Tumor Cell Binding Research Articles

Fully human monoclonal antibody targeting the cysteine-rich substrate-interacting region of ADAM17 on cancer cells

ADAM17 sheds EGFR/erbB ligands and triggers oncogenic pathways that lead to the progression of solid tumors. We targeted the ADAM17 disintegrin and cysteine rich domain region (D+C) to generate a panel of single-chain antibody fragments (scFvs) that selectively bind to the D or C domains of ADAM17, but not of ADAM10 or ADAM19. From the panel, we selected one scFv, referred to as C12, based on its high binding affinity towards the target, and re-formatted it to a full IgG for further studies. High-resolution cryo-electron microscopy studies documented that the mAb binds to the ADAM17 C-domain that in ADAM proteases, notably ADAM10 and ADAM17, is known to impart substrate-specificity. The C12 mAb significantly inhibited EGFR phosphorylation in cancer cell lines by hindering the cleavage of EGFR ligands tethered to the cell surface. This inhibition provides a mechanism for potential anti-tumor effects, and indeed C12 diminished the viability of a variety of EGFR-expressing cancer cell lines. Cell-based ELISA studies revealed that C12 preferentially bound to activated ADAM17 present on tumor cells, as compared to the autoinhibited ADAM17 that is the predominant form on HEK293 and other non-tumor cells. C12 also exhibited tumor growth inhibition in an ovarian cancer xenograft mouse model. Consistent with its selective tumor cell binding in vitro, radioimmuno PET (positron emission tomography) imaging with 89Zr-DFO-C12 in mouse xenograft models confirmed tumoral accumulation of the C12 mAb.

Dual-Engineered Macrophage-Microbe Encapsulation for Metastasis Immunotherapy.

Lung metastases are the leading cause of death among cancer patients. The challenges of inefficient drug delivery, compounded by a robust immunosuppressive microenvironment, make effective treatment difficult. Here, an innovative dual-engineered macrophage-microbe encapsulation (Du-EMME) therapy is developed that integrates modified macrophages and engineered antitumor bacteria. These engineered macrophages, termed R-GEM cells, are designed to express RGD peptides on extracellular membranes, enhancing their tumor cell binding and intratumor enrichment. R-GEM cells are cocultured with attenuated Salmonella typhimurium VNP20009, producing macrophage-microbe encapsulation (R-GEM/VNP cells). The intracellular bacteria maintain bioactivity for more than 24h, and the bacteria released from R-GEM/VNP cells within the tumor continue to exert bacteria-mediated antitumor effects. This is further supported by macrophage-based chemotaxis and camouflage, which enhance the intratumoral enrichment and biocompatibility of the bacteria. Additionally, R-GEM cells loaded with IFNγ-secreting strains (VNP-IFNγ) form R-GEM/VNP-IFNγ cells. Treatment with these cells effectively halts lung metastatic tumor progression in three mouse models (breast cancer, melanoma, and colorectal cancer). R-GEM/VNP-IFNγ cells vigorously activate the tumor microenvironment, suppressing tumor-promoting M2-type macrophages, MDSCs, and Tregs, and enhancing tumor-antagonizing M1-type macrophages, mature DCs, and Teffs. Du-EMME therapy offers a promising strategy for targeted and enhanced antitumor immunity in treating cancer metastases.

In Vitro Study of Tumor-Homing Peptide-Modified Magnetic Nanoparticles for Magnetic Hyperthermia.

Cancer cells have higher heat sensitivity compared to normal cells; therefore, hyperthermia is a promising approach for cancer therapy because of its ability to selectively kill cancer cells by heating them. However, the specific and rapid heating of tumor tissues remains challenging. This study investigated the potential of magnetic nanoparticles (MNPs) modified with tumor-homing peptides (THPs), specifically PL1 and PL3, for tumor-specific magnetic hyperthermia therapy. The synthesis of THP-modified MNPs involved the attachment of PL1 and PL3 peptides to the surface of the MNPs, which facilitated enhanced tumor cell binding and internalization. Cell specificity studies revealed an increased uptake of PL1- and PL3-MNPs by tumor cells compared to unmodified MNPs, indicating their potential for targeted delivery. In vitro hyperthermia experiments demonstrated the efficacy of PL3-MNPs in inducing tumor cell death when exposed to an alternating magnetic field (AMF). Even without exposure to an AMF, an additional ferroptotic pathway was suggested to be mediated by the nanoparticles. Thus, this study suggests that THP-modified MNPs, particularly PL3-MNPs, hold promise as a targeted approach for tumor-specific magnetic hyperthermia therapy.

Balancing the Affinity and Tumor Cell Binding of a Two-in-One Antibody Simultaneously Targeting EGFR and PD-L1.

The optimization of the affinity of monoclonal antibodies is crucial for the development of drug candidates, as it can impact the efficacy of the drug and, thus, the dose and dosing regimen, limit adverse effects, and reduce therapy costs. Here, we present the affinity maturation of an EGFR×PD-L1 Two-in-One antibody for EGFR binding utilizing site-directed mutagenesis and yeast surface display. The isolated antibody variants target EGFR with a 60-fold-improved affinity due to the replacement of a single amino acid in the CDR3 region of the light chain. The binding properties of the Two-in-One variants were confirmed using various methods, including BLI measurements, real-time antigen binding measurements on surfaces with a mixture of both recombinant proteins and cellular binding experiments using flow cytometry as well as real-time interaction cytometry. An AlphaFold-based model predicted that the amino acid exchange of tyrosine to glutamic acid enables the formation of a salt bridge to an arginine at EGFR position 165. This easily adaptable approach provides a strategy for the affinity maturation of bispecific antibodies with respect to the binding of one of the two antigens.

Abstract 6721: AI-guided generation of novel GUCY2C-targeting T-cell engagers for the treatment of colorectal cancer

Abstract Colorectal cancer (CRC) ranks as one of the most lethal cancers worldwide. Given the advances in immunotherapy and targeted therapy, the survival time in CRC has been improving recently. However, the development of novel and effective therapies for CRC still remains an unmet medical need. Guanylyl cyclase C (GUCY2C) is a transmembrane receptor expressed on the surface of intestinal epithelial cells. Recent studies have demonstrated that GUCY2C is causally linked to intestinal inflammation, dysbiosis, and tumorigenesis. To this end, GUCY2C has emerged as a promising target for immunotherapy and a diagnostic marker of primary and metastatic cancer. Here we describe the discovery and optimization of a novel panel of GUCY2C-targeting T cell engagers (TCEs) developed via our streamlined AI-guided TCE development platform. We have identified a number of anti-GUCY2C antibodies with various binding epitopes on GUCY2C, and subsequently generated a panel of novel GUCY2C-targeting multispecific antibodies with favorable developability and functionalities via our AI-driven engineering technology. Our data demonstrate that a biparatopic GUCY2C x CD3 TCE engineered as an asymmetric ‘1+2’ format can recognize two distinct epitopes on GUCY2C. This biparatopic GUCY2C x CD3 TCE shows enhanced tumor cell binding affinity and superior T cell-mediated cytotoxicity compared to the ‘1+1’ GUCY2C x CD3 TCE and ‘1+2’ bivalent GUCY2C x CD3 TCE where the two GUCY2C targeting arms can only bind to an identical epitope on GUCY2C. In addition, we have engineered a novel GUCY2C × CD3 x CD28 trispecific TCE, in which the CD3-targeting arm in tandem with the co-stimulating CD28-targeting arm mediate the interaction with T cells. This anti-GUCY2C trispecific TCE induces favorable synergistic T cell activation and tumor cell killing in vitro compared to the bispecific counterparts. In vivo anti-tumor activity assessments of these multispecific TCE molecules in various tumor xenograft mouse models are currently ongoing. Citation Format: Liang Tian, Jinxiu Hou, Chenpeng Su, Xiaoou Xu, Xiaoqian Chen, Dandan Liu, Jiyuan Tian, Rui Shen, Yi Pan, Yuan Yuan, Lisha Dong, Tingting Gu, Jian Peng. AI-guided generation of novel GUCY2C-targeting T-cell engagers for the treatment of colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6721.

EZH2 Inhibitors Enhance CART Cell Quality, Efficacy, In Vivo Homing, Tumor Cell Binding and Killing of Fully Syngeneic Primary B Cell Lymphomas, As Well As Reprogramming Lymphoma Cells to a Highly Immunogenic and T Cell Adherent Phenotype

Although having efficacy in a subset of diffuse large B cell lymphoma (DLBCL) patients, cellular immunotherapies are currently administered without guidance as to whether the underlying biology or immunology are conducive to durable response. Moreover, mechanisms explaining CART resistance in DLBCLs remain largely unknown. One critical missing link to address these unmet needs are syngeneic animal models precisely reflecting the genetics and immunology of human DLBCLs. To overcome these hurdles, we developed a suite of genetically engineered mouse models (GEMM) faithfully recapitulating different types of DLBCL. A retrospective study showed that C3/EZB DLBCLs, featuring EZH2 mutations and BCL2 translocation, experienced inferior outcomes after CART therapy. We reported that Ezh2 mutations cause aberrant repression of genes required for productive immune synapses with T cells. We hypothesized that this phenotype limits CART cell activity in vivo, and that EZH2 inhibitors could overcome CART resistance. We generated EZB GEMMs with conditional expression of Ezh2 Y641F and BCL2 in germinal center (GC) B cells. These mice developed T cell depleted lymphomas, histologically similar to human C3/EZB DLBCLs. We generated several EZB cell lines from these mice, which recapitulated the epigenetic, transcriptional, histological, and immune microenvironment of EZB DLBCLs in humans when engrafted in immunocompetent recipients. Strikingly, treating EZB cells with EZH2 inhibitor tazemetostat (taz) ex vivo reprogrammed them to re-express the full spectrum of T cell engagement genes such as ICOSL, ITGB7, 4-1BBL, and OX40L and rendered them highly immunogenic. For example, mixing endogenous T cells with taz pre-treated EZB cells yielded markedly enhanced killing (p<0.001). Taz pre-treated EZB cells failed to engraft in syngeneic mice due to immunological rejection, but caused fatal DLBCL in Rag1-/- mice. EZB cell lines did not undergo proliferation arrest after taz ex vivo, but showed a significant reduction in tumor burden (p<0.05) when treated in vivo, along with a 3-fold increase in tumor CD4 and CD8 cells (p<0.05), and 50% reduction in Tregs (p<0.01). Taz also increased the proportion of naïve/memory CD8 and reduced effector CD8 cells. EZH2 inhibitors therefore mediate their effects at least in part through immunological mechanisms. We next examined whether taz had any deleterious effect on CART cells. Pre-treating donor mice with taz prior to harvesting T cells yielded significant increase in memory CD8 CART cells after transduction (p<0.05). Taz pre-treated CART cells displayed superior killing of EZB cells (p<0.001) and proliferative capacity ex vivo (p<0.001). Infusing taz pre-treated CART cells in EZB mice resulted in significantly reduced tumor burden in vivo (p<0.001) and reduction in PD1+CD38+ exhausted CD8 CART cells (p<0.001). On the other hand, pre-treatment of EZB cells (but not CART cells) with taz ex vivo enhanced CART cell killing by 3-fold (p<0.001). Administering CART cells in vivo in EZB mice after taz treatment significantly prolonged their survival (100% vs 30%, p<0.01) and was associated with significant reduction in tumor burden assessed by IVIS and post-mortem examination. Therefore, EZH2 inhibition enhances CART cell activity through direct effects on CART cells, in addition to making EZB immunogenic. Finally, to directly demonstrate the functional consequence of rendering EZB lymphomas immunogenic on CART performance in vivo, we performed intravital 2-photon imaging of popliteal lymph nodes using dTomato labeled CART cells and GFP+ EZB lymphomas. Remarkably, pre-treatment of EZB cells with taz, doubled the recruitment of CART cells into lymphomas (p<0.001) and significantly enhanced the CART to EZB lymphoma cell surface engagement index (strength and duration of binding, p<0.001). This was associated with increased killing, and CART-engaged lymphoma cells were observed to be ingested/engulfed or phagocyted by tumor resident macrophages. Collectively, EZH2 inhibitors yield a potent boost to CART mediated anti-lymphoma effects by enhancing CART cell functions and B cell immunogenicity, which would likely yield a significant clinical benefit for these patients where CART cells are less active. These results prompted us to initiate a clinical trial evaluating the safety and efficacy of this combination in B cell lymphomas (NCT05934838).

Biomaterial-Mediated Exogenous Facile Coating of Natural Killer Cells for Enhancing Anticancer Efficacy toward Hepatocellular Carcinoma.

Natural killer (NK) cells exhibit a good therapeutic efficacy against various malignant cancer cells. However, the therapeutic efficacy of plain NK cells is relatively low due to inadequate selectivity for cancer cells. Therefore, to enhance the targeting selectivity and anticancer efficacy of NK cells, we have rationally designed a biomaterial-mediated ex vivo surface engineering technique for the membrane decoration of cancer recognition ligands onto NK cells. Our designed lipid conjugate biomaterial contains three major functional moieties: (1) 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) lipid for cell membrane anchoring, (2) polyethylene glycol for intracellular penetration blocker, and (3) lactobionic acid (LBA) for cancer recognition. The biomaterial was successfully applied to NK cell surfaces (LBA-NK) to enhance recognition and anticancer functionalities, especially toward asialoglycoprotein receptor (ASGPR)-overexpressing hepatocellular carcinoma. Highly efficient and homogeneous NK cell surface editing was achieved with a simple coating process while maintaining intrinsic properties of NK cells. LBA-NK cells showed potential ASGPR-mediated tumor cell binding (through LBA-ASGPR interaction) and thereby significantly augmented anticancer efficacies against HepG2 liver cancer cells. Thus, LBA-NK cells can be a novel engineering strategy for the treatment of liver cancers via facilitated immune synapse interactions in comparison with currently available cell therapies.

Doxorubicin-Loaded Platelet Decoys for Enhanced Chemoimmunotherapy Against Triple-Negative Breast Cancer in Mice Model.

Triple-negative breast cancer (TNBC) is a highly aggressive subtype with a poor prognosis. Current single-agent checkpoint therapy has limited effectiveness in TNBC patients. In this study, we developed doxorubicin-loaded platelet decoys (PD@Dox) for chemotherapy and induction of tumor immunogenic cell death (ICD). By combining PD-1 antibody, PD@Dox has the potential to enhance tumor therapy through chemoimmunotherapy in vivo. Platelet decoys were prepared using 0.1% Triton X-100 and co-incubated with doxorubicin to obtain PD@Dox. Characterization of PDs and PD@Dox was performed using electron microscopy and flow cytometry. We evaluated the properties of PD@Dox to retain platelets through sodium dodecyl sulfate-polyacrylamide gel electrophoresis, flow cytometry, and thromboelastometry. In vitro experiments assessed drug-loading capacity, release kinetics, and the enhanced antitumor activity of PD@Dox. The mechanism of PD@Dox was investigated through cell viability assays, apoptosis assays, Western blot analysis, and immunofluorescence staining. In vivo studies were performed using a TNBC tumor-bearing mouse model to assess the anticancer effects. Electron microscopic observations confirmed that platelet decoys and PD@Dox exhibited a round shape similar to normal platelets. Platelet decoys demonstrated superior drug uptake and loading capacity compared to platelets. Importantly, PD@Dox retained the ability to recognize and bind tumor cells. The released doxorubicin induced ICD, resulting in the release of tumor antigens and damage-related molecular patterns that recruit dendritic cells and activate antitumor immunity. Notably, the combination of PD@Dox and immune checkpoint blockade therapy using PD-1 antibody achieved significant therapeutic efficacy by blocking tumor immune escape and promoting ICD-induced T cell activation. Our results suggest that PD@Dox, in combination with immune checkpoint blockade therapy, holds promise as a potential strategy for TNBC treatment.

Hyaluronic acid-engineered Bcl-2 inhibitor nanocrystals for site-specific delivery to breast tumor cells.

Aim: This investigation aims to repurpose venetoclax using hyaluronic acid-coated venetoclax nanocrystals (HA-VEN-NCs) to target breast cancer. Materials & methods: An antisolvent precipitation method was used to fabricate the nanocrystals and optimize them using central composite design. Hyaluronic acid (HA)-coated and -uncoated nanocrystals were compared in terms of in vitro drug release, cell line studies, CD44-expressing breast tumor cell binding capability and anticancer activity. Results: HA-VEN-NCs and venetoclax nanocrystals (VEN-NCs) showed pH-responsive drug-release behavior, exhibiting sustained release at pH 6.8. Our extensive in vitro cell line investigation showed that HA-VEN-NCs efficiently bind to CD44-expressing breast tumor cells and possess excellent anticancer activity (half maximal inhibitory concentration: 2.00μg/ml) compared with VEN-NCs. Conclusion: Our findings anticipate that HA-VEN-NCs could serve as valuable nanoplatforms for cancer treatments in the future.

DNA Damage Inducer Mitoxantrone Amplifies Synergistic Mild-Photothermal Chemotherapy for TNBC via Decreasing Heat Shock Protein 70 Expression.

Patients with triple-negative breast cancer (TNBC) have the worst clinical outcomes when compared to other subtypes of breast cancer. Nanotechnology-assisted photothermal therapy (PTT) opens new opportunities for precise cancer treatment. However, thermoresistance caused by PTT, as well as uncertainty in the physiological metabolism of existing phototherapeutic nanoformulations, severely limit their clinical applications. Herein, based on the clinically chemotherapeutic drug mitoxantrone (MTO), a multifunctional nanoplatform (MTO-micelles) is developed to realize mutually synergistic mild-photothermal chemotherapy. MTO with excellent near-infrared absorption (≈669nm) can function not only as a chemotherapeutic agent but also as a photothermal transduction agent with elevated photothermal conversion efficacy (ƞ = 54.62%). MTO-micelles can accumulate at the tumor site through the enhanced permeability and retention effect. Following local near-infrared irradiation, mild hyperthermia (<50°C) assists MTO in binding tumor cell DNA, resulting in chemotherapeutic sensitization. In addition, downregulation of heat shock protein 70 (HSP70) expression due to enhanced DNA damage can in turn weaken tumor thermoresistance, boosting the efficacy of mild PTT. Both in vitro and in vivo studies indicate that MTO-micelles possess excellent synergetic tumor inhibition effects. Therefore, the mild-photothermal chemotherapy strategy based on MTO-micelles has a promising prospect in the clinical transformation of TNBC treatment.

Abstract 5040: Novel HER3 targeting antibody radioconjugates, 225Ac-HER3 ARC and 177Lu-HER3 ARC, exhibit potent antitumor efficacy in HER3-positive solid tumors

Abstract Background: HER3 is a unique member of the EGFR family that collaborates with other EGFR receptors to induce tumorigenesis and drug resistance. Moreover, HER3 expression is linked to poor survival for patients with solid tumors. Despite HER3 being a rational cancer therapeutic target, no HER3-directed therapies have been approved for clinical use. However, new therapeutic strategies such as antibody drug conjugates (ADCs) are being investigated. Targeted radiotherapy, including radiolabeled antibodies (ARCs), is unique mechanistically by inducing cell death independent of biologic pathway inhibition, and can be efficacious with less toxicity relative to other therapeutic modalities. Therefore, we hypothesized that the cytotoxic effects of alpha (225Ac) or beta (177Lu) emitting radionuclides combined with the specificity of anti-HER3 antibody targeting is a compelling therapeutic approach for HER3-expressing tumors. Here we evaluated the antitumor effects of 225Ac or 177Lu armed HER3 ARCs across multiple HER3-expressing cancer models such as ovarian, colorectal, prostate, and renal cancer. Methods: ARCs were prepared by radiolabeling AT-02, an anti-HER3 antibody, with 225Ac or 177Lu using p-SCN-Bn-DOTA to yield 225Ac or 177Lu-HER3 ARC. The binding activity and tumor cell cytotoxicity of HER3 ARCs were assessed by ELISA using human recombinant HER3, flow cytometry on HER3-expressing cells, and colony forming assays. To evaluate the antitumor growth effects of 225Ac-HER3 and 177Lu-HER3 ARCs in vivo, preclinical human tumor xenograft models were developed. Mice bearing HER3-positive tumors were dosed with 225Ac-HER3 ARC (0.2 or 0.4 µCi), or 177Lu-HER3 ARC (200 or 400 µCi) and tumor growth and body weight was monitored. Results: The pharmacological binding properties of HER3 antibody radiolabeled with 225Ac or 177Lu were similar to that of unmodified antibody as demonstrated by HER3 binding ELISA and flow cytometry. HER3 ARCs induced cytotoxicity and inhibited colony formation of HER3-positive tumor cell lines. Significant in vivo human tumor xenograft growth inhibition was observed in response to 225Ac or 177Lu HER3 ARCs compared to control groups (unmodified AT02 or IgG ARCs) in the models studied. No significant loss of body weight was observed in mice treated with HER3 ARCs suggesting that all treatments were well tolerated. Conclusions: In this study, both 225Ac-HER3 ARC and 177Lu-HER3 ARC demonstrated significant antitumor activity against HER3-expressing tumors in a dose-dependent manner. The HER3 targeted radiotherapy approach that we have undertaken could potentially overcome the limitations of current solid tumor therapies in resistance settings and warrants further evaluation in patients with HER3-expressing tumors. Citation Format: Denis Beckford-Vera, Megan McCloskey, Jason Li, Caroline Jennings, Le-Cun Xu, Debbie Lewis, Patrik Brodin, Amanda Chin, Monideepa Roy, Mary Chen, Helen Kotanides. Novel HER3 targeting antibody radioconjugates, 225Ac-HER3 ARC and 177Lu-HER3 ARC, exhibit potent antitumor efficacy in HER3-positive 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 5040.

Abstract 6295: Belzupacap sarotalocan, an investigational virus-like drug conjugate, preferentially binds cancer cell glycosaminoglycans associated with the epithelial to mesenchymal transition

Abstract Human papillomaviruses (HPVs) are non-enveloped, double-stranded DNA viruses that utilize modified glycosaminogycans (GAGs) such as heparan sulfate proteoglycans, within the epidermal basement membrane as initial attachment factors prior to cell entry and infection. Similarly modified GAGs are also commonly found on the surface of tumor cells, enabling HPV capsids with tumor-selective binding properties. Belzupacap sarotalocan (bel-sar, formerly AU-011), is a virus-like drug conjugate composed of an HPV virus-like particle (VLP) conjugated to a cytotoxic payload (phthalocyanine dye) which upon light activation, causes rapid immunogenic tumor necrosis of bound tumor cells in vivo and has the potential to induce long-term anti-tumor immunity. Bel-sar has been shown to bind to and kill a wide variety of tumor types in preclinical models and is entering a Phase 3 clinical trial for the treatment of early-stage choroidal melanoma. To investigate the precise GAG modifications as well as the overall genetic signature which mediates the binding specificity of bel-sar, a large, multi-tumor type binding screen of bel-sar on 124 cancer cell lines was performed in vitro, where we identified a strong relationship between drug binding and pathways involved in the epithelial to mesenchymal transition (EMT). Additionally, through dose escalation treatments, HeLa subclones were generated which fail to bind and are resistant to the cytotoxic effects of bel-sar. Gene expression profiling and biochemical characterization of these resistant HeLa cell lines demonstrated a strong reduction in the expression of GAGs and a marked down-regulation of TGF-β signaling, ultimately resulting in the induction of a mesenchymal to epithelial transition (MET). Furthermore, gene-gene correlation studies showed that a variety of GAG biosynthesis genes are associated with TGF-β signaling and EMT progression, and that these genes are important determinates of bel-sar binding. Overall, these data suggest that the binding of bel-sar and HPV VLPs is strongly dependent on the expression and modification of GAGs that occurs during the EMT process. As many cancers, especially metastatic tumors, are thought to undergo at least partial EMT, these data provide mechanistic insights into the tumor-targeting ability of HPV VLP-conjugates such as bel-sar and suggest possible applications for such therapies across a wide variety of other tumor types. Citation Format: Nathan R. Fons, Rhonda C. Kines, John T. Schiller. Belzupacap sarotalocan, an investigational virus-like drug conjugate, preferentially binds cancer cell glycosaminoglycans associated with the epithelial to mesenchymal transition [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 6295.

Abstract 4075: A modified FcγRI expressing-T cell, SolidT, enables antibody-mediated cytotoxicity to overcome the limitations of CAR-T cell therapy against solid tumors

Abstract The pioneering design of chimeric antigen receptor T-cell (CAR-T) therapy demonstrated the potential of reprogramming the immune system. Nonetheless, T-cell exhaustion, toxicity, and suppressive microenvironment limit their efficacy in solid tumors. Recently, we characterized a novel subset of tumor-infiltrating CD4+ T-cells expressing the FcγRI receptor (Rasoulouniriana et al, 2019). Herein we detail the engineering of a novel receptor, based on the FcγRI structure, which is retrovirally transduced into PBMCs (both CD4 and CD8 T-cells). These cells, named SolidT cells, can target tumor cells using antibody intermediates. They show effective and specific cytotoxicity only when an appropriate antibody is added. Only target-bound antibodies activate these cells, while free antibodies are internalized without activation. Their cytotoxic activity is correlated to target protein density, therefore targeting tumor cells with high antigen density while sparing normal cells with low or no expression. This activation mechanism prevents premature exhaustion. Furthermore, during ADCC these cells secrete attenuated cytokine levels compared to CAR-T, thereby enhancing their safety profile. These cells eradicate established melanoma tumors, infiltrate the tumor microenvironment and facilitate host immune cell recruitment in immunocompetent mice. In NSG mice, they infiltrate, persist, and eradicate tumors. As opposed to CAR therapies, which require changing the receptor across different types of cancer, our engineered T-cells remain the same throughout tumor types, while only the injected antibody changes. As such, can be adapted to treat a wide range of solid cancers without changing the manufacturing program. Overall, SolidT cells are capable of binding tumor cells with high affinity, while preserving the cytotoxic specificity only to cells expressing a high density of tumor-associated antigens. Citation Format: Diana Rasoulouniriana, Nadine Santana-Magal, Amit Gutwillig, Leen Farhat-Younis, Hana Shpilt, Shahar Dotan, Noam Pilpel, Peleg Rider, Yaron Carmi. A modified FcγRI expressing-T cell, SolidT, enables antibody-mediated cytotoxicity to overcome the limitations of CAR-T cell therapy against 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 4075.

Oncolytic vaccinia virus expressing a bispecific T-cell engager enhances immune responses in EpCAM positive solid tumors.

Insufficient intratumoral T-cell infiltration and lack of tumor-specific immune surveillance in tumor microenvironment (TME) hinder the progression of cancer immunotherapy. In this study, we explored a recombinant vaccinia virus encoding an EpCAM BiTE (VV-EpCAM BiTE) to modulate the immune suppressive microenvironment to enhance antitumor immunity in several solid tumors. VV-EpCAM BiTE effectively infected, replicated and lysed malignant cells. The EpCAM BiTE secreted from infected malignants effectively mediated the binding of EpCAM-positive tumor cells and CD3ϵ on T cells, which led to activation of naive T-cell and the release of cytokines, such as IFN-γ and IL-2. Intratumoral administration of VV-EpCAM BiTE significantly enhanced antitumor activity in malignancies with high other than with low EpCAM expression level. In addition, immune cell infiltration was significantly increased in TME upon VV-EpCAM BiTE treatment, CD8+ T cell exhaustion was reduced and T-cell-mediated immune activation was markedly enhanced. Taken together, VV-EpCAM BiTE sophistically combines the antitumor advantages of bispecific antibodies and oncolytic viruses, which provides preclinical evidence for the therapeutic potential of VV-EpCAM BiTE.

Abstract 259: Study of a low anticoagulant bovine heparin as a drug capable of disrupting tumor cell-platelet interaction and preventing hematogenous metastasis

Abstract Metastasis is the leading cause of death in cancer patients. During metastatic dissemination, circulating tumor cells need to survive in the bloodstream and their interaction with platelets through P-selectin-ligand seems to be crucial. It has been demonstrated that heparin (from porcine mucosa, porcine UFH), a glycosaminoglycan composed by repetitive disaccharides of uronic acid and glucosamine, may interfere with P-selectin interaction. Recently our research group purified by ion exchange chromatography a fraction of bovine heparin displaying very low anticoagulant potential, named LABH (~20 IU/mg LABH x ~200 IU/mg porcine UFH x ~100 IU/mg bovine UFH). The advantage of bovine UFH and, especially of LABH, is their reduced risk of bleeding side effect. Our aim in this work was to investigate the potential use of LABH as an antimetastatic agent in vitro and in vivo, evaluating its ability to interfere with tumor cell-platelet interaction, tumor cell binding to P-selectin and preventing induced metastasis in mice. For this, we challenged 8-12 weeks C57Bl/6 mice with 4 or 8 mg/kg of porcine UFH, bovine UFH or LABH followed by intravenous injection of B16F10 cells (murine melanoma cell line). After 21 days, lungs were analyzed and metastatic foci were counted. The number of metastatic foci was significantly reduced when animals were pretreated with heparins (50-70 foci in control mice and 10-15 in treated mice). In other attempt, isolated platelets from healthy volunteers were incubated for 1h with MV3 cells (human melanoma cell line) in vitro in the presence or absence of heparin. All three heparins tested were efficient in blocking MV3-platelet interaction in a dose-response manner, but LABH required higher doses than porcine and bovine UFH. When analyzing the direct binding of U937 cell (human lymphoma cell line) to immobilized P-selectin, we could observe that porcine and bovine UFH inhibited at the same level (~ 60% of inhibition with 100 μg/mL), while LABH was less effective (~ 45% of inhibition with 100 μg/mL). Collectively our results demonstrate that despite presenting distinct patterns of sulfation and anticoagulant potential, all heparins significantly reduced lung metastasis, platelet-tumor cell adhesion and P-selectin binding. On the other hand, we hypothesize that LABH may act suppressing metastasis also by other mechanisms since its effect on in vitro experiments was less pronounced in comparison to porcine and bovine UFH. Citation Format: Juliana Maria Motta, Carlos Roberto-Fernandes, Kayene Vitória Micheli, Ana Maria Tovar, Paulo Antônio Mourão, Mariana Sá Pereira. Study of a low anticoagulant bovine heparin as a drug capable of disrupting tumor cell-platelet interaction and preventing hematogenous metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 259.

Abstract 2913: Personalized ovarian cancer therapy using in vivo assembled bi-specific antibodies targeting Her2 and FSHR

Abstract According to The American Cancer Society, ovarian cancer will cause ~14,000 deaths in the US in 2021. Despite recent advances in therapy, clinical outcomes remain poor and there is a great unmet need for newer therapies for ovarian cancer patients. Bispecific T cell engagers (BiTEs) consist of two single chain variable fragments (scFVs) linked via a peptide linker and are being studied as a tool against many different cancers. BiTEs redirect T cell activity towards tumor cells in an MHC independent manner by bringing T cells close to a tumor associated antigen (TAA). A BiTE targeting CD19 has been approved for treating ALL, however the success of BiTEs in treating solid tumor remains limited. Two reasons that reduce the efficacy of BiTEs in solid tumors are 1) their short half-life which drastically reduces their availability 2) tumor heterogeneity preventing targeting by a single BiTE. Tumors downregulate TAA expression in response to targeted therapy allowing treatment escape. Development of novel BiTEs with longer serum half-lives and in vivo delivery strategies which allow rapid customization of BiTEs is important. Here we describe a strategy for in vivo assembly using the Knob-in-hole (KIH) approach. We designed two different DNA encoded KIH (dKIH), targeting Her2 and FSHR, and studied their ability to be assembled in vivo into functional therapeutics. The knobs encoded full length antibodies engineered to target either Her2 or FSHR. We designed a third construct as the hole to target CD3. Following in vitro characterization, we injected NSG mice with KIH pairs for Her2+CD3 or FSHR+ CD3 and studied their expression and function. Sera from mice injected with Her2 or FSHR dKIH bound tumor cells expressing Her2 or FSHR respectively. Sera from mice from both groups also bound to T cells. We observed that both dKIH resulted in mouse sera with high potency (pg level) killing of ovarian cancer cell lines expressing Her2 and FSHR in an in vitro killing assay. A single injection of either dKIH allowed for in vivo expression and tumor killing for over a month post injection which is a significant improvement over delivery of protein KIH. This study demonstrates the potential of in vivo assembly of KIH. The patients produce their own therapy, which allows to skip complex bioprocessing steps and makes KIH delivery simpler and cheaper. This study illustrates the potential to improve immunotherapy outcomes for ovarian cancer patients via in vivo assembly of multiple BiTEs. Citation Format: Pratik Sanjivkumar Bhojnagarwala, Ali Raza Ali, Drew Frase, Abhijeet Kulkarni, Devivasha Bordoloi, David B. Weiner. Personalized ovarian cancer therapy using in vivo assembled bi-specific antibodies targeting Her2 and FSHR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2913.

Bispecific Antibodies Progression in Malignant Melanoma.

The discovery of oncogenes and immune checkpoints has revolutionized the treatment of melanoma in the past 10 years. However, the current PD-L1 checkpoints lack specificity for tumors and target normal cells expressing PD-L1, thus reducing the efficacy on malignant melanoma and increasing the side effects. In addition, the treatment options for primary or secondary drug-resistant melanoma are limited. Bispecific antibodies bind tumor cells and immune cells by simultaneously targeting two antigens, enhancing the anti-tumor targeting effect and cytotoxicity and reducing drug-resistance in malignant melanoma, thus representing an emerging strategy to improve the clinical efficacy. This review focused on the treatment of malignant melanoma by bispecific antibodies and summarized the effective results of the experiments that have been conducted, also discussing the different aspects of these therapies. The role of the melanoma epitopes, immune cell activation, cell death and cytotoxicity induced by bispecific antibodies were evaluated in the clinical or preclinical stage, as these therapies appear to be the most suitable in the treatment of malignant melanoma.

Synthesis, Radiolabeling, and Preclinical Evaluation of 68Ga/177Lu-Labeled Leuprolide Peptide Analog for the Detection of Breast Cancer.

Objectives: The expansion of novel and potent tumor receptor binding peptides is a promising approach for the precise targeting of various cancer. Leuprolide is a 9-residue peptide analog of gonadotropin-releasing hormone and is extensively used in the treatment of sex hormone-dependent tumors, including prostate, breast, and ovarian cancer. This preclinical study was undertaken to prepare a new radiolabeled leuprolide peptide for the detection of breast carcinoma. Methods: A 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA)-coupled 9-amino acid leuprolide peptide was synthesized after typical 9-fluorenylmethyl-oxycarbonyl-based solid-phase peptide synthesis and radiolabeled with both 68Ga and 177Lu radionuclides for theranostic use. The systemic pharmacokinetics was done in healthy balb/c mice. The in vitro tumor cell binding affinity was determined on MCF7, T47D, and MDA-MB-231 breast cancer cell lines. In vivo tumor targeting and micro positron-emission tomography imaging was performed on nude mice with MCF7 breast tumor xenografts. Results: The leuprolide peptide was conveniently synthesized by solid-phase synthesis strategy and its identity and purity were validated by mass spectrometry and high-performance liquid chromatography. The peptide radiolabeled efficiently (˃94%) with both diagnostic (68Ga) and therapeutic (177Lu) radionuclides and displayed nanomolar binding potency to all three tested MCF7, T47D, and MDA-MB-231 cell lines. Fast and favorable pharmacokinetics was observed for 68Ga/177Lu-leuprolide in healthy Balb/c mice. In nude mice, 68Ga-leuprolide peptide exhibited rapid clearance from the blood circulation with low to moderate (up to 5% ID/g) uptake/retention by the major body organs. The accumulation in the estrogen receptor-positive MCF7 tumor was 2.24% ± 0.62% ID/g at 45 min p.i, with good tumor to blood and muscle uptake ratios. The radiolabeled peptide was excreted primarily through the renal pathway. Conclusion: The encouraging results of this initial study demonstrate that additional testing of this leuprolide peptide seems to be indicated because of its convincing potential to be a new agent for the management of breast carcinoma.

Directing Multivalent Aptamer‐Receptor Binding on the Cell Surface with Programmable Atom‐Like Nanoparticles

AbstractMultivalent interactions of biomolecules play pivotal roles in physiological and pathological settings. Whereas the directionality of the interactions is crucial, the state‐of‐the‐art synthetic multivalent ligand–receptor systems generally lack programmable approaches for orthogonal directionality. Here, we report the design of programmable atom‐like nanoparticles (aptPANs) to direct multivalent aptamer–receptor binding on the cell interface. The positions of the aptamer motifs can be prescribed on tetrahedral DNA frameworks to realize atom‐like orthogonal valence and direction, enabling the construction of multivalent molecules with fixed aptamer copy numbers but different directionality. These directional‐yet‐flexible aptPAN molecules exhibit the adaptability to the receptor distribution on cell surfaces. We demonstrate the high‐affinity tumor cell binding with a linear aptPAN oligomer (≈13‐fold improved compared to free aptamers), which leads to ≈50 % suppression of cell growth.

Directing Multivalent Aptamer-Receptor Binding on the Cell Surface with Programmable Atom-Like Nanoparticles.

Multivalent interactions of biomolecules play pivotal roles in physiological and pathological settings. Whereas the directionality of the interactions is crucial, the state-of-the-art synthetic multivalent ligand-receptor systems generally lack programmable approaches for orthogonal directionality. Here, we report the design of programmable atom-like nanoparticles (aptPANs) to direct multivalent aptamer-receptor binding on the cell interface. The positions of the aptamer motifs can be prescribed on tetrahedral DNA frameworks to realize atom-like orthogonal valence and direction, enabling the construction of multivalent molecules with fixed aptamer copy numbers but different directionality. These directional-yet-flexible aptPAN molecules exhibit the adaptability to the receptor distribution on cell surfaces. We demonstrate the high-affinity tumor cell binding with a linear aptPAN oligomer (≈13-fold improved compared to free aptamers), which leads to ≈50 % suppression of cell growth.