<p>L2 CAR T cells reduce mammosphere formation when compared to irrelevant CAR control cells.</p>

L2 CAR T cells provide a survival advantage and disrupt vessels and tumor architecture in TNBC xenografts .

<p>Mice that received systemic administration of L2 CAR T cells have normal pathology.</p>

<p>TEM8 distribution in normal tissues</p>

<p>Mice that received systemic administration of L2 CAR T cells have normal pathology.</p>

<p>Information about methods and data that further support the claims made in this article. This includes, but is not limited to TEM8 distribution in normal tissues, CAR T cell normalization, additional control groups and CAR toxicity studies.</p>

L2 CAR T cells provide a survival advantage and disrupt vessels and tumor architecture in TNBC xenografts .

L2 2G and L2 3G CAR T cell normalization and functional testing.

<p>No overt toxicity detected in mice receiving TEM8 CAR T cells.</p>

Collagen I, the most abundant protein in humans, is ubiquitous in solid tumors where it provides a rich source of exploitable metabolic fuel for cancer cells. While tumor cells were unable to exploit collagen directly, here we show they can usurp metabolic byproducts of collagen-consuming tumor-associated stroma. Using genetically engineered mouse models, we discovered that solid tumor growth depends upon collagen binding and uptake mediated by the TEM8/ANTXR1 cell surface protein in tumor-associated stroma. Tumor-associated stromal cells processed collagen into glutamine, which was then released and internalized by cancer cells. Under chronic nutrient starvation, a condition driven by the high metabolic demand of tumors, cancer cells exploited glutamine to survive, an effect that could be reversed by blocking collagen uptake with TEM8 neutralizing antibodies. These studies reveal that cancer cells exploit collagen-consuming stromal cells for survival, exposing an important vulnerability across solid tumors with implications for developing improved anticancer therapy.

Although nonmalignant stromal cells facilitate tumor growth and can occupy up to 90% of a solid tumor mass, better strategies to exploit these cells for improved cancer therapy are needed. Here, we describe a potent MMAE-linked antibody-drug conjugate (ADC) targeting tumor endothelial marker 8 (TEM8, also known as ANTXR1), a highly conserved transmembrane receptor broadly overexpressed on cancer-associated fibroblasts, endothelium, and pericytes. Anti-TEM8 ADC elicited potent anticancer activity through an unexpected killing mechanism we term DAaRTS (drug activation and release through stroma), whereby the tumor microenvironment localizes active drug at the tumor site. Following capture of ADC prodrug from the circulation, tumor-associated stromal cells release active MMAE free drug, killing nearby proliferating tumor cells in a target-independent manner. In preclinical studies, ADC treatment was well tolerated and induced regression and often eradication of multiple solid tumor types, blocked metastatic growth, and prolonged overall survival. By exploiting TEM8 tumor stroma fortargeted drug activation, these studies reveal a drug delivery strategy with potential to augment therapies against multiple cancer types.

Abstract Triple-negative breast cancer (TNBC) is an aggressive disease lacking targeted therapy. In this study, we developed a CAR T cell–based immunotherapeutic strategy to target TEM8, a marker initially defined on endothelial cells in colon tumors that was discovered recently to be upregulated in TNBC. CAR T cells were developed that upon specific recognition of TEM8 secreted immunostimulatory cytokines and killed tumor endothelial cells as well as TEM8-positive TNBC cells. Notably, the TEM8 CAR T cells targeted breast cancer stem–like cells, offsetting the formation of mammospheres relative to nontransduced T cells. Adoptive transfer of TEM8 CAR T cells induced regression of established, localized patient-derived xenograft tumors, as well as lung metastatic TNBC cell line–derived xenograft tumors, by both killing TEM8+ TNBC tumor cells and targeting the tumor endothelium to block tumor neovascularization. Our findings offer a preclinical proof of concept for immunotherapeutic targeting of TEM8 as a strategy to treat TNBC. Significance: These findings offer a preclinical proof of concept for immunotherapeutic targeting of an endothelial antigen that is overexpressed in triple-negative breast cancer and the associated tumor vasculature. Cancer Res; 78(2); 489–500. ©2017 AACR.

Lacking marked expression of human epidermal growth factor receptor 2 (HER2), estrogen receptor (ER), and progesterone receptor (PR), triple-negative breast cancer (TNBC) is a breast cancer subtype in desperate need of targeted therapy options. Tumor endothelial marker 8 (TEM8), initially identified as a tumor endothelium marker in colon cancer, has been shown to be upregulated in TNBC. To confirm this, we stained primary TNBC tissues for TEM8; in all cases TEM8 was expressed with no expression in normal breast tissue. TEM8 is expressed by TNBC cell lines as indicated by flow cytometry and Western blot. We thus engineered chimeric antigen receptor (CAR) T cells to specifically target TEM8 in TNBC. TEM8 CAR T cells distinctly recognized TEM8, secreted immunostimulatory cytokines, and killed TEM8-positive TNBC cells in vitro. In vivo, the adoptive transfer of TEM8 CAR T cells induced regression against orthotopic patient-derived xenograft (PDX) models, including the aggressive claudin-low TNBC PDX, WHIM12. Systemic administration of TEM8 CAR T cells also induced regression against a lung metastasis TNBC model. In all models, treatment with TEM8 CAR T cells resulted in a survival advantage in mice compared to controls. Hence, TEM8 may serve as an attractive targeted immunotherapy of TNBC. Citation Format: Tiara Byrd, Kristen Fousek, Antonella Pignata, Christopher Szot, Heba Samaha, Lacey Dobrolecki, Htoo Zarni Oo, Poul Sorensen, Matthew Ellis, Michael Lewis, Meenakshi Hegde, Bradley Fletcher, Brad St. Croix, Nabil Ahmed. TEM8 specific CAR T cells induce regression of patient-derived xenograft and metastatic models of triple-negative breast cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A25.

Targeting the tumor vasculature with antibody-drug conjugates (ADCs) is a promising anti-cancer strategy that in order to be realized must overcome several obstacles, including identification of suitable targets and optimal warheads. Here, we demonstrate that the cell-surface protein CD276/B7-H3 is broadly overexpressed by multiple tumor types on both cancer cells and tumor-infiltrating blood vessels, making it a potentially ideal dual-compartment therapeutic target. In preclinical studies CD276 ADCs armed with a conventional MMAE warhead destroyed CD276-positive cancer cells, but were ineffective against tumor vasculature. In contrast, pyrrolobenzodiazepine-conjugated CD276 ADCs killed both cancer cells and tumor vasculature, eradicating large established tumors and metastases, and improving long-term overall survival. CD276-targeted dual-compartment ablation could aid in the development of highly selective broad-acting anti-cancer therapies.

Background: Triple Negative Breast Cancer (TNBC) refers to an aggressive subtype of breast cancer negative for HER2, estrogen and progesterone receptors. Lacking these receptors, individuals with TNBC do not benefit from many of the targeted therapies for breast cancer. Tumor endothelial marker 8 (TEM8), originally identified as a tumor endothelium associated antigen, has more recently been implicated in TNBC pathogenesis and as a marker of breast cancer stem like cells. Here we report that T cells expressing a TEM8-specific chimeric antigen receptor (CAR) serve as a novel approach to target both TNBC cells and its supporting endothelium. CARs combine the specificity of a monoclonal antibody with the signaling properties of a T cell. Methods: We designed two novel TEM8-specific CAR molecules. A CAR molecule containing an exodomain derived from the anti-TEM8 L2 antibody, followed by CD28 and CD3-zeta signaling domains (second generation CAR) and CD28, 41BB and CD3-zeta signaling domains (third generation CAR), respectively. Retroviral transduction was used to express the TEM8 CAR transgene constructs on HEK 293T cells, then on primary T cells. Results: Immunofluorescence staining revealed that in a panel of primary TNBC breast cancer samples, TEM8 was overexpressed in comparison to normal adjacent breast tissue (6/6). Costaining with the pan-endothelial cell marker CD31 revealed that this overexpression was not confined to the endothelial compartment, but also present on tumor parenchymal cells. The immortalized TNBC cell lines (MDA-MB-231, MDA-MB-436, MDA-MB-468 and Hs578T) expressed endogenous levels of TEM8 protein as revealed by western blot. Greater than 90% transduction of primary human T cells was achieved using both of our CAR constructs, as detected by flow cytometry. TEM8 specific T cells displayed significantly higher killing of TEM8 positive TNBC and tumor endothelial cells (2H11 and bEND.3) in standard four hour chromium release assays when compared to both non-transduced or irrelevant (CD19) CAR T cells and secreted immunomostimulatory cytokines upon encounter of TEM8 positive cells in coculture assays. In a vascularized xenograft model, MDA MB468 cells were injected subcutaneously with matrigel into athymic nude mice and followed via bioluminescence imaging over the course of two months. Established tumors were treated with either, second or third generation TEM8 specific T cells, HER2 specific T cells, non-transduced T cells or left untreated. Relative to non-transduced T cells, TEM8 specific second and third generation CAR T cells significantly delayed tumor growth by 36 days and 50 days, respectively. Conclusion: TEM8 specific CAR T cells could serve as a novel targeted therapy for TNBC and supporting endothelium. Citation Format: Byrd T, Fousek K, Pignata A, Szot C, Bielamowicz K, Wakefield A, Koch J, Landi D, Seaman S, Wels W, Fletcher B, Hegde M, St Croix B, Ahmed N. TEM8 specific CAR T cells serve as a novel targeted therapy for triple negative breast cancer and its supporting endothelium. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr PD3-07.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with no approved targeted therapies. Tumor endothelial marker 8 (TEM8), initially identified as a marker of tumor endothelial cells in colorectal cancer and other solid tumors has recently been shown to be upregulated in TNBC and breast cancer stem cells (BCSCs). We investigated whether TEM8 specific chimeric antigen receptor (CAR) T cells recognize and kill both tumor endothelial cells as well as TNBC tumor cells. TEM8 specific CAR molecules were generated using single chain variable fragment derived from the monoclonal antibody, L2. L2 CAR T cells selectively recognized TEM8, secreted immunostimulatory cytokines and effectively killed both TEM8 positive TNBC and tumor endothelial cell lines. Moreover, L2 CAR T cells targeted breast cancer stem cells significantly reducing the number of mammospheres relative to non-transduced T cells. In vivo, adoptive transfer of L2 CAR T cells induced regression of established vascularized TNBC xenografts. Hence, TEM8 may serve as an attractive target for immunotherapy of TNBC. Citation Format: Tiara Byrd, Kristen Fousek, Antonella Pignata, Christopher Szot, Kevin Bielamowicz, Steven Seaman, Daniel Landi, Nino Rainusso, Poul Sorensen, Joachim Koch, Winfried Wels, Bradley Fletcher, Meenakshi Hegde, Brad St Croix, Nabil Ahmed. TEM8/ANTXR1 specific T cells co-target tumor stem cells and tumor vasculature in triple-negative breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2312.

Triple-negative breast cancer (TNBC) refers to a subset of breast cancers that are HER2, estrogen-receptor and progesterone-receptor negative. Associated with an aggressive phenotype and high incidence of recurrence, and devoid of the aforementioned receptors, there are currently no targeted therapies for TNBC. Tumor Endothelial Marker 8 (TEM8) is one of nine gene products up-regulated in the tumor vs. normal endothelium and is overexpressed in TNBC. To investigate whether TEM8 could serve as a viable target for TNBC therapy. To validate the target antigen we conducted double immunofluorescence staining of a cohort of 6 TNBC specimens for TEM8 and the pan-endothelial cell marker (CD31). In all 6 cases TEM8 was overexpressed, when compared to normal adjacent breast tissue. TEM8 staining was not restricted to the CD31 positive cells, but also detected in tumor parenchymal cells. The immortalized TNBC cell lines (MDA-MB-231, MdA-MB-436, MDA-MB-468 and Hs578T) expressed endogenous levels of TEM8 protein as revealed by western blot. To target TNBC using T cells we designed in silico a novel TEM8-specific chimeric antigen receptor (CAR) molecule. This CAR consisted of a TEM8 recognition extracellular domain single-chain variable fragment (scFv) derived from the monoclonal antibody, L2, a connecting transmembrane chain, followed by CD28, 41BB and CD3-zeta chain intracellular signaling domains. The TEM8 CAR-encoding DNA construct was synthesized and then sequence verified. Retroviral transduction was used to integrate the TEM8 CAR transgene in HEK 293T, then on primary T cells. Over 90% of primary human T cells expressed the TEM8 specific CAR, as indicated by flow cytometry. TEM8 specific T cells recognized and killed TEM8 positive cells in standard 4 hour 51Cr release cytotoxicity assays, effectively targeting both TNBC and tumor endothelium cell lines. Further, TEM8 CAR T cells secreted immunostimulatory cytokines in coculture. In contrast, there was minimal reactivity against TEM8 negative targets or by non-transduced T cells from the same blood donor. Adoptively transferred TEM8 specific CAR T cells resulted in a significant decline in the tumor growth of vascularized orthotopic breast cancer xenografts in a murine model, compared to nontransduced T cells (p=0.03). We conclude that TEM8 specific CAR T cells could serve as a tumor and vascular targeted therapy for TNBC.

Irreversible electroporation (IRE) is an emerging focal therapy which is demonstrating utility in the treatment of unresectable tumors where thermal ablation techniques are contraindicated. IRE uses ultra-short duration, high-intensity monopolar pulsed electric fields to permanently disrupt cell membranes within a well-defined volume. Though preliminary clinical results for IRE are promising, implementing IRE can be challenging due to the heterogeneous nature of tumor tissue and the unintended induction of muscle contractions. High-frequency IRE (H-FIRE), a new treatment modality which replaces the monopolar IRE pulses with a burst of bipolar pulses, has the potential to resolve these clinical challenges. We explored the pulse-duration space between 250 ns and 100 μs and determined the lethal electric field intensity for specific H-FIRE protocols using a 3D tumor mimic. Murine tumors were exposed to 120 bursts, each energized for 100 μs, containing individual pulses 1, 2, or 5 μs in duration. Tumor growth was significantly inhibited and all protocols were able to achieve complete regressions. The H-FIRE protocol substantially reduces muscle contractions and the therapy can be delivered without the need for a neuromuscular blockade. This work shows the potential for H-FIRE to be used as a focal therapy and merits its investigation in larger pre-clinical models.

Tumor endothelial marker 8 (TEM8) is a cell surface receptor that is highly expressed in a variety of human tumors and promotes tumor angiogenesis and cell growth. Antibodies targeting TEM8 block tumor angiogenesis in a manner distinct from the VEGF receptor pathway. Development of a TEM8 imaging agent could aid in patient selection for specific antiangiogenic therapies and for response monitoring. In these studies, L2, a therapeutic anti-TEM8 monoclonal IgG antibody (L2mAb), was labeled with 89Zr and evaluated in vitro and in vivo in TEM8 expressing cells and mouse xenografts (NCI-H460, DLD-1) as a potential TEM8 immuno-PET imaging agent. 89Zr-df–L2mAb was synthesized using a desferioxamine–L2mAb conjugate (df–L2mAb); 125I-L2mAb was labeled directly. In vitro binding studies were performed using human derived cell lines with high, moderate, and low/undetectable TEM8 expression. 89Zr-df–L2mAb in vitro autoradiography studies and CD31 IHC staining were performed with cryosections from human tumor xenografts (NCI-H460, DLD-1, MKN-45, U87-MG, T-47D, and A-431). Confirmatory TEM8 Western blots were performed with the same tumor types and cells. 89Zr-df–L2mAb biodistribution and PET imaging studies were performed in NCI-H460 and DLD-1 xenografts in nude mice. 125I-L2mAb and 89Zr-df–L2mAb exhibited specific and high affinity binding to TEM8 that was consistent with TEM8 expression levels. In NCI-H460 and DLD-1 mouse xenografts nontarget tissue uptake of 89Zr-df–L2mAb was similar; the liver and spleen exhibited the highest uptake at all time points. 89Zr-L2mAb was highly retained in NCI-H460 tumors with <10% losses from day 1 to day 3 with the highest tumor to muscle ratios (T:M) occurring at day 3. DLD-1 tumors exhibited similar pharmacokinetics, but tumor uptake and T:M ratios were reduced ∼2-fold in comparison to NCI-H460 at all time points. NCI-H460 and DLD-1 tumors were easily visualized in PET imaging studies despite low in vitro TEM8 expression in DLD-1 cells indicating that in vivo expression might be higher in DLD-1 tumors. From in vitro autoradiography studies 89Zr-df–L2mAb specific binding was found in 6 tumor types (U87-MG, NCI-H460, T-47D MKN-45, A-431, and DLD-1) which highly correlated to vessel density (CD31 IHC). Westerns blots confirmed the presence of TEM8 in the 6 tumor types but found undetectable TEM8 levels in DLD-1 and MKN-45 cells. This data would indicate that TEM8 is associated with the tumor vasculature rather than the tumor tissue, thus explaining the increased TEM8 expression in DLD-1 tumors compared to DLD-1 cell cultures. 89Zr-df–L2mAb specifically targeted TEM8 in vitro and in vivo although the in vitro expression was not necessarily predictive of in vivo expression which seemed to be associated with the tumor vasculature. In mouse models, 89Zr-df–L2mAb tumor uptakes and T:M ratios were sufficient for visualization during PET imaging. These results would suggest that a TEM8 targeted PET imaging agent, such as 89Zr-df–L2mAb, may have potential clinical, diagnostic, and prognostic applications by providing a quantitative measure of tumor angiogenesis and patient selection for future TEM8 directed therapies.

Although successful remission has been achieved when cancer is diagnosed and treated during its earliest stages of development, a tumor that has established neovascularization poses a significantly greater risk of mortality. The inability to recapitulate the complexities of a maturing in vivo tumor microenvironment in an in vitro setting has frustrated attempts to identify and test anti-angiogenesis therapies that are effective at permanently halting cancer progression. We have established an in vitro tumor angiogenesis model driven solely by paracrine signaling between MDA-MB-231 breast cancer cells and telomerase-immortalized human microvascular endothelial (TIME) cells co-cultured in a spatially relevant manner. The bilayered bioengineered tumor model consists of TIME cells cultured as an endothelium on the surface of an acellular collagen I hydrogel under which MDA-MB-231 cells are cultured in a separate collagen I hydrogel. Results showed that TIME cells co-cultured with the MDA-MB-231 cells demonstrated a significant increase in cell number, rapidly developed an elongated morphology, and invasively sprouted into the underlying acellular collagen I layer. Comparatively, bioengineered tumors cultured with less aggressive MCF7 breast cancer cells did not elicit an angiogenic response. Angiogenic sprouting was demonstrated by the formation of a complex capillary-like tubule network beneath the surface of a confluent endothelial monolayer with lumen formation and anastomosing branches. In vitro angiogenesis was dependent on vascular endothelial growth factor secretion, matrix concentration, and duration of co-culture. Basic fibroblast growth factor supplemented to the co-cultures augmented angiogenic sprouting. The development of improved preclinical tumor angiogenesis models, such as the one presented here, is critical for accurate evaluation and refinement of anti-angiogenesis therapies.