Tony Polverino

3134 Background: AMG 706 is a potent, oral, multi-kinase inhibitor with anti-angiogenic and anti-tumor activity achieved by selectively targeting all known VEGF, PDGF, Kit and Ret receptors. In previous experiments, AMG 706 treatment resulted in regression of large, established A431 epidermoid carcinoma tumors within seven days of the initiation of treatment. The present experiments were carried out to relate the therapeutic effect to changes in vascular permeability and blood flow, with the intent of identifying a pharmacodynamic marker for use in clinical trials. Methods: After an initial dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) baseline scan, mice bearing A431 xenografts (300 mm3) were dosed with either vehicle or AMG 706 at dose levels ranging between 10 and 75 mg/kg. DCE-MRI was repeated after 24, 48 or 96 hrs of treatment. All MRI studies were performed at 4.7 Tesla (Bruker Biospin, Billerica, MA). During the DCE-MRI scans, 0.2 mmol/kg gadopentetate dimeglumine was injected intravenously via a tail vein catheter. Results: DCE-MRI analysis identified a statistically significant (p = 0.0235) decrease in contrast agent uptake (AUC relative to baseline), occurring in a dose dependent fashion in the interior region of the tumor after only 24 hrs of treatment with AMG 706. The effect seen at 24 hrs is consistent with the previous observation that tumor endothelial cell apoptosis occurs within 12 hrs of initiation of treatment, and indicates a statistically significant treatment related reduction in tumor blood flow and vascular permeability in the A431 tumor model. Conclusions: Oral administration of AMG 706 produced a statistically significant reduction in vascular blood flow and/or permeability in the A431 human tumor xenograft model as early as 24 hrs of treatment. These results are consistent with previous observations indicating the destruction of tumor blood vessels as the mechanism of action of AMG 706, and support the use of DCE-MRI to obtain pharmacodynamic information in clinical trials of AMG 706. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Amgen Amgen Amgen

Abstract Background: Multiple myeloma (MM) is a usually fatal malignancy of plasma cells, with no current therapy considered curative. About 15% of patients diagnosed with MM are stratified as high risk with poor treatment outcomes and short (2-3 years) survival from diagnosis. Standard risk patients tend to live longer but undergo chronic and/or high intensity therapy and likely experience a relapsing and remitting disease pattern. Therefore, there is still a considerable unmet need for innovative therapies that improve outcomes in MM. One such approach is to use adoptive transfer of engineered autologous T cells expressing a chimeric antigen receptor (CAR) directed against malignant cells. The efficacy of CAR T cells directed against hematological malignancies, particularly CD19-expressing B cell leukemia and lymphomas, has been demonstrated in multiple clinical studies. KITE-585 was developed as a CAR T cell immunotherapy product candidate directed against B cell maturation antigen (BCMA). BCMA is nearly ubiquitously expressed on MM cells, plasma cells and subsets of mature B cells, but with limited or absent expression on other tissues. Methods: We generated >50 fully human IgGs directed against BCMA using the BCMA protein as antigen and selection criteria including affinity, cross-reactivity and poly-specificity. Following assessment of the binding of the IgGs to a MM cell line known to express BCMA, >10 IgGs were identified that met the criteria for affinity and selectivity and had a >50-fold binding over background. The 8 IgGs that demonstrated the highest specific binding were then sequence-converted to single-chain variable fragments (scFvs) and incorporated into CARs. Results: In all but one case, human T cells engineered to express these CAR constructs exhibited specific cytolytic activity against MM cell lines (NCI-H929 and MM.1s). These CAR T cells demonstrated killing efficiencies of >95% at effector:target ratios of 1:1 over a 24-hour period. Similarly antigen-specific production of inflammatory cytokines was observed in response to target cell lines in vitro. Assessment of antigen-dependent proliferation over a 5 day period revealed >80% proliferation in the 7 constructs that showed cytolytic activity in vitro. Multiple different anti-BCMA CAR constructs representing distinct epitope binding bins of BCMA were then selected for in vivo evaluation. In two disseminated tumor models of luciferase labeled NCI-H929 or MM.1s cells injected intravenously (i.v.), a single i.v. injection of anti-BCMA CAR T-cells delayed the progression of disease and significantly increased survival when compared to control treatment. Conclusions: The results of these studies highlight the potential of targeting BCMA with adoptive transfer of engineered T cells for the treatment of MM. Given these positive findings, progress towards Phase 1 clinical studies in MM patients with KITE-585 is continuing. Citation Format: Gregor B. Adams, Jun Feng, Atefah Ghogha, Armen Mardiros, Jodi Murakami, Tammy Phung, Ruben Rodriguez, Stuart Sievers, Tassja J. Spindler, Jed Wiltzius, Clare Yarka, Sean C. Yoder, Tony Polverino. Development of KITE-585: A fully human BCMA CAR T-cell therapy for the treatment of multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4979. doi:10.1158/1538-7445.AM2017-4979

Abstract Background: Immunotherapy has provided treatment options for cancers that are otherwise refractory to standard approaches. One such technique is to use adoptive transfer of engineered autologous T cells expressing a chimeric antigen receptor (CAR) directed against a tumor antigen. The efficacy of CAR T cells directed against hematological malignancies, particularly CD19-expressing B cell leukemia and lymphomas, has been demonstrated in multiple clinical studies. The success of this approach has prompted development of CAR T cells directed to different tumor antigens for other tumor types. To ensure the selectivity and specificity of the CAR T cells against their intended target, screening methods need to be employed. Multiple myeloma is an incurable malignancy of plasma cells. B-cell maturation antigen (BCMA), also known as tumor necrosis factor superfamily member 17 (TNFRSF17) is nearly ubiquitously expressed on multiple myeloma cells, plasma cells and subsets of mature B cells. Methods: In order to screen for the specificity of novel CAR T cells directed against BCMA, we utilized a cell microarray platform developed by Retrogenix. In this screen, approximately 4500 human plasma membrane proteins (representing up to 75% of the human plasma membrane proteome) are individually expressed in human HEK293 cells. Fluorescently labeled CAR T cells, which showed cytolytic activity against MM cell lines expressing BCMA, were applied to the cell microarray and specific binding of the CAR T cells to target cells was determined. Results: Primary hits were sequenced to confirm identity and secondary specificity screens were performed on the identified hits. Specific binding of both mock transduced and BCMA CAR transduced T cells were confirmed for different plasma membrane proteins expressed from the HEK293 cells. These included known T cell interactors, such as ICOSLG, CD244 and CD86, where binding is proposed to be independent of CAR expression. Subtracting the hits of the mock transduced T cells from the BCMA CAR T cells demonstrated specific binding of the CAR T cells to BCMA. Utilizing the fully human IgGs directed against BCMA from which the single-chain variable fragments (scFvs) of the CARs were derived, we further confirmed specific binding to BCMA in additional secondary screens. Additionally, a lack of off-target binding of the fully human IgGs to normal tissue was demonstrated in a tissue cross reactivity screen. Conclusions: These studies highlight the tractability of this cell microarray approach for determining the specificity of novel CAR constructs expressed in T cell. Demonstrating the selectivity and specificity of anti-BMCA CAR T cells further supports the progression of KITE-585 towards Phase 1 clinical studies in MM patients. Citation Format: Gregor B. Adams, Jun Feng, Atefeh Ghogha, Armen Mardiros, Ruben Rodriguez, Tassja J. Spindler, Jed Wiltzius, Tony Polverino. Selectivity and specificity of engineered T cells expressing KITE-585, a chimeric antigen receptor targeting B-cell maturation antigen (BCMA) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2135. doi:10.1158/1538-7445.AM2017-2135

Proc Amer Assoc Cancer Res, Volume 47, 2006 3792 Background: AMG 706 is a potent, oral, multi-kinase inhibitor that selectively targets VEGF, PDGF, and Kit receptors. Treatment with AMG 706 has been shown to cause regression of established disease in A431 xenografts. The present studies were designed to address the mechanism of action of this molecule. Methods: Female CD1 nu/nu mice were challenged subcutaneously with 1x107 tumor cells. Treatment with AMG 706 (75 mg/kg, po, bid) or vehicle (n = 10) was initiated after the tumors became established. At various time points thereafter, animals were sacrificed and tumors were collected and evaluated for blood vessel area (CD31), endothelial cell apoptosis (CD31 and TUNEL), viable tumor area (hematoxylin) and tumor cell apoptosis (cleaved caspase-3). Results: Treatment with AMG 706 resulted in a significant increase in % endothelial cell apoptosis and decrease in % blood vessel area starting at 12 hours after initiation of treatment ( p = 0.0257 and 0.0005, respectively). Subsequently, a significant increase in tumor cell apoptosis was observed at 48 hrs ( p = 0.009), followed by a significant decline in % viable tumor area at 72 hours ( p = 0.0002). These effects occurred immediately prior to the statistically significant regression of disease at 96 hrs ( p = 0.0327). Conclusions: The effect of AMG 706 upon tumor blood vessels was found to temporally precede the destruction of tumor in the A431 xenograft model. The temporal sequence associated with these observations support the concept that tumor blood vessels are the primary target of AMG 706 and illustrate a mechanism of action consistent with the inhibition of VEGF receptors.