhibitor receptor or activation of a costimulatory receptor, thus reflecting the mechanisms of action for the therapeutic antibodies of interest. The bioassays are sensitive and quantitative, are prequalified according to ICH guidelines, and demonstrate the performance characteristics (specificity, precision, accuracy, and linearity) to be used as potency assays for product release and stability studies during immunotherapy drug development. Citation Format: Jun Wang, Julia Gilden, Denise Garvin, Pete Stecha, Jim Hartnett, Frank Fan, Mei Cong, Jamison Grailer. Novel cell-based bioassays for mono- and multi-specific antibodies targeting PD-1/PD-L1 for combination immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3274.

Modeling the interaction between donor-derived regulatory T cells and effector T cells early after allogeneic hematopoietic stem cell transplantation

While allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potential curative therapy against hematological malignancies, modulation of donor T cell alloreactivity is required to enhance the graft-versus-leukemia (GVL) effect and control graft-versus-host-disease (GVHD) after allo-HSCT. Donor-derived regulatory CD4+CD25+Foxp3+ T cells (Tregs) play a central role in establishing of immune tolerance after allo-HSCT. They could be a key target to be modulated for increasing the GVL effect and control of GVHD. We constructed an ordinary differential equation model incorporating bidirectional interactions between Tregs and effector CD4+ T cells (Teffs) as a mechanism for control of Treg cell concentration. The goal is to elucidate how the interaction between Tregs and Teffs is modulated in order to get insights into fine tuning of alloreactivity after allo-HSCT. The model was calibrated with respect to published Treg and Teff recovery data after allo-HSCT. The calibrated model exhibits perfect or near-perfect adaptation to stepwise perturbations between Treg and Teff interactions, as seen in Treg cell populations when patients with relapsed malignancy were treated with anti-CTLA-4 (cytotoxic T lymphocyte-associated antigen 4). In addition, the model predicts observed shifts of Tregs and Teffs concentrations after co-stimulatory receptor IL-2R or TNFR2 blockade with allo-HSCT. The present results suggest simultaneous blockades of co-stimulatory and co-inhibitory receptors as a potential treatment for enhancing the GVL effect after allo-HSCT without developing GVHD.

Abstract 2983: Tumor-specific CD28 costimulatory bispecific antibodies enhance T cell activation in solid tumors

Abstract T cells in the tumor micro-environment require TCR/MHC engagement and co-stimulatory receptor engagement to achieve optimal activation. Solid tumor cells lack expression of CD28 ligands, so we hypothesized that activation of CD28 signaling at the T cell/tumor cell interface could enhance anti-tumor activity. We developed a CD28 bispecific platform that allows for the rapid generation of tumor-associated antigen (TAA) x CD28 bispecific antibodies that conditionally provide CD28 costimulation only in the presence of TAA and TCR engagement. Here, we present results for a set of bispecific antibodies with broad applicability across a range of solid tumors, including CEACAM5 x CD28, Trop-2 x CD28, STEAP1 x CD28, and mesothelin x CD28. We developed optimized CD28 scFv and Fabs that cover a range of affinities and are only agonistic in the context of TCR engagement. A matrix of bispecifics pairing these CD28 binders with CEACAM5, Trop-2, STEAP1, and mesothelin antibodies of varying affinities and epitopes were generated using Xencor’s XmAb® bispecific antibody platform. Activities of these bispecifics were assessed in vitro by measuring T cell proliferation, cytokine production, and cytotoxicity in co-cultures of human cancer cell lines mixed with primary human T cells. CEACAM5 x CD28, Trop-2 x CD28, STEAP1 x CD28, and mesothelin x CD28 bispecifics enhanced T cell degranulation, cytokine secretion, and cancer cell cytotoxicity only in concert with TCR engagement. IHC was used to compare TAA expression on cancer cell lines versus that found on cancer tissues, allowing categorization of cell lines as high density (tumor surrogate) or low density (normal tissue surrogate). CD28 bispecifics were identified with selective potency on high versus low expressing cell lines, suggesting a favorable therapeutic index. CD28 bispecific antibodies co-targeting CEACAM5, Trop-2, STEAP1, and mesothelin show promising activity and warrant further development across a range of solid tumors. Citation Format: Matthew A. Dragovich, Viralkumar Davra, Matthew S. Faber, Yoon Kyung Kim, Alex Nisthal, Veronica G. Zeng, Jonathan Jacinto, Juan E. Diaz, Thuy Truong, Jing Qi, Kendra N. Avery, Rumana Rashid, Sung-Hyung Lee, Seung Y. Chu, Christine Bonzon, Ruschelle Love, Matthew J. Bernett, James A. Ernst, Rena Bahjat, Norman J. Barlow, John R. Desjarlais, Michael Hedvat, Gregory L. Moore. Tumor-specific CD28 costimulatory bispecific antibodies enhance T cell activation in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2983.

Abstract 1807: CB307: a novel targeted CD137 agonist for enhancement of immune cell responses to PSMA+ tumors

Abstract Introduction: CD137 (HGNC:TNFRSF9, 4-1BB) is a TNF receptor superfamily costimulatory receptor expressed by T cells and NK cells. Upon engagement, CD137 enhances immune cell survival, proliferation, cytokine production and memory formation. CD137 agonists in the clinic have shown tantalizing anti-tumor effects, however, hepatic toxicity has hampered clinical utility. To circumvent this limitation, we developed CB307, a half-life extended heavy-chain only Humabody® that selectively agonizes CD137 only in the presence of prostate specific membrane antigen (PSMA, HGNC:FOLH1). PSMA is highly upregulated on tumor cells in prostate cancer and tumor neovasculature in other solid tumors including lung (NSCLC), kidney (clear cell RCC), bladder and colorectal cancers. CB307 enhances immune cell activation in a PSMA-dependent manner. We present here preclinical mechanistic differentiators of CB307 with respect to non-targeted CD137 agonists. Methods: CD137 agonism, immune cell activation and cytokine secretion were evaluated in vitro using coculture assays in the presence/absence of PSMA-expressing tumor cells and in vivo with a transgenic mouse syngeneic tumor model. The ability of CB307 or non-targeted CD137 agonists to enhance tumor cell killing was additionally characterized in 3D spheroid cocultures. In vitro cocultures were used to determine the impact of PSMA expression level on CB307 activity. CB307 was evaluated as a single agent and in combination with PD-1 or PD-L1 inhibitors; tumoral checkpoint contribution was investigated using isogenic PD-L1 positive vs. PD-L1 CRISPR-deleted tumor cells. Results: CB307 or PD-1/PD-L1 inhibition alone induced IL-2 secretion in PBMC/PSMA-expressing tumor cell cocultures; this effect was synergistically enhanced when CB307 was combined with either PD-1 or PD-L1 inhibition. CB307 augmented tumor cell killing in 3D spheroids containing PSMA-expressing cells. Enhanced tumor cell killing was observed in spheroid cultures containing CB307 in combination with PD-1/PD-L1 inhibition. In in vivo pharmacology studies in a syngeneic mouse model, CB307 demonstrated statistically significant inhibition of tumor growth versus control without peripheral T cell activation - consistent with pharmacology restricted to the PSMA+ tumor microenvironment, whereas an anti-CD137 monoclonal antibody induced tumor growth inhibition accompanied by peripheral immune cell proliferation and cytokine production. Summary: CB307 is a novel CD137 agonist that selectively enhances immune cell activity only in the presence of PSMA-positive cells. Our data demonstrate PSMA-targeted CB307 immunological pharmacology both in vitro and in vivo which has supported the ongoing Phase 1 ‘POTENTIA’ clinical trial (NCT04839991). Citation Format: Andrew J. Pierce, Phillip M. Brailey, Sophie Archer, Clare Song, Anca-Giuilia Cionca, Joanne Fernando, Phillip D. Bartlett, Philip Bland-Ward. CB307: a novel targeted CD137 agonist for enhancement of immune cell responses to PSMA+ tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1807.

Abstract 6346: Discovery of HX011, a novel OX40 mAb, for potential cancer immunotherapy

Abstract OX40, tumor necrosis factor receptor superfamily member 4, is an immune co-stimulatory receptor, together with its ligand OX40L, forming OX40-OX40L trimer-trimer complex between the surface of T-cells (OX40) and APCs/NKs (OX40L). OX40 expresses constitutively on Treg as well as on conventional Teff, particularly within TME. OX40/OX40L interactions directly triggers conventional T cell activation via NF-kB1 pathway and inhibits bcl-x and survivin to prevent apoptosis; and it also downregulates FoxP3/CTLA4, thus Treg function. OX40 agonistic antibodies have also been shown to activate tumor immunity and increase anti-tumor activity in preclinical models. Here we report that we identified/constructed a novel OX40-agonistic humanized IgG1 monoclonal antibody (mAb), HX011, with high affinity through hybridoma technology. HX011 was subsequently confirmed of the similar binding properties and functions as the original mouse mAb clone, 9C12: 1) binding to human OX40 (EC50 ~0.012 nM; 2) similar function binding in the reporter assay (EC50 ~6.6 nM); but 3) has little blockade of the OX40L binding to OX40. In another word, HX011 is similar to BMS-986178 for the first two assays, but contrastingly different for the third assay. Non-competing with OX40L may actually maximize T-cell activation, a potential advantage over a competing antibody. In vivo pharmacology modeling of HX011 in the humanized syngeneic CRC model, MC38, where syngeneic C57BL/6 mouse mice were knocked in with huOX40 (“HuGEMM-OX40”), demonstrated strong anti-tumor activity (TGI ~100% at 1 mg/kg, twice weekly). HX011 is being further investigated as a potential new immunotherapy for the treatment of cancers. Citation Format: Hang Ke, Faming Zhang, Jialin Li, Feiyu Peng, Cen Chen, Henry Qixiang Li. Discovery of HX011, a novel OX40 mAb, for potential cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6346.