Tumor-specific Surface Antigen Research Articles

Cathepsin B Nuclear Flux in a DNA-Guided "Antinuclear Missile" Cancer Therapy.

Some antinuclear antibodies (ANAs) bind extracellular nucleic acids released into tumor environments and are pulled into the nuclei of live cancer cells through nucleoside salvage pathways, independent of tumor-specific surface antigens. Here we show that ANA nuclear penetration induces nuclear flux by the lysosomal protease cathepsin B and leverage this mechanism to design an antinuclear antibody-drug conjugate (ANADC) with cathepsin B-labile drug linker. The ANADC targets nucleic acid exhaust from necrotic tumors and crosses membrane barriers through nucleoside salvage as a DNA-seeking and tumor agnostic "antinuclear missile" cancer therapy.

A novel SLC3A2-targeting antibody-drug conjugate exerts potent antitumor efficacy in head and neck squamous cell cancer

The development of innovative therapeutic strategies for head and neck squamous cell carcinoma (HNSCC) is a critical medical requirement. Antibody-drug conjugates (ADC) targeting tumor-specific surface antigens have demonstrated clinical effectiveness in treating hematologic and solid malignancies. Our investigation revealed high expression levels of SLC3A2 in HNSCC tissue and cell lines. This study aimed to develop a novel anti-SLC3A2 ADC and assess its antitumor effects on HNSCC both in vitro and in vivo. This study developed a potent anti-SLC3A2 ADC (19G4-MMAE) and systematically investigated its drug delivery potential and antitumor efficacy in preclinical models. This study revealed that 19G4-MMAE exhibited specific binding to SLC3A2 and effectively targeted lysosomes. Moreover, 19G4-MMAE induced a significant accumulation of reactive oxygen species (ROS) and apoptosis in SLC3A2-positive HNSCC cells. The compound demonstrated potent antitumor effects derived from MMAE against SLC3A2-expressing HNSCC in preclinical models, displaying a favorable safety profile. These findings suggest that targeting SLC3A2 with an anti-SLC3A2 ADC could be a promising therapeutic approach for treating HNSCC patients.

Abstract 1236: Characterization of CBX-250, a first-in-class TCR-mimetic-based T-cell engager targeting a cathepsin G peptide-HLA complex for the treatment of myeloid leukemia

Abstract While T-cell engaging therapies have demonstrated clear clinical benefit in the treatment of B-cell malignancies, their application to myeloid malignancies remains a challenge due to the limited repertoire of suitable tumor specific surface antigens. Targeting HLA-restricted peptides (pHLA) derived from intracellular cancer antigens provides an opportunity to explore the entire cancer proteome. CG1 (FLLPTGAEA) has been validated as an HLA-A*02:01 restricted leader peptide from Cathepsin G (CTSG) and is abundantly presented by leukemic versus normal myeloid cells. TCR-mimetic (TCRm) antibodies are ideally suited to target pHLA with high potency and specificity and can be engineered and manufactured using standard antibody technologies. Here we report the preclinical characterization of CBX-250, a novel CG1/HLA-A2 TCRm-CD3 bispecific T-Cell Engager (TCE). CBX-250 induced potent T-cell activation and T-cell mediated killing of leukemia cell lines with varying levels of target antigen density with sub-nM EC50 in vitro. In vivo, a closely related precursor of CBX-250 demonstrated potent tumor control in various AML and CML CDX models at doses as low as 0.01mpk. Although CTSG is a serine protease stored in neutrophil azurophilic granules, the CG1 peptide is preferentially presented by leukemia cells, as validated by mass spectrometry. Moreover, when co-culturing PBMCs with HLA-A2 neutrophils, we did not observe any CBX-250-mediated T-cell activation or IFNγ production. In silico predictions identified HLA-A2-restricted human peptides that share sequence or structural similarity to CG1. We determined the cross-reactivity risk of CBX-250 to be low, based on T-cell activation and T-cell mediated cytotoxicity assays against cells pulsed with supra-physiological concentrations of these peptides. Moreover, we observed no activity against target-negative cancer cells, including a CTSG-KO cell line. To further assess the specificity of CBX-250, we screened a panel of normal cells and found no meaningful T-cell activation or IFNγ production at CBX-250 concentrations exceeding its EC90. Finally, the CBX-250 precursor molecule demonstrated favorable safety in a double transgenic mouse model expressing human HLA-A2 and CG1. Following sequence optimization, CBX-250 demonstrated robust serum and pH stress stability and a favorable melting temperature, while retaining excellent potency and specificity. A full suite of analytical and biophysical assessments supports CBX-250’s favorable developability profile. In summary, these data provide strong preclinical evidence of the potency, specificity, safety, and developability of CBX-250, a novel, first-in-class, off-the-shelf, TCRm-based TCE for the treatment of myeloid malignancies. Citation Format: Geraldine Paulus, Benjamin Lee, Preethi Sankaran, Tanzila Rahman, Jessica Jimenez, Delainey O'Connor, Simon Yue, Yu Huang, Melissa Bikowitz, Sarah Jaffe, Shawn O'Malley, Bhupal Ban, Galina Gabriely, Tao Wang, Amanda Mak, Michael Princiotta, Chunhua Shi, Helen He, Ningping Feng, Jun Yan, Timothy Heffernan, Gheath Alatrash, Jeffrey Molldrem, Dmitri Wiederschain. Characterization of CBX-250, a first-in-class TCR-mimetic-based T-cell engager targeting a cathepsin G peptide-HLA complex for the treatment of myeloid leukemia [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 1236.

Abstract 6225: Developing a novel combination therapy using engineered chimeric antigen receptor natural killer cells targeting avsialidase with avsialidase-armed oncolytic vaccinia virus in solid tumor models

Abstract Chimeric antigen receptor (CAR) - engineered natural killer (NK) cell therapy has emerged as a promising platform for adoptive immunotherapy for cancer. However, CAR-NK or CAR-T therapy so far has achieved limited efficacy in solid tumors compared with hematologic malignancies. One of the challenges is the lack of prevalent tumor-specific surface antigen target in solid tumors. Hence, we are developing a novel oncolytic vaccinia virus (VV) to deliver a dual functional glycol-immune checkpoint inhibitor and universal tumor cell marker combined with CAR-NK cell therapy to increase anti-tumor efficacy. Using the vaccinia virus deleted in both viral TK and VGF genes (vvDD) to drive the expression of a membrane-bound sialidase derived from actinomyces viscosus (avSial) under the viral late promoter, we observed efficient desialylation of both VV-infected and non-infected tumor cells, which would alleviate the suppression of sialic acid on NK and other immune cells within TME. Further, the surface bound avSial on VV-infected tumor cells can also serve as a universal target for avSial-CAR NK cells, with less concern for cross-reactivity to normal human tissues and antigen loss. We developed anti-avSial antibodies through mice immunization study. The selected single chain variable fragments (scFV) were constructed in CAR format containing CD28 CD3 ζ for co-stimulation and human IL-15 gene for better NK persistence and function. The binding affinity of anti-avSial scFv to avSialidase were further screened in CAR constructs-transfected 293T cells. The selected anti-avSial scFv CAR were packaged into gamma retroviral vectors to transduce activated and expanded NK cells derived from healthy donor peripheral blood. For proof-of-concept, target tumor cell lines A375, A549 and HT-29 expressing transmembrane sialidase and GFP were also generated. Compared with CD19 CAR NK and none transduced (NT) NK cells, avSial CAR NK had markedly increased cytotoxicity against avSial-expressing tumor cells in co-culture assays at low E:T ratios <= 1:2. Upon tumor cell rechallenge, avSial CAR NK also controlled the tumor growth significantly better than CD19 CAR NK or NT NK cells. Furthermore, avSial CAR NK completely eliminated A375 tumor spheroids expressing transmembrane sialidase whereas CD19 CAR NK or NT NK cells only transiently controlled the tumor spheroids growth. Taken together, we have developed an effective avSial targeting CAR IL15 NK that will be used to demonstrate enhanced efficacy and versatility in combination with avSial-armed VV for treatment of solid tumors. Citation Format: Xiaomei Wang, Guowei Wei, Keshav B. Karki, Winnie Chan, Mariya Viskovska, Andrew Williams, Nancy Chang, Haiyan Jiang. Developing a novel combination therapy using engineered chimeric antigen receptor natural killer cells targeting avsialidase with avsialidase-armed oncolytic vaccinia virus in solid tumor models [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 6225.

Abstract IAP0301: Bispecific T cell engagers (TCEs) for treatment of solid tumors: Challenges and opportunities

Abstract T cell-engaging bispecific antibody constructs (TCEs) have the potential to engage all cytotoxic T cells for redirected cancer cell lysis, including CD8, CD4, gamma/delta, NKT and regulatory T cells. TCEs transiently connect T cells with target cells leading to formation of a cytolytic synapse. Once activated by TCEs, T cells can adopt a serial lysis mode and increase troop size by their local proliferation. In numerous animal models, TCEs can eradicate large tumors as single-agent therapy. TCEs show a remarkable single-agent activity in hematological diseases, including ALL, NHL (DLBCL and FL) and AML. A CD19/CD3-bispecific TCE called blinatumomab (Blincyto) has been approved in 2014, and six other TCEs targeting CD20, BCMA and CD123 are in late-stage development. With complete response rates >50% and durability of responses, TCEs are in par with autologous CD19 CAR-T cells in NHL but are easier to manufacture and allow for a faster treatment start. Treatment of solid tumor indications with TCEs is not as successful as treatment of blood-borne cancers. This may be due to issues with tumor penetration, the hostile tumor microenvironment and the scarcity of T cells in cold tumors. A variety of TCEs explore their single-agent activity against solid tumors by targeting surface antigen like PSMA, GPC3, GD2, or HER-2. Enhancements in the form of combination therapy with checkpoint inhibitors, or 4-1BB and CD28 agonists are being explored as ways of augmenting solid tumor activity of TCEs. The first TCE showing robust anti-tumor activity in a solid tumor indication is targeting a gp100 peptide/MHC (developed by Immunocore). It is made from a soluble TCR fragment of very high affinity for the gp100 peptide HLA*0201 complex fused to an anti-CD3 scFv fragment. Although only few partial responses were observed in uveal melanoma patients, the single-agent treatment showed a very robust improvement in overall survival with a hazard ratio of 0.51 compared to investigators’ choice. This data will inspire the development of more TCEs that are specific for peptide/MHC complexes. This class of targets can also be addresses by TCR-mimetic antibodies. TCEs using such antibodies showed high anti-tumor activity in mouse models against MAGE-A4, p53 mutant or tyrosinase peptide/MHC complexes, all targets that are otherwise not amenable to classical TCEs. Another emerging opportunity for treating cancers are conditional TCEs. These are infused as inactive precursors. Inside tumors, the locally produced proteases convert the precursors into highly active TCEs. One example is the so called ProTriTACs, where a half-life extending albumin-binding domain masks the CD3-binding domain. The mask confers a 500-fold reduction in the cytolytic activity of the TCE. Inside tumors, the mask is cleaved off and a TCE is activated that has a very short serum half-life once it is released from the tumor. Conditional TCEs promise not only a wider therapeutic index but can also leverage as targets tumor-associated antigens that are widely expressed on tumors but also on healthy normal tissues, such as EGFR or EpCAM/TROP1. On-target toxicity of TCEs relates to the quality of target antigens. Finding more tumor-specific surface antigens, appropriate pMHC targets (e.g., mutant oncogene epitopes) and conditional T cell engagers are ways to address this challenge. Off-target toxicity of TCEs relates to T cell activation because of redirected target cell lysis. In the worst case, a cytokine release syndrome is induced. In the best case, a low-level cytokine release is contributing to the efficacy of TCEs. CRS can be managed by steroids, step dosing, anti-IL6 antibody and/or dasatinib. Another promising means is subcutaneous administration of TCEs. A dozen TCE programs currently explore this route, which allows reduction of Cmax and of the initial cytokine release. TCEs will soon become a major weapon in the arsenal of cancer therapy. They will not only work in blood-borne cancers but will soon conquer select solid tumor indications. Peptide/MHC-targeting and conditional TCEs are very promising approaches to achieve solid tumor activity of TCEs. Citation Format: Patrick A. Baeuerle. Bispecific T cell engagers (TCEs) for treatment of solid tumors: Challenges and opportunities [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr IAP0301.

Mitotropic triphenylphosphonium doxorubicin-loaded core-shell nanoparticles for cellular and mitochondrial sequential targeting of breast cancer

HypothesesTargeted therapy exploits cancerous niches’ properties including acidic extracellular environment, hypoxic tumor core, and over expression of tumor-specific surface antigens. The present study aims to develop and evaluate a sequential targeted core–shell nanoparticulate (NPs) system for treatment of breast cancer. Sequential (double-stage) targeting was achieved at the cellular-level through employing the selective CD44- receptor binding hyaluronic acid (HA), followed by subcellular mitochondrial drug-delivery using the mitotropic triphenylphosphonium-conjugated doxorubicin (DOX-TPP+). ExperimentsNPs were prepared through incorporation of the electrostatic-complexes of DOX.HCl/DOX-TPP+ with tripolyphosphate (STPP-) into chitosan (CS) forming the core that was further coated with HA shell. Physicochemical characterization techniques namely; FTIR, DSC, DLS, morphological evaluation and spectroscopic assessments were implemented. Moreover, the drug entrapment efficiency (EE%), loading capacity (LC%), drug release profile and kinetics were investigated. Lastly, to validate the biological efficiency of the developed NPs, cytotoxic activity was evaluated as well as flow cytometric analyses to assess apoptosis induction and cell-cycle arrest were studied. FindingsResults showed that, the obtained core–shell NPs possessed a spherical shape with a mean size of 220–280 nm and attained high EE% and LC%. In-vitro cytotoxicity evaluations demonstrated successful apoptosis induction and cell-cycle abrogation. Moreover, in-vivo studies on Solid Ehrlich carcinoma (SEC)-bearing mice confirmed the efficient anticancer activity of the mitotropic DOX-TPP+-loaded NPs. Conclusively, the developed core–shell NPs proved efficient in sequential targeting of DOX to breast cancer.

Treating Relapsed / Refractory (RR) AML with Biodegradable Anti-CD123 CAR Modified T Cells

Treating Relapsed / Refractory (RR) AML with Biodegradable Anti-CD123 CAR Modified T Cells

The Combination of 41BB and CD28 Costimulatory Signals Endows Lower Affinity CAR T Cells with the Most Optimal Properties for Selective Targeting of the Tumor Associated Antigen CD38

T cells equipped with Chimeric Antigen Receptors (CARs) have been emerged as a potent therapy for cancer. The scarcity of tumor-specific surface antigens in several cancer types, however, prompted the research towards improved CAR designs to optimally target tumor associated antigens (TAA), which are also expressed on normal cells at lower densities. To this end, we recently demonstrated the feasibility of targeting CD38, a multiple myeloma (MM) associated TAA, without on-target/off-tumor effects, by designing CARs with optimally lowered affinity for the antigen. It is, however, largely unknown whether lowering the antigen affinity affects the costimulatory requirements of CAR T cells for optimal expansion and in vivo persistence. Therefore we now evaluated the impact of different costimulation designs on CD38-CARs with lower affinity. Previous reports indicate that a CAR design including CD28 costimulation improves the cytotoxic potential of CAR T cells whereas a configuration involving the 4-1BB-costimulation confers more persistent responses. Therefore, we used CARs with 5 different affinities (Kd range 1.8 - >1915 nM), in combination with 3 different described costimulatory designs (28z, 4-1BBz and 28z + 4-1BBL). Lowering the affinity of CAR gradually diminished the cytolytic activity of CAR T cells with a 4-1BB costimulatory domain. Such an effect was however not observed with CARs containing the CD28 costimulatory domain, alone or expressed in combination with 4-1BBL. In vitro as well as in vivo experiments revealed that 28z + 4-1BBL expressing low affinity CD38-CAR T cells possessed a significantly better discriminative capacity between tumor and normal cells, displayed a more clear central memory phenotype, showed better in vitro expansion and improved in vivo persistence compared to the other two configurations tested. More importantly, the 28z + 4-1BBL design potentiated even CARs bearing scFvs with immeasurable affinity (>1915nM) and brought them to the functional level of 4-1BB- and 28z-based CARs of higher affinities. In conclusion, we show that the type of costimulatory domain is of importance when fine-tuning the CAR affinity. A combination of 4-1BB and CD28 signals provides the most optimal costimulation to affinity-tuned CAR T cells and suggests the careful construction of TAA-targeting CARs to enhance their clinical potential. DisclosuresVan De Donk:Janssen, Celgene, Bristol-Myers Squibb, Amgen: Research Funding. Zweegman:Celgene: Other: advisory board participation, Research Funding; Janssen: Other: advisory board participation, Research Funding; Takeda: Other: advisory board participation, Research Funding; Amgen: Other: advisory board participation. Lokhorst:Genmab: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; OncoImmune: Research Funding. Mutis:Novartis: Research Funding; OncoImmune: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genmab: Research Funding; Gilead: Research Funding; Celgene: Research Funding.

Combination of Oncolytic Virotherapy and CAR T/NK Cell Therapy for the Treatment of Cancer

Multiple lines of evidence indicate that CAR-T cell based therapy and oncolytic virotherapy display robust performance in both immunocompetent and immunodeficient mouse models. Rare, yet highly successful attempts to combine these therapeutic platforms have also been reported. Interestingly, both approaches have shown pronounced efficacy in human trials, albeit these were limited to just a handful of malignancies. Specifically, CD19-specific CAR-T cell products (Kymriah and Yescarta) have been highly effective against B cell lymphomas and leukemias, whereas administering oncolytic viruses resulted in pronounced responses in melanoma (Imlygic and Rigvir) and nasopharyngeal carcinoma (Oncorine) patients. It is well established that efficacy of virotherapy as a standalone approach is largely restricted by the pre-existing and mounting immune response against viral antigens, and requires a relatively functional immune system, which is not typical for cancer patients, with the current antitumor therapy schemes. On the other hand, the most important challenges faced by the current CAR-T cell therapy formats include the lack of targetable tumor-specific surface antigens, tumor cell heterogeneity, and immunosuppressive tumor microenvironment, not to mention the unacceptably high costs. Remarkably, combining the two approaches may help address their individual bottlenecks. Namely, local acute inflammatory reaction induced by the viral infection may reverse tumor-associated immunosuppression and lead to more efficient homing and penetration of CAR-expressing lymphocytes into the tumor stroma; combined viral and CAR-mediated cytotoxicity may ensure the production of immunogenic cell debris and efficient presentation of tumor neoantigens, and potently recruit the patient's own bystander immune cells to attack cancer cells. Thus, testing the combinations of CAR-based and virolytic approaches in the clinical setting appears both logical and highly promising.

Redirecting T-Cells Against AML in a Multidimensional Targeting Space Using T-Cell Engaging Antibody Circuits (TEAC)

Background T cell redirection strategies, such as CAR-T and bispecific antibodies (bsAb), are rapidly changing the way in which we approach and treat cancer. While CAR-T and bsAb have shown impressive clinical efficacy in a limited number of cancers, both strategies are ultimately limited by on-target toxicity that currently restricts application to B-cell lineage tumors as the number of genuinely tumor-specific surface antigens is extremely limited. BsAb also suffer from off-target toxicity relating their ability to directly active T-cells severely restricting the therapeutic window. We sought to solve these inherent problems with the current generation bsAb by re-designing the molecule to alter the mechanism of T-cell activation. By splitting the T-cell engaging VHVL antibody paratope between two separate molecules we created two molecules that formed an active T-cell engaging unit through protein domain complementation following proteolytic activation. Each antibody could target independent surface antigens vastly increasing targeting permutations. Thus, these two antibodies functioned as an "antibody circuit" permitting Boolean type logic to precisely control T-cell activation in multi-dimensional targeting space. We selected AML as model cancer to develop T-cell Engaging Antibody Circuits (TEACs) due to the highly characterized surface antigen landscape and the clear challenges and limitations of single-antigen targeting approaches. Results We first screened 10 AML cell lines for candidate surface antigens based upon prior studies of surface antigen display (Perna F et al, 2016) and identified CD33, CD123, CD49d, CD70, CD71, CD38, CLEC12A, Flt3, CD24, CD244, TIM3 and CCR1 as promising targets. We developed a secondary TEACs screening assay where the two TEAC molecules contained either a FITC or biotin binding domain and paired these to commercial FITC or biotin conjugated antibodies targeting the antigens above. We screened 72 TEAC pairs against the 10 cell lines which identified optimal antigen target combinations which included CD33xCD123, CD33xCLEC12A, CD33xCD49d and CD33xCD24. Using a FRET-based fluorescent peptide assay to identify peptide linkers susceptible to proteases we found MMP2 to be highly expressed in AML samples and thus designed all our TEACs with this cleavage site. We next generated IgG4 format TEACs targeting CD33, CD123, CLEC12A and CD24 that included the MMP2 cleavage activation site and tested these as TEAC pairs in vitro. This screen identified the CD123xCD33 as the most active TEAC pair which was active in 9/10 cells lines. To assess potential safety concerns, we tested TEACs and CD123 and CD33 BiTEs individually and as pairs on PBMCs and on plate-immobilized molecules. These data demonstrated that BiTEs were extremely active against healthy monocytes and also activate T-cells non-specifically once plate-immobilized. In contrast CD123xCD33 IgG TEACs pairs did not activate T-cells when plate-immobilized and did not target healthy monocytes.Finally, we examined the activity of both CD33 BiTEs and CD123xCD33 TEACs on primary patient AML samples. We conducted FRET based assays which confirmed high activity of MMP2 cleavage site on all primary AML samples. When we examined T-cell activation, CD123xCD33 TEACs were active in all CD123+ CD33+ AML samples evaluated with an EC50 of 30ug/ml. Conclusion These data suggest T-cell engaging antibody circuits is a new approach that could be safely applied toward AML. TEAC agents do not directly activate T-cells and CD123xCD33 TEAC pairs do not activate PBMC or monocytes. However, CD123xCD33 TEACs show strong activity against AML cell lines and primary CD123+CD33+ AML cells. Disclosures Millar: Revitope Oncology: Equity Ownership. Minshull:Atum Biotechnology: Employment, Equity Ownership. Narayan:Takeda: Other: Employment (spouse); Genentech: Other: Equity ownership (spouse); Merck: Other: Equity ownership (spouse). Graubert:Biogen: Other: Spouse Employee; Calico Life Sciences: Other: Research Support; Janssen Pharmaceuticals: Other: Research Support. Cobbold:Gritstone Oncology: Equity Ownership; Revitope Oncology: Consultancy; Revitope Oncology: Equity Ownership.

The Generation of CAR-Transfected Natural Killer T Cells for the Immunotherapy of Melanoma

Natural killer T (NKT) cells represent a cell subpopulation that combines characteristics of natural killer (NK) cells and T cells. Through their endogenous T-cell receptors (TCRs), they reveal a pronounced intrinsic anti-tumor activity. Thus, a NKT cell transfected with a chimeric antigen receptor (CAR), which recognizes a tumor-specific surface antigen, could attack tumor cells antigen-specifically via the CAR and additionally through its endogenous TCR. NKT cells were isolated from peripheral blood mononuclear cells (PBMCs), expanded, and electroporated with mRNA encoding a chondroitin sulfate proteoglycan 4 (CSPG4)-specific CAR. The CAR expression on NKT cells and their in vitro functionality were analyzed. A transfection efficiency of more than 80% was achieved. Upon stimulation with melanoma cells, CAR-NKT cells produced cytokines antigen-specifically. Compared with conventional CAR-T cells, cytokine secretion of CAR-NKT cells was generally lower. Specific cytotoxicity, however, was similar with CAR-NKT cells showing a trend towards improved cytotoxicity. Additionally, CAR-NKT cells could kill target cells through their endogenous TCRs. In summary, it is feasible to generate CAR-NKT cells by using mRNA electroporation. Their CAR-mediated cytotoxicity is at least equal to that of conventional CAR-T cells, while their intrinsic cytotoxic activity is maintained. Thus, CAR-NKT cells may represent a valuable alternative to conventional CAR-T cells for cancer immunotherapy.

Chimeric Antigen Receptor Therapeutic Strategies: The Future of Glioblastoma Management

The field of adoptive cellular therapy, using autologous T-cells modified ex vivo to specifically target tumor cells prior to being reintroduced to the patient, has become a new focus of research endeavors searching for a novel and efficacious treatment for oncologic disease, including glioblastoma. Chimeric Antigen Receptor (CAR)-T-cells consist of a single chain variable fragment of a monoclonal antibody coupled with extant T-cell intracellular signaling cascade systems using a viral vector ex vivo. This provides the advantage of targeting tumor specific surface markers, while minimizing off-target effects and potential toxicity. Additionally, the CAR T-cells bypass the need for MHC-restricted presentation, a system which is frequently down-regulated in tumor cells. Among the surface antigens described as targets for CAR T-cell therapy for GBMs, Epidermal growth factor variant III (EGFRvIII), HER2 (HER2/neu, ERBB2), interleukin-13 receptor α2 subunit (IL-13Rα2), and erythropoietin-producing hepatocellular carcinoma A2 (EphA2) are the leading options for tumor specific surface antigens to target with CAR-T cells. This article reviews history and advantages of CAR-T cell therapies, and discuss future directions.

Isolation of a novel IgG from a patient with lung cancer using immunoassays and cloning.

e22163 Background: Cancer cells express novel antigens that are characteristic of the transformed state and may engender an immune response ( i.e., antibody production) in patients. These antibodies may identify novel immune targets for antibody-based immunotherapies and diagnostics. Furthermore, the presence of these auto-antibodies could be followed as biomarkers of therapeutic response or relapse. Previous studies of the antibody response to tumors have been hampered by inefficient methods of cloning the human antibodies in their native configurations. We collected blood samples from a group of lung cancer patients with diverse clinical and pathologic features and are characterizing the anti-tumor antibody response using a variety of whole-cell binding assays. Our approach also uses an optimized hybridoma method to clone tumor-specific IgG antibodies from primary memory B-cells. Methods: We obtained serum and peripheral blood mononuclear cell samples from 50 patients with advanced lung cancer. We characterized the immune response of these patients to tumor cell lines using fluorescence microscopy with the Operetta (Perkin Elmer) and by whole-cell ELISA. We performed a cell fusion using B-cells from one of the immunoreactive patients, screening the hybridomas for antibodies specific for binding in the whole cell ELISA. We characterized the antibody activity using flow cytometry and immunoblotting, and we identified the antigen using mass spectroscopy analysis of a gel-purified, immunoreactive band. We confirmed the identity of the antigen bound by immunoblotting to recombinant protein. Results: Human IgG antibodies immunoreactive with human cancer cell lines are prevalent in our panel of lung cancer patients. Using our hybridoma method, we isolated a human IgG that binds a tumor-specific surface antigen expressed in a variety of tumors, but only minimally expressed by normal cells. Conclusions: Novel IgG antibodies against specific tumor antigens can be isolated from lung cancer patients’ serum.Using our novel hybridoma method for cloning human antibodies, we have identified an antibody that binds a tumor-specific antigen. The study of these antibodies may lead to novel biomarkers for cancer diagnosis and therapy.

Effect of lenalidomide and pomalidomide combined with IgG1-isotype antibodies on antibody-dependent cellular cytotoxicity (ADCC) via cytokine signaling and effector cell granzyme B and FasL expression

3058 Background: Lenalidomide and pomalidomide (CC-4047) display anti-angiogenic and immunomodulatory activities in a variety of in vitro assay systems. Evidence from single agent patient studies indicate enhancement of T cell and NK cell function in patients either with hematological or solid tumors. There is also in vitro and preclinical evidence that both drugs can enhance NK cell mediated ADCC. Methods: We have utilized an in vitro ADCC system to investigate the mechanism(s) whereby lenalidomide and pomalidomide are able to enhance human NK cell and monocyte effector function in response to FCγ receptor signaling initiated by therapeutic antibodies. Results: Pre-treatment of NK cells or monocytes with pomalidomide or lenalidomide (but not thalidomide) greatly enhanced ADCC of Her2/neu overexpressing breast cancer cells pre-coated with trastuzumab and EGFR positive colorectal cancer cells pre-coated with cetuximab. NK cell and monocyte-mediated killing of antibody coated tumor cells was dose-dependent. There was minimal tumor cell killing by antibody alone or by either drug alone or in the presence of panitumumab, an anti-EGFR IgG2a antibody that does not efficiently engage NK cell FCγ receptors. NK cell-mediated killing is associated with distinct changes in intracellular signaling (decreased pSHIP-1, increased PLCγ2, pERK) and increased granzyme B and FasL expression. Monocyte-mediated ADCC takes longer to initiate (16 hrs vs 4 hrs) is also associated with increased granzyme B and FasL and is blocked by anti-IL-12mAb. ADCC is completely blocked by an inhibitor of granzyme B activity and partially blocked by an antibody that blocks FasL-Fas interaction. Conclusions: Pomalidomide and lenalidomide (but not thalidomide) strongly enhance the ability of therapeutic IgG1 antibodies to induce ADCC via NK cell/monocyte-mediated killing of tumor cells in vitro. Furthermore, granzyme B activity and concurrent cytokine signaling is crucial to enable the synergistic combination of either drug with antibodies to tumor-specific surface antigens the potential utility of which is being assesses in upcoming clinical studies. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Celgene Corp Celgene Corp

Lenalidomide and pomalidomide strongly enhance tumor cell killing in vitro during antibody-dependent cellular cytotoxicity (ADCC) mediated by trastuzumab, cetuximab and rituximab

3023 Background: Potential mechanisms of action of lenalidomide and pomalidomide (CC-4047) include anti-angiogenic, anti- proliferative and immunomodulatory activities, e.g., enhancement of T cell and NK cell function. Both drugs appear to enhance T cell activation and Th1-type cytokines in cancer patients. Also, both drugs have been shown to enhance rituximab-mediated protection in a mouse lymphoma model. Methods: We have utilized an in vitro ADCC system to assess the ability of these drugs to enhance human NK cell function in response to the approved therapeutic antibodies trastuzumab, cetuximab and rituximab. Results: Pre-treatment of NK cells with either pomalidomide or lenalidomide greatly enhanced IFN-γ production by NK cells in response to IgG in the presence of either IL-2 or IL- 12. In a series of functional ADCC assays, Her2/neu overexpressing breast cancer cells (SKBR3 & MCF-7) pre-coated with trastuzumab, EGFR positive colorectal cancer cells (HCT-116) pre-coated with cetuximab and NHL cell lines (Namalwa, Farage & Raji) pre-coated with rituximab, were exposed to NK cells pre-treated with pomalidomide, lenalidomide or thalidomide. Both pomalidomide and lenalidomide synergistically increased (up to 6-fold) NK cell-mediated killing of antibody coated tumor cells in a dose-dependent manner. Thalidomide had no effect in this system. There was minimal tumor cell killing by antibody alone or in the absence of antibody. The presence of IL-2 or IL-12 was required to see enhanced killing by either drug. Enhanced ADCC was associated with increased signaling in NK cells, specifically with inhibition of the negative regulator SHIP-1. Other downstream effects on signaling pathways are also being investigated. Monocyte-mediated tumor cell ADCC was also enhanced by both drugs. Conclusions: We have shown that pomalidomide and lenalidomide (but not thalidomide) strongly enhance the ability of therapeutic antibodies to induce ADCC via NK cell/monocyte-mediated killing of tumor cells in vitro. These results provide a strong rationale for combination of either lenalidomide or pomalidomide with antibodies to tumor-specific surface antigens in cancer patients. No significant financial relationships to disclose.

Structure and function of ETAA16: a novel cell surface antigen in Ewing's tumours.

Immunoscreening of an Ewing's family of tumour (EFT)-derived cDNA library using formerly described EFT-specific antibodies led to the isolation of a 3.5 kb cDNA, named Ewing's tumour-associated antigen 16 (ETAA16). The ETAA16 cDNA shows no homology to any functionally characterised human gene. Only a bovine cDNA expressed in bovine testis and hepatocytes is functionally characterised as it encodes for a junction plaque associated protein and showed a homology of 69.9% at amino acid level to ETAA16. The human cDNA encodes for a 926 amino acid tumour antigen with a calculated molecular weight of 103 kDa. The epitope of the ETAA16-specific antibody, Ak16, covers the central region of the protein which is part of an extra cellular domain. The human ETAA16 gene locus has been assigned to chromosome 2p13-15 by FISH analyses and is confirmed by the human genome sequencing project. As demonstrated by flow cytometry, the cell surface expression of ETAA16 antigen is restricted to ET cell lines and not expressed on other small blue round cell tumours or other kind of tumour. RT-PCR analysis revealed a high expression of ETAA16 in brain, liver and kidney while lung and heart were negative. Immunohistochemistry showed an intracellular expression of ETAA16 in different kind of non-Ewing tumour tissues. These results suggest that ETAA16 may function as a tumour-specific cell surface antigen in EFTs.

Future approaches for treating hematologic disease.

The approval of monoclonal antibodies for therapy of hematologic malignacies (Rituxan, Mylotarg, Campath) renewed the interest in antibodies as potential new treatment options for cancer patients. Antibodies are effective in inhibiting tumor cell growth , inducing apoptosis, and activating host effector mechanisms for tumor cell killing. Monoclonal antibodies can be clinically effective as monotherapy, as targeting agents delivering either potent cytotoxic drugs or radionuclides as well as in combination with conventional chemotherapies. Advances in antibody engineering provided new capabilities to reduce immunogenicity, alter half life, increase effector functions, and increase tumor targeting for optimal therapeutic modalities requiring chronic dosing regimens. During the next decade, as new tumor-specific surface antigens are discovered and the linkage between genes and function is better understood, new targets will be identified for regulating tumor cell growth by engineered antibodies with agonist or antagonist activity. Additionally, antibody engineering will allow for more efficient radionuclide or cytotoxic drug targeting or lead to more selective activation of relevant host effector mechanisms, leading to a safe and effective therapy of cancer.

Multiple pre-neoplastic events and clonal selection of radiation induced mouse thymic lymphomas shown by TCR gene rearrangements

After split-dose irradiation, pre-lymphoma cells develop from a tumor-specific surface antigen TL-2 + thymocyte subpopulation. To analyze the clonality of pre-lymphoma cells, various numbers of TL-2 + thymocytes from a single irradiated mouse were intra-thymically injected to Thyl congenic recipient mice. The incidence of donor type thymic lymphoma(s) was subsequently examined in a group of recipient mice. We chose several lymphomas derived from a single donor mouse and analyzed the TCR gene rearrangements and V(D)J junctional diversity as genetic markers of clonality. These results indicate multiple initial neoplastic events and clonal selection into lymphoma.

Use of colloidal gold cytochemistry in the study of the basic cell biology of cancer

We are currently investigating the morphologic aspects of two areas of the basic cell biology of cancer: tumor-specific surface antigens as targets for immunotoxins, and the phenomenon of multidrug resistance in chemotherapy of human tumors. Colloidal gold cytochemistry has provided a useful method for the electron-microscopic cytochemical detection of materials endocytosed by cells in culture. This technique has been used to study the internalization pathway of ligands bound to the surface of cancer cells, particularly antibodies for use as immunologic targeting reagents for the construction of immunotoxins. These colloidal gold conjugates with monoclonal antibodies have demonstrated the internalization of these immunologic reagents through coated pits and receptosomes, which is a necessary step in the delivery of immunotoxins into the cell where they can mediate their cell-killing functions. Morphologic methods have been employed for the screening and selection of monoclonal antibodies reactive with the surface of human ovarian cancer cells for use as immunotoxins and have demonstrated the in vivo activity of immunotoxins made with these antibodies and Pseudomonas exotoxin in a nude mouse model system. In other studies, we have employed such reagents for the immunocytochemical detection of the surface expression of P170, the cell-surface efflux pump protein responsible for the phenotype of multidrug resistance in tumor cells, and to investigate the distribution of this protein by using immunocytochemistry in normal human tissues. These results have suggested a role for P170 in normal cell membrane transport of metabolites in various organ systems.

Anticancer effect of liposome incorporated with methotrexate and antibody against tumor specific surface antigen of rat hepatoma.

antibody against tumor specific surface membrane protein was produced by immunizing a New Zealand White rabbit with antigen (66 kDa) prepared from the plasma membrane of rat hepatoma induced by feeding a diet containing 3'-methyl-4-dimethylaminoazobenzene, and was purified by protein A-Sepharose 6MB affinity chromatography. The purified antibody was incorporated into liposomes by a reverse phase evaporation vesicle method in order to prepare a tumor specific anticancer drug carrier. The effect of the antibody against tumor specific antigen was evaluated by comparing the inhibition of DNA synthesis in hepatoma cells with different preparations of methotrexate. Methotrexate encapsulated into liposome showed a stronger inhibitory effect on DNA synthesis (1.4-1.7 times) than free methotrexate. Liposomes having the antibody showed stronger inhibitory effect (3.1 times) on DNA synthesis than free methotrexate group in hepatic nodular area. From these results, it is concluded that tumor specific antibody inserted into liposomal membrane would be recognized by surface antigens which were expressed on the plasma surface membrane of rat hepatoma cells and thereby increase the carrying efficiency of drugs to the target cells. This could be useful in cancer chemotherapy.