Tumor-associated Surface Antigen Research Articles

Developmental Therapeutics in Metastatic Prostate Cancer: New Targets and New Strategies.

There is an unmet need to develop new treatments for metastatic prostate cancer. With the development of targeted radioligand therapies, bispecific T cell engagers, antibody-drug conjugates and chimeric antigen receptor T cell (CAR T) therapies, tumor-associated cell surface antigens have emerged as new therapeutic targets in metastatic prostate cancer. Ongoing and completed clinical trials targeting prostate-specific membrane antigen (PSMA), six transmembrane epithelial antigens of the prostate 1 (STEAP1), kallikrein-related peptidase 2 (KLK2), prostate stem cell antigen (PSCA), and delta-like protein 3 (DLL3) in metastatic prostate cancer were reviewed. Strategies for sequential or combinational therapy were discussed.

Longitudinal evaluation of the biodistribution and cellular internalization of the bispecific CD3xTRP1 antibody in syngeneic mouse tumor models

BackgroundCD3 bispecific antibodies (CD3-bsAbs) require binding of both a tumor-associated surface antigen and CD3 for their immunotherapeutic effect. Their efficacy is, therefore, influenced by the tumor uptake and the extracellular...

Antibody-drug conjugates: the paradigm shifts in the targeted cancer therapy.

Cancer is one of the deadliest diseases, causing million of deaths each year globally. Conventional anti-cancer therapies are non-targeted and have systemic toxicities limiting their versatile applications in many cancers. So, there is an unmet need for more specific therapeutic options that will be effective as well as free from toxicities. Antibody-drug conjugates (ADCs) are suitable alternatives with the right potential and improved therapeutic index for cancer therapy. The ADCs are highly precise new class of biopharmaceutical products that covalently linked a monoclonal antibody (mAb) (binds explicitly to a tumor-associated surface antigen) with a customized cytotoxic drug (kills cancer cells) and tied via a chemical linker (releases the drug). Due to its precise design, it brings about the target cell killing sparing the normal counterpart and free from the toxicities of conventional chemotherapy. It has never been so easy to develop potential ADCs for successful therapeutic usage. With relentless efforts, it took almost a century for scientists to advance the formula and design ADCs for its current clinical applications. Until now, several ADCs have passed successfully through preclinical and clinical trials and because of proven efficacy, a few are approved by the FDA to treat various cancer types. Even though ADCs posed some shortcomings like adverse effects and resistance at various stages of development, with continuous efforts most of these limitations are addressed and overcome to improve their efficacy. In this review, the basics of ADCs, physical and chemical properties, the evolution of design, limitations, and future potentials are discussed.

Molecular insights into recognition of GUCY2C by T-cell engaging bispecific antibody anti-GUCY2CxCD3

The intestinal epithelial receptor Guanylyl Cyclase C (GUCY2C) is a tumor-associated cell surface antigen expressed across gastrointestinal malignancies that can serve as an efficacious target for colorectal cancer immunotherapy. Here, we describe a yeast surface-display approach combined with an orthogonal peptide-based mapping strategy to identify the GUCY2C binding epitope of a novel anti-GUCY2CxCD3 bispecific antibody (BsAb) that recently advanced into the clinic for the treatment of cancer. The target epitope was localized to the N-terminal helix H2 of human GUCY2C, which enabled the determination of the crystal structure of the minimal GUCY2C epitope in complex with the anti-GUCY2C antibody domain. To understand if this minimal epitope covers the entire antibody binding region and to investigate the impact of epitope position on the antibody’s activity, we further determined the structure of this interaction in the context of the full-length extracellular domain (ECD) of GUCY2C. We found that this epitope is positioned on the protruding membrane-distal helical region of GUCY2C and that its specific location on the surface of GUCY2C dictates the close spatial proximity of the two antigen arms in a diabody arrangement essential to the tumor killing activity of GUCY2CxCD3 BsAb.

Identification of immunotherapy and radioimmunotherapy targets on desmoplastic small round cell tumors.

Development of successful antibody-based immunotherapeutic and radioimmunotherapeutic strategies rely on the identification of cell surface tumor-associated antigens (TAA) with restricted expression on normal tissues. Desmoplastic small round cell tumor (DSRCT) is a rare and generally neglected malignancy that primarily affects adolescent and young adult males. New therapies capable of treating disseminated disease are needed for DSRCT, which is often widespread at diagnosis. We used immunohistochemistry (IHC) on fresh frozen surgical specimens and patient-derived xenograft (PDX) tumors and flow cytometry on DSRCT cell lines to evaluate expression of TAAs in these tumors. In vitro cytotoxicity assays were used to evaluate the efficacy of T cell-engaging bispecific antibodies (T-BsAbs) directed at these targets. In vivo, we used an intraperitoneal xenograft mouse model of DSRCT to test T-BsAbs against several TAAs. In DSRCT specimens we found widespread expression of B7-H3, EGFR, GD2, HER2, mesothelin, and polysialic acid, clinical targets for which specific antibody therapeutics are available. The expression of B7-H3, EGFR, HER2, and mesothelin was confirmed on the cell surface of DSRCT cell lines. In vitro cytotoxicity assays confirmed the efficacy of T cell-engaging bispecific antibodies (T-BsAbs) directed at these targets against DSRCT cells. Remarkably, a HER2xCD3 T-BsAb was capable of completely shrinking established tumors in an intraperitoneal mouse model of DSRCT. We propose that these TAAs should be further investigated in preclinical models as targets for immunotherapy and radioimmunotherapy with the hope of providing a rationale to extend these therapies to patients with advanced DSRCT.

Abstract 2752: Targeting intracellular tumor antigens using fully human TCR mimic antibodies derived from HLA transgenic RenMiceTM

Abstract Therapeutic antibodies have ushered in a new age of cancer immunotherapy. Historically, these therapies have targeted a limited subset of soluble and cell surface tumor-associated antigens (TAAs). T cell receptors (TCRs) on cytotoxic CD8+ T cells recognize peptide antigens bound to major histocompatibility class I (MHC-I) proteins, called HLA-A/B/C in humans. By this pathway, antigen is regularly sampled from the intracellular peptidome, processed, and presented to cytotoxic T cells. Expanding TCR-based recognition of soluble, intracellular TAAs presented on the surface of malignant cells by this mechanism is a propitious therapeutic strategy. Here we describe a novel platform for generating T cell receptor mimic (TCRm) antibodies using our humanized immunoglobulin (RenMabTM) mice engineered to express HLA. TCRm antibodies have the same binding properties as endogenous TCRs and recognize processed, HLA-bound peptides including intracellular tumor-associated antigens, viral oncoproteins, and cancer-testis antigen (CTA). TCRm antibodies bind peptide-HLA with high specificity and up to nanomolar affinity. Our optimized immunization protocols and high-throughput screening methods allow for one-step TCRm antibody generation. TCRm antibodies can also be used to assemble bispecific T cell engaging antibodies (BiTEs) to enhance tumor targeting of cytotoxic T cells. Biocytogen’s TCRm antibodies are a flexible and powerful tool for cancer immunotherapy. By enabling TCR-mediated recognition of an unrestricted repertoire of cancer neoantigens, TCRm antibodies may find broad clinical application. Citation Format: Jun Du, Taolin Liu, Wanbo Tang, Yue Zhang, Limin Zhao, Xin Jiao, Chao Sun, Pengfei Du, Yuqi Zhang, Baihong Liu, Qingcong Lin, Yi Yang. Targeting intracellular tumor antigens using fully human TCR mimic antibodies derived from HLA transgenic RenMiceTM [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 2752.

Abstract 4089: PanCAR-specific antibody-cytokine fusion proteins

Abstract CAR-T cells are a potent immunotherapy that redirect T cell specificity towards cell surface tumor-associated antigens. While this therapy has been effective in hematological malignancies, therapies against solid tumors not achieved the same efficacy due to a multitude of additional challenges, including a lack of tumor specific antigens and an immunosuppressive tumor microenvironment. One strategy to increase T cell potency is to use fourth generation CAR-T cells that also secrete an immunostimulatory factor, such as a cytokine. We have created CAR-T cells targeting Tn-MUC1 utilizing a scFv from the 5E5 antibody that also secrete interleukin-12 (IL-12), interleukin-18 (IL-18), and interleukin-23 (IL-23). These cytokine-secreting CAR-T cells have shown an increase in efficacy against multiple epithelial based tumors in impedance-based killing assays. In a xenograft model of human breast cancer, CAR-T cells secreting IL-12 and IL-23 were able to significantly delay tumor growth compared to non-transduced T cells, CAR-T cells alone, and CAR-T cells secreting IL-18. While these cells showed good efficacy in vivo, clinical translation is difficult due to concerns over cytokine storm due to the constitutive secretion of cytokines. To overcome this, we created a fusion protein utilizing an antibody specific to an invariant region found in majority of CAR molecules, including those utilized commercially available therapies and under pre-clinical development, fused to the three immunostimulatory cytokines. In impedance-based killing assays, antibody-cytokine fusions stimulated non-cytokine secreting CAR-T cells to completely clear the tumor at an E:T ratio in which the CAR-T cells incubated with only the antibody alone were unable to control the tumor growth. Interestingly, combination therapy of CAR-T cells and antibody-cytokine fusions outperformed CAR-T cells constitutively secreting the same cytokines in in vitro assays. This work highlights the use of a single reagent capable of increasing the efficacy of the majority of CAR-T cell therapies and may be an important contribution towards treating solid tumor malignancies with CAR-T cell therapies. Citation Format: John T. Keane, Avery D. Posey. PanCAR-specific antibody-cytokine fusion proteins. [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 4089.

Multivalent adaptor proteins specifically target NK cells carrying a universal chimeric antigen receptor to ErbB2 (HER2)-expressing cancers

Chimeric antigen receptor (CAR)-engineered immune effector cells constitute a promising approach for adoptive cancer immunotherapy. Nevertheless, on-target/off-tumor toxicity and immune escape due to antigen loss represent considerable challenges. These may be overcome by adaptor CARs that are selectively triggered by bispecific molecules that crosslink the CAR with a tumor-associated surface antigen. Here, we generated NK cells carrying a first- or second-generation universal CAR (UniCAR) and redirected them to tumor cells with so-called target modules (TMs) which harbor an ErbB2 (HER2)-specific antibody domain for target cell binding and the E5B9 peptide recognized by the UniCAR. To investigate differential effects of the protein design on activity, we developed homodimeric TMs with one, two or three E5B9 peptides per monomer, and binding domains either directly linked or separated by an IgG4 Fc domain. The adaptor molecules were expressed as secreted proteins in Expi293F cells, purified from culture supernatants and their bispecific binding to UniCAR and ErbB2 was confirmed by flow cytometry. In cell killing experiments, all tested TMs redirected NK cell cytotoxicity selectively to ErbB2-positive tumor cells. Nevertheless, we found considerable differences in the extent of specific cell killing depending on TM design and CAR composition, with adaptor proteins carrying two or three E5B9 epitopes being more effective when combined with NK cells expressing the first-generation UniCAR, while the second-generation UniCAR was more active in the presence of TMs with one E5B9 sequence. These results may have important implications for the further development of optimized UniCAR and target module combinations for cancer immunotherapy.

Chimeric antigen receptor T cells applied to solid tumors.

Chimeric antigen receptor T cell (CAR-T) therapy is novel tumor immunotherapy that enables autologous T to express synthetic receptors to specifically recognize the surface tumor-associated antigens for exerting subsequent antitumor effects, and eliminating the resistance, metastases and recurrence of cancer. Although CAR T cells have exhibited success in eradicating hematologic malignancies, their applications to solid tumors has not yet been achieved due to obstacles such as the immune-suppressor tumor microenvironment and lack of tumor specific target antigens. In this review, we presented advancements in the development of CAR T cell therapy in solid tumors, and offered a brief summary of the challenges, as well as novel engineering and pharmaceutical interventions to overcome these barriers. Looking forward, we discussed the latest studies which are expected to reach the clinicals in the next few years, including CRISPR screens-based CAR modification and CAR T cells driven from progenitor-like T cells. Collectively, this review may inspire researchers and clinicians to develop clinical available strategies of CAR T cell therapies in solid tumor.

Construction of tandem diabody (IL-6/CD20)-secreting human umbilical cord mesenchymal stem cells and its experimental treatment on diffuse large B cell lymphoma

BackgroundMore than 40% patients with diffuse large B cell lymphoma (DLBCL) experienced relapse or refractory (R/R) lymphoma after the standard first R-CHOP therapy. IL-6 was reportedly associated with chemotherapy resistance of rituximab. Further, mesenchymal stem cells (MSCs) are known as the potential cell vehicle for their tropism toward tumor. A MSCs-based tandem diabody for treating DLBCL is currently lacking.MethodsWe constructed a tandem diabody (Tandab(IL-6/CD20)) with modified umbilical cord MSCs (UCMSCs) and designed a cell-based Tandab releasing system. Western blot, qPCR and immunofluorescence were used to confirm the construction and expression of lentivirus-infected UCMSCs. The vitality, apoptosis and homing abilities of UCMSCs were examined via CCK-8 assay, apoptosis, wound healing and migration analysis. Cell binding assay was used to demonstrate the targeting property of Tandab binding to CD20-positive DLBCL cells. Furthermore, we evaluated the viability of SU-DHL-2 and SU-DHL-4 by using CCK-8 and EDU assay after the treatment of UCMSCs-Tandab(IL-6/CD20).ResultsTandab protein peaked at 6273 ± 487 pg/ml in the medium on day 7 after cell culture. The proliferation and homing ability of UCMSCs did not attenuate after genetically modification. Immunofluorescence images indicated the Tandab protein bound to the lymphoma cells. UCMSCs-Tandab(IL-6/CD20) inhibited the growth of SU-DHL-2 or SU-DHL-4 cells in vitro.ConclusionsUCMSCs-Tandab(IL-6/CD20), which bound with both tumor-associated surface antigens and pro-tumor cytokines in tumor microenvironment, might serve as a potential treatment for DLBCL, evidenced by inhibiting the growth of SU-DHL-2 or SU-DHL-4 cells.

Bispecific Antibodies on Hematological Malignancies: A Novel Mechanism of Action That May Contribute to Therapeutic Efficacy

Bispecific antibodies (BsAbs) act through the formation of an immunologic synapse between T-cells (CD3) and a tumor-associated surface antigen (TAA) leading to T-cell activation and serial lysis of tumor cells. The aim of the present study is to explore the mechanism of action (MOA) and the in vitro effect of BsAbs on hematological samples with the PharmaFlow platform.For this purpose, whole bone marrow (BM) and peripheral blood (PB) from 44 samples from 3 different hematological diseases (34 AML, 3 ALL and 7 CLL) and two AML cell lines were tested with CD3-CD123 (for AML patients and cell lines) or CD3-CD19 (for ALL and CLL patients) BsAbs in the PharmaFlow platform, an innovative proprietary method that uses flow cytometry (FCM) to efficiently count the number of tumor cells killed by activated T-cells. We analyzed the populations of leukemic cells, activated T-cells, and residual normal cells. Additional key parameters were also used to explore the MOA after BsAb exposure at different time incubations (24h-144h), such as the effective E:T ratio (the number of T-cells that kill a number of leukemic cells), real basal E:T ratio, tumor antigen expression, T-cell expansion, and expression of immune checkpoint proteins on target and effector cells before and after cell culture. For some experiments, fluorescence-activated cell sorting (FACS) was performed to evaluate T-cell cytotoxicity after BsAb exposure.Most of the samples demonstrated T-cell activation and effective lysis of tumor cells after BsAb exposure independent of TAA expression and in a dose-response manner. Once sorted, these T-cells could kill tumor cells in the absence of BsAb, as well as tumor cells that did not express the TAA target. Interestingly, these activated T-cells selectively killed tumor cells with low cytotoxicity in residual normal cells from the same patients. Moreover, differential T-cell cytotoxicity was observed between samples. We observed samples with leukemic resistance or no T-cell activity (especially in CLL with CD3-CD19), as well as others with higher T-cell cytotoxicity and minimal number of activated T-cells (especially in AML with CD3-CD123). The integration of all the predictive parameters (E:T ratios, Tumor-Specific Antigen (TSA) expression, etc.) allowed us to generate an in vitro response model and select samples with higher T-cell cytotoxicity after the BsAb exposure.Our findings are consistent with a model where, in addition to the standard MOA inducing tumor cells lysis by proximity, BsAbs can highly enrich cytotoxic clonal T-cell subsets with TSA and induce strong activation and proliferation of T-cells capable of killing tumor cells in an effective and selective manner. This differential in vitro T-cell cytotoxicity effect between patients could allow us to select better candidates for adoptive anti-tumor immunotherapy with BsAbs DisclosuresPrimo:Vivia Biotech: Employment. Hernandez:Vivia Biotech: Employment. Ribera:Amgen Inc.: Research Funding, Speakers Bureau; Pfizer: Research Funding, Speakers Bureau; Incyte: Research Funding, Speakers Bureau; ARIAD: Research Funding, Speakers Bureau; Roche: Honoraria; Janssen: Honoraria; Gilead: Honoraria; Celgene: Honoraria. Gorrochategui:Vivia Biotech: Employment. María Luisa:Vivia Biotech: Employment. Montesinos:Celgene Corporation: Honoraria, Research Funding. Ballesteros:Vivia Biotech: Employment.

“In Vivo Vaccination” Effect in Clinical Responders to Anti-Myeloma Monoclonal Antibody Isatuximab

Background: Isatuximab is a monoclonal antibody directed against surface molecule CD38, which is expressed on the tumor cells of patients with Multiple Myeloma (MM). In clinical trials, isatuximab was well tolerated and demonstrated good efficacy as monotherapy and in combination with standards of care. Isatuximab targets a unique epitope and exhibits anti-myeloma activity primarily through innate immunity via antibody-dependent cellular cytotoxicity (ADCC). It has been proposed that cytotoxic monoclonal antibodies can also induce an adaptive immune response against the antibody's respective target antigen as well as other tumor-associated antigens in the host, a so-called “in vivo vaccination” effect, but this has never been demonstrated in patients with MM undergoing treatment.Methods: Immunomonitoring was performed on 4 patients with relapsed / refractory MM, who had received 3-5 (median: 3) prior therapies, enrolled in a Phase I clinical study (NCT02514668) at the Huntsman Cancer Institute. The patients received 20 mg/kg intravenous single-agent isatuximab weekly for the first cycle (4 weeks) and every 2 weeks for all subsequent cycles (4 weeks) until progression or intolerable toxicity. Blood samples were collected before treatment and at weeks 2, 4, 8, 12 and/or end-of-treatment under isatuximab therapy. Additional samples from clinical responders were obtained monthly. Patients' serum samples were analyzed by Enzyme-Linked Immunosorbent Assay (ELISA) for the presence of autologous antibody responses against a total of 45 myeloma-associated antigens. In addition, we determined the number of peripheral blood T cells specific for CD38, the target for isatuximab, as well as for other myeloma-associated antigens using an Enzyme-Linked ImmunoSpot (ELISPOT) assay.Results: We observed clinical responses in 2 out of 4 patients consecutively enrolled in our immunologic analyses. One of these patients (840002003/ID210), a quadruple refractory patient who had received 5 lines of prior therapy, achieved a complete response (CR) and the other patient (840002002/ID246) who was double refractory after 3 lines of prior therapy achieved a stringent complete remission (sCR, Fig. 1). These two patients with an objective clinical response had new or increasing autologous IgG antibody responses against tumor antigens: one patient (ID210) against MAGE-C2, NY-ESO-1, and against the target for isatuximab CD38; the second patient (ID246) against MAGE-C1 (Fig. 1). Both antibody-positive patients had also developed polyclonal T cell responses against CD38. In addition, one of the clinical responders had T cell responses against 2 other myeloma-associated antigens, MAGE-C2 and PRAME (Fig. 1). We did not observe any new or increasing autologous antibody responses against myeloma-associated antigens in the 2 patients who had not clinically responded to isatuximab.Conclusions: Here, we demonstrate for the first time that treatment with a monoclonal antibody directed against a tumor-associated surface antigen, such as CD38, can lead to an “in vivo vaccination” effect where patients with MM develop autologous T cell responses against the therapeutic target as well as antigen spreading to tumor-associated antigens independent of the target for the monoclonal antibody. Among the limited number of patients analyzed, these autologous adaptive immune responses were observed only in patients with clinical responses to isatuximab treatment.This work was supported by Sanofi. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Retargeting of NK-92 Cells against High-Risk Rhabdomyosarcomas by Means of an ERBB2 (HER2/Neu)-Specific Chimeric Antigen Receptor.

Simple SummaryIn this study, we apply the ERBB2-chimeric antigen receptor (CAR)-modified natural killer (NK) cell line NK-92 (NK-92/5.28.z), a well-defined, good manufacturing practice (GMP)-compliant, third-party, off-the-shelf immune effector cell product as a novel immunotherapeutic approach for the treatment of high-risk rhabdomyosarcomas. Our preclinical in vitro data show enormous potential to improve immunotherapy of ERBB2-positive high-risk rhabdomyosarcoma a still incurable, rapidly lethal disease, assigning to NK-92/5.28.z cells rather than to unmodified parental NK-92 cells a multifarious role as ERBB2-specific CAR-targeted killers and modulators of endogenous adaptive immunity of the host, justifying the further evaluation of this approach in in vivo mouse xenograft models as a prerequisite for a possible future phase I/II clinical trial in defined subsets of high-risk rhabdomyosarcoma patients.The dismal prognosis of pediatric and young adult patients with high-risk rhabdomyosarcoma (RMS) underscores the need for novel treatment options for this patient group. In previous studies, the tumor-associated surface antigen ERBB2 (HER2/neu) was identified as targetable in high-risk RMS. As a proof of concept, in this study, a novel treatment approach against RMS tumors using a genetically modified natural killer (NK)-92 cell line (NK-92/5.28.z) as an off-the-shelf ERBB2-chimeric antigen receptor (CAR)-engineered cell product was preclinically explored. In cytotoxicity assays, NK-92/5.28.z cells specifically recognized and efficiently eliminated RMS cell suspensions, tumor cell monolayers, and 3D tumor spheroids via the ERBB2-CAR even at effector-to-target ratios as low as 1:1. In contrast to unmodified parental NK-92 cells, which failed to lyse RMS cells, NK-92/5.28.z cells proliferated and became further activated through contact with ERBB2-positive tumor cells. Furthermore, high amounts of effector molecules, such as proinflammatory and antitumoral cytokines, were found in cocultures of NK-92/5.28.z cells with tumor cells. Taken together, our data suggest the enormous potential of this approach for improving the immunotherapy of treatment-resistant tumors, revealing the dual role of NK-92/5.28.z cells as CAR-targeted killers and modulators of endogenous adaptive immunity even in the inhibitory tumor microenvironment of high-risk RMS.

The surface dominant antigen MUC1 is required for colorectal cancer stem cell vaccine to exert anti-tumor efficacy

Colorectal cancer (CRC), initiated and maintained by colorectal cancer stem cells (CCSCs), ranks the third most common cancers and has drawn wide attentions worldwide. Therefore, targeting clearance of CCSCs has become an important strategy of CRC immunotherapy. Mucin1 (MUC1) is a tumor-associated cell surface antigen of CRC, but its role in CCSC vaccine remains unclear. In the study, we demonstrated that MUC1 may be a dominant antigen to exert antitumor immunity in CCSC vaccine. First, CCSCs were enriched from CT26 cell line via a serum-free sphere formation approach, and were identified by detecting expression of CD133, ALDH, and ALCAM. Then, the isolated CCSCs were frozen for 30 min and thawed for 30 min to prepare the cell lysate. The specific anti-MUC1 antibody was added to the cell lysate to neutralize the dominant antigen MUC1. Finally, mice were subcutaneously immunized with the cell lysate, followed by a challenge with CT26 cells at one week after final vaccination. Attractively, CCSC vaccine significantly activated the NK cells, T cells, and B cells, resulting in inhibiting the tumor growth via a target killing of CCSCs as evidenced by a decrease of CD133+cells in tumor compared to CCSC vaccine with specific anti-MUC1 antibody. In addition, CCSC vaccine reduced expression of inflammatory factors in vaccinated mice. As expected, neutralizing antibody against MUC1 significantly impaired the antitumor efficacy of CCSC vaccine. Overall, CCSC vaccine could serve as a potent vaccine for CRC immunotherapy. The surface dominant antigen MUC1 may play a key role in regulating immunogenicity of CCSCs.

Abstract IA20: Circulating tumor cells: Biology and clinical applications as liquid biopsy

Abstract The analysis of circulating tumor cells (CTCs) in blood may provide clinically relevant information as “liquid biopsy” and provide new insights into tumor biology (Alix-Panabieres, Pantel, Nature Rev Cancer 2014; Keller, Pantel, Nature Rev Cancer 2019). Various technologies have been developed over the past 10 years, which include label-dependent assays based on the expression of tumor-associated cell surface antigens and label-independent assays based on physical properties of tumors cells distinct from the surrounding leukocytes (Pantel, Alix-Panabieres, Nature Rev Cancer 2019). After detection, CTCs can be further analyzed at the DNA, RNA, and protein level to obtain global information on tumor biology and targets relevant to cancer therapy (Pantel, Alix-Panabieres, Nature Rev Clin Oncol 2019). Liquid biopsy analyses with validated platforms provide reliable information on prognosis and may serve to identify therapeutic targets or mechanisms of resistance on metastatic cells. Metastatic cells might have unique characteristics that can differ from the bulk of cancer cells in the primary tumor currently used for stratification of patients to systemic therapy. Moreover, monitoring of blood samples before, during, and after systemic therapy (e.g., chemotherapy or targeted therapy) provides unique information for the future clinical management of the individual cancer patient and serves as surrogate marker for response to therapy (Alix-Panabieres, Pantel, Cancer Discovery 2016; Bardelli, Pantel, Cancer Cell 2017). Citation Format: Klaus Pantel. Circulating tumor cells: Biology and clinical applications as liquid biopsy [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr IA20.

Mass spectrometry-based identification of a B-cell maturation antigen-derived T-cell epitope for antigen-specific immunotherapy of multiple myeloma

The B-cell maturation antigen (BCMA) is currently being evaluated as promising tumor-associated surface antigen for T-cell-based immunotherapy approaches, such as CAR T cells and bispecific antibodies, in multiple myeloma (MM). Cytotoxic T cells bearing BCMA-specific T-cell receptors might further allow targeting HLA-presented antigens derived from the intracellular domain of BCMA. By analyzing a mass spectrometry-acquired immunopeptidome dataset of primary MM samples and MM cell lines for BCMA-derived HLA ligands, we identified the naturally presented HLA-B*18-restricted ligand P(BCMA)B*18. Additionally, P(BCMA)B*18 was identified on primary CLL samples, thereby expanding the range for possible applications. P(BCMA)B*18 induced multifunctional BCMA-specific cells de novo from naïve CD8+ T cells of healthy volunteers. These T cells exhibited antigen-specific lysis of autologous peptide-loaded cells. Even in the immunosuppressive context of MM, we detected spontaneous memory T-cell responses against P(BCMA)B*18 in patients. By applying CTLA-4 and PD-1 inhibition in vitro we induced multifunctional P(BCMA)B*18-specific CD8+ T cells in MM patients lacking preexisting BCMA-directed immune responses. Finally, we could show antigen-specific lysis of autologous peptide-loaded target cells and even MM.1S cells naturally presenting P(BCMA)B*18 using patient-derived P(BCMA)B*18-specific T cells. Hence, this BCMA-derived T-cell epitope represents a promising target for T-cell-based immunotherapy and monitoring following immunotherapy in B-cell malignancy patients.

Cell Surface Notch Ligand DLL3 is a Therapeutic Target in Isocitrate Dehydrogenase–mutant Glioma

Isocitrate dehydrogenase (IDH)-mutant glioma is a distinct glioma molecular subtype for which no effective molecularly directed therapy exists. Low-grade gliomas, which are 80%-90% IDH-mutant, have high RNA levels of the cell surface Notch ligand DLL3. We sought to determine DLL3 expression by IHC in glioma molecular subtypes and the potential efficacy of an anti-DLL3 antibody-drug conjugate (ADC), rovalpituzumab tesirine (Rova-T), in IDH-mutant glioma. We evaluated DLL3 expression by RNA using TCGA data and by IHC in a discovery set of 63 gliomas and 20 nontumor brain tissues and a validation set of 62 known IDH wild-type and mutant gliomas using a monoclonal anti-DLL3 antibody. Genotype was determined using a DNA methylation array classifier or by sequencing. The effect of Rova-T on patient-derived endogenous IDH-mutant glioma tumorspheres was determined by cell viability assay. Compared to IDH wild-type glioblastoma, IDH-mutant gliomas have significantly higher DLL3 RNA (P < 1 × 10-15) and protein by IHC (P = 0.0014 and P < 4.3 × 10-6 in the discovery and validation set, respectively). DLL3 immunostaining was intense and homogeneous in IDH-mutant gliomas, retained in all recurrent tumors, and detected in only 1 of 20 nontumor brains. Patient-derived IDH-mutant glioma tumorspheres overexpressed DLL3 and were potently sensitive to Rova-T in an antigen-dependent manner. DLL3 is selectively and homogeneously expressed in IDH-mutant gliomas and can be targeted with Rova-T in patient-derived IDH-mutant glioma tumorspheres. Our findings are potentially immediately translatable and have implications for therapeutic strategies that exploit cell surface tumor-associated antigens.

Exceptionally Potent Cytotherapy Using T Cells Armed with Novel Tetravalent Recombinant Bispecific Antibodies Specific for GD2 and HER2

▪Introduction: T-cell based therapies have emerged as one of the major breakthroughs in anticancer treatment: Immune checkpoint inhibitors, chimeric antigen receptor gene-modified T-cells (CAR-T-cells), and T-cell engaging bispecific antibodies (BsAb) are leading the advances. In the era of personalized medicine, T-cells offer alternative strategies to overcome resistance to chemotherapy or small molecules. Yet, hurdles for such therapy can be crippling, such as inability of T cells to infiltrate “cold tumors”, cytokine release syndrome following T cell-based therapies, neurologic toxicity, and on-target off-tumor effects. To address these hurdles, polyclonal T-cells armed with GD2xCD3 or HER2xCD3 BsAb for cytotherapy hold promise. Ganglioside GD2 and HER2 are tumor associated surface antigens expressed in a broad spectrum of aggressive malignancies, while being restricted in normal tissues. Phase I trials of T-cells armed with a chemical conjugate of hu3F8 x mouse OKT3 (NCT02173093) or trastuzumab x mouse OKT3 (NCT00027807) demonstrated the safety of 160 x 106/kg/injection x 8 doses (or 1.28 x 109/cycle) with suggestion of clinical benefit. Here, we report the safety and efficacy of adoptive T-cell therapy armed with the recombinant forms of these BsAb for the treatment of GD2(+) and/or HER2(+) tumors in preclinical models.Methods: Recombinant anti-GD2 BsAb and anti-HER2 BsAb were made using the IgG(L)-scFv platform (Can Immunol Res, 3:266, 2015, Oncoimmunology, PMID:28405494). T-cells from normal volunteer donors were isolated, activated and expanded by CD3/CD28 beads in the presence of 100 IU/mL of interleukin 2 (IL-2). Between day 7 and day 14, activated T cells (ATCs) were harvested and armed for 20 minutes at room temperature with -GD2-BsAb or HER2-BsAb. After washing, armed ATCs were tested for cell surface density of BsAb and antibody dependent T cell mediated cytotoxicity (ADTC) in vitro. In vivo anti-tumor potencies of armed T cells were tested against GD2(+) or HER2(+) cell lines or patient derived xenografts (PDXs) in BALB-Rag2-/-IL-2R-γϲ-KO (DKO) mice.Results: GD2-BsAb of the IgG(L)-scFv form showed superior potency over other bispecific platforms in vitro and in vivo. GD2-BsAb or HER2-BsAb armed ATCs showed potent antigen-specific cytotoxicity against GD2 or HER2 positive tumors such as neuroblastoma, melanoma and osteosarcoma in vitro over a range of antibody dose (5 to 500 ng/106 cells). Optimal arming per T cell required 25,000 to 45,000 idiotype(+) molecules. There was no evidence of activation induced cell death when confronted by antigen or tumor target. In vivo GD2-BsAb or HER2-BsAb armed ATCs could ablate neuroblastoma, malignant melanoma, and osteosarcoma tumors over a range of cell doses (10x106, 20x106 and 40x106 per injection, one to three times a week for 2 to 4 weeks) with a range of BsAb doses (5 ng to 500 ng/million of T-cells) without significant toxicities in DKO mice. By immunohistochemistry, the frequency of tumor infiltrating CD3(+) T-cells strongly correlated with tumor response.Conclusions:Using the IgG(L)-scFv format, GD2-BsAb or HER2-BsAb armed ATCs could provide a potent and economical cytotherapy platform against GD2(+) or HER2(+) tumors without the complexity of gene modification (as in chimeric antigen receptor modified T cells). At such low arming doses, where BsAb is T cell bound, where ADTC is not induced, and T cell expansion is not required for anti-tumor effect, clinical toxicity is expected to be low. DisclosuresCheung:Ymabs: Patents & Royalties.

Abstract B38: Tetravalent bispecific antibodies specific for HER2 and disialoganglioside GD2 to engage polyclonal T cells for osteosarcoma therapy

Abstract Introduction: Osteosarcoma is the most common pediatric high-grade bone tumor. Although multimodal therapeutic approaches have significantly improved patient survival to more than 60%, the prognosis for patients with metastatic or relapsed disease remains dismal—an urgent unmet need. Its genomic complexity and higher mutational burden compared to other pediatric cancers should provide neoantigens as potential targets for T cell-based immunotherapy; however, these tumors are in general “cold,” with insufficient or inactive tumor-infiltrating lymphocytes. Ganglioside GD2 is a tumor-associated surface antigen expressed in a broad spectrum of pediatric malignancies, including neuroblastoma, brain tumors, Ewing’s sarcoma, rhabdomyosarcoma, and osteosarcoma, while being restricted in normal tissues. Another promising target, HER2 or ErbB2, is overexpressed in many aggressive malignancies, including pediatric medulloblastoma, nephroblastoma, osteosarcoma, and desmoplastic small round cell tumor (DSRT). Fully humanized tetravalent bispecific antibodies (BsAb) specific for human GD2 and CD3 (hu3F8-BsAb) or HER2 and CD3 (HER2-BsAb), built on an IgG(L)-scFv platform, have been developed (Oncoimmunology 2017; Can Immunol Res 2015). They induce rapid and quantitative T-cell homing to tumors, mediating potent T-cell dependent cytotoxicity (TDCC) against GD2 or HER2 expressing tumor cells, effecting cures of xenografts in SCID mice. In this study, we evaluated the in vitro and in vivo antitumor properties of these BsAbs in the treatment of osteosarcoma. Methods: Hu3F8-BsAb and HER2-BsAb were tested both in vitro and in vivo against a panel of osteosarcoma cell lines (RG143B, U2OS, CRL1427, HOS, and SaOS2). Using these cell lines, FACS analysis for surface antigen (GD2 and HER2) expression (MFI), sensitivity to TDCC (EC50), and antitumor activity in vivo were evaluated. Two in vivo tumor models with different effector routes were used to simulate different clinical situations: 1) subcutaneous (sc) tumor cells/sc effector peripheral blood mononuclear cells (PBMCs), and 2) sc tumor cells/intravenous (iv) PBMCs. In vivo tumor responses were measured by Peira TM900 imaging device or by bioluminescence. Results: The majority of osteosarcoma cell lines express GD2 and HER2 on their surface, and their in vitro sensitivity to TDCC (EC50) was inversely correlated with MFI of their respective antigen. Both hu3F8-BsAb and HER2-BsAb mediated potent TDCC against osteosarcoma cell lines. In vivo, both hu3F8-BsAb and HER2-BsAb exerted a significant antitumor effect compared to control BsAb (P=0.001). Conclusions: Hu3F8-BsAb and HER2-BsAb induced strong TDCC and had significant antitumor effect against osteosarcoma cell lines both in vitro and in vivo. Considering the limited therapeutic options currently available in advanced osteosarcomas, these results support their further clinical development as potential T cell-based immunotherapeutics. Citation Format: Jeong A. Park, Hong Xu, Irene Cheung, Nai-Kong V. Cheung. Tetravalent bispecific antibodies specific for HER2 and disialoganglioside GD2 to engage polyclonal T cells for osteosarcoma therapy [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr B38.

Abstract 4631: Expression of ganglioside GD2 in Ewing sarcoma cells is not associated with specific functional characteristics or stem cell features

Abstract Safe and effective targeting of cancer with chimeric antigen receptor (CAR) engineered T cells relies on the presence of adequate tumor-associated surface antigens. We have found that Ewing sarcomas (EwS) express ganglioside GD2 and are recognized by T cells engineered with GD2-specific CARs. In an extended series of 97 EwS patient samples, 52 were GD2pos by immunohistochemistry. Based on reports that GD2 in breast cancer defines a malignant population with stem cell characteristics, we hypothesized that GD2 expression in EwS is associated with high capacity to self-renew and reinitiate tumor growth. Among 15 individual EwS cell lines with variable surface expression of GD2 by flow cytometry, GD2 expression levels were not associated with the capacities to proliferate and expand in vitro, form colonies in semi-solid medium, nor with their chemosensitivity, assessed by cell viabilities in the presence of increasing concentrations of the cytotoxic drug doxorubicin. Subpopulations with GD2hi phenotype selected from GD2pos and from GD2low EwS cell lines by cell sorting maintained their GD2hi and GD2low expression status during subsequent cultures over several weeks. The two subpopulations did not have different in vitro growth, colony-formation capacity, or chemosensitivity. In xenografting experiments, GD2hi and GD2low subpopulations of two EwS cell lines initiated tumors with comparable efficacies. To obtain direct evidence that GD2 surface expression is irrelevant for the biology of EwS cells, we performed genetic knockdown of the GD3S gene which drives biosynthesis of GD2 by CRISPR/Cas9 gene editing. GD3S gene editing resulted in effective elimination of GD2 surface expression in the GD2hi EwS cell lines TC-71, VH-64 and A4573. The knockdown did not affect the capacity of the cells to proliferate, form colonies in soft agar in vitro, nor their chemosensitivity compared to wild-type EwS cells from the individual cell lines. We conclude that GD2 expression in EwS cells, other than reported in breast cancer, is not associated with distinct functional features. Specifically, GD2 does not affect the growth characteristics, clono- and tumorigenicity and chemosensitivity of the tumor cells. Elimination of GD2pos subpopulations from heterogeneous tumors by CAR T cell targeting is therefore unlikely to eradicate the disease and will have to be combined with additional targeting strategies. Citation Format: Sareetha Kailayangiri, Bianca Altvater, Silke Jamitzky, Stefanie Lesch, Jan-Henrik Mikesch, Sonja Schelhaas, Schaefers Michael, Jutta Meltzer, Nicole Farwick, Wolfgang Hartmann, Eva Wardelmann, Petra Fischer, Jendrik Hardes, Claudia Rossig. Expression of ganglioside GD2 in Ewing sarcoma cells is not associated with specific functional characteristics or stem cell features [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4631.