ChingLam W. Wong

A transgene-encoded cell surface polypeptide for selection, in vivo tracking, and ablation of engineered cells

An unmet need in cell engineering is the availability of a single transgene encoded, functionally inert, human polypeptide that can serve multiple purposes, including ex vivo cell selection, in vivo cell tracking, and as a target for in vivo cell ablation. Here we describe a truncated human EGFR polypeptide (huEGFRt) that is devoid of extracellular N-terminal ligand binding domains and intracellular receptor tyrosine kinase activity but retains the native amino acid sequence, type I transmembrane cell surface localization, and a conformationally intact binding epitope for pharmaceutical-grade anti-EGFR monoclonal antibody, cetuximab (Erbitux). After lentiviral transduction of human T cells with vectors that coordinately express tumor-specific chimeric antigen receptors and huEGFRt, we show that huEGFRt serves as a highly efficient selection epitope for chimeric antigen receptor(+) T cells using biotinylated cetuximab in conjunction with current good manufacturing practices (cGMP)-grade anti-biotin immunomagnetic microbeads. Moreover, huEGFRt provides a cell surface marker for in vivo tracking of adoptively transferred T cells using both flow cytometry and immunohistochemistry, and a target for cetuximab-mediated antibody-dependent cellular cytotoxicity and in vivo elimination. The versatility of huEGFRt and the availability of pharmaceutical-grade reagents for its clinical application denote huEGFRt as a significant new tool for cellular engineering.

Engraftment of human central memory-derived effector CD8+ T cells in immunodeficient mice

In clinical trials of adoptive T-cell therapy, the persistence of transferred cells correlates with therapeutic efficacy. However, properties of human T cells that enable their persistence in vivo are poorly understood, and model systems that enable investigation of the fate of human effector T cells (T(E)) have not been described. Here, we analyzed the engraftment of adoptively transferred human cytomegalovirus pp65-specific CD8(+) T(E) cells derived from purified CD45RO(+)CD62L(+) central memory (T(CM)) or CD45RO(+)CD62L(-) effector memory (T(EM)) precursors in an immunodeficient mouse model. The engraftment of T(CM)-derived effector cells (T(CM/E)) was dependent on human interleukin-15, and superior in magnitude and duration to T(EM)-derived effector cells (T(EM/E)). T-cell receptor Vβ analysis of persisting cells demonstrated that CD8(+) T(CM/E) engraftment was polyclonal, suggesting that the ability to engraft is a general feature of T(CM/E.) CD8(+) T(EM/E) proliferated extensively after transfer but underwent rapid apoptosis. In contrast, T(CM/E) were less prone to apoptosis and established a persistent reservoir of functional T cells in vivo characterized by higher CD28 expression. These studies predict that human CD8(+) effector T cells derived from T(CM) precursors may be preferred for adoptive therapy based on superior engraftment fitness.

Phenotypic and functional attributes of lentivirus-modified CD19-specific human CD8+ central memory T cells manufactured at clinical scale.

A key determinant of the therapeutic potency of adoptive T-cell transfer is the extent to which infused cells can persist and expand in vivo. Ex vivo propagated virus-specific and chimeric antigen receptor (CAR)-redirected antitumor CD8+ effector T cells derived from CD45RA− CD62L+ central memory (TCM) precursors engraft long-term and reconstitute functional memory after adoptive transfer. Here, we describe a clinical scale, closed system, immunomagnetic selection method to isolate CD8+ TCM from peripheral blood mononuclear cells (PBMC). This method uses the CliniMACS device to first deplete CD14+, CD45RA+, and CD4+ cells from PBMC, and then to positively select CD62L+ cells. The average purity and yield of CD8+CD45RA− CD62L+ TCM obtained in full-scale qualification runs were 70% and 0.4% (of input PBMC), respectively. These CD8+ TCM are responsive to anti-CD3/CD28 bead stimulation, and can be efficiently transduced with CAR encoding lentiviral vectors, and undergo sustained expansion in interleukin (IL)-2/IL-15 over 3–6 weeks. The resulting CD8+ TCM-derived effectors are polyclonal, retain expression of CD62L and CD28, exhibit CAR-redirected antitumor effector function, and are capable of huIL-15-dependent in vivo homeostatic engraftment after transfer to immunodeficient NOD/Scid IL-2RgCnull mice. Adoptive therapy using purified TCM cells is now the subject of a Food and Drug Administration-authorized clinical trial for the treatment of CD19+ B-cell malignancies, and 3 clinical cell products expressing a CD19-specific CAR for IND #14645 have already been successfully generated from lymphoma patients using this manufacturing platform.

CMVpp65 Vaccine Enhances the Antitumor Efficacy of Adoptively Transferred CD19-Redirected CMV-Specific T Cells

Purpose: T cells engineered with chimeric antigen receptors (CAR) recognizing CD19 can induce complete remission of B-cell malignancies in clinical trials; however, in some disease settings, CAR therapy confers only modest clinical benefit due to attenuated persistence of CAR T cells. The purpose of this study was to enhance persistence and augment the antitumor activity of adoptively transferred CD19CAR T cells by restimulating CAR+ T cells through an endogenous cytomegalovirus (CMV)-specific T-cell receptor. Experimental Design: CMV-specific T cells from CMV seropositive healthy donors were selected after stimulation with pp65 protein and transduced with clinical-grade lentivirus expressing the CD19R:CD28:ζ/EGFRt CAR. The resultant bispecific T cells, targeting CMV and CD19, were expanded via CD19 CAR-mediated signals using CD19-expressing cells. Results: The bispecific T cells proliferated vigorously after engagement with either endogenous CMVpp65 T-cell receptors or engineered CD19 CARs, exhibiting specific cytolytic activity and IFNγ secretion. Upon adoptive transfer into immunodeficient mice bearing human lymphomas, the bispecific T cells exhibited proliferative response and enhanced antitumor activity following CMVpp65 peptide vaccine administration. Conclusions: We have redirected CMV-specific T cells to recognize and lyse tumor cells via CD19CARs, while maintaining their ability to proliferate in response to CMV antigen stimulation. These results illustrate the clinical applications of CMV vaccine to augment the antitumor activity of adoptively transferred CD19CAR T cells in patients with B-cell malignancies. Clin Cancer Res; 21(13); 2993–3002. ©2015 AACR.

Comparison of naïve and central memory derived CD8+ effector cell engraftment fitness and function following adoptive transfer

abstract Human CD8+ effector T cells derived from CD45RO+CD62L+ precursors enriched for central memory (TCM) precursors retain the capacity to engraft and reconstitute functional memory upon adoptive transfer, whereas effectors derived from CD45RO+CD62L− precursors enriched for effector memory precursors do not. Here we sought to compare the engraftment fitness and function of CD8+ effector T cells derived from CD45RA+CD62L+ precursors enriched for naïve and stem cell memory precursors (TN/SCM) with that of TCM. We found that cytotoxic T cells (CTLs) derived from TCM transcribed higher levels of CD28, FOS, INFγ, Eomesodermin (Eomes), and lower levels of BCL2L11, maintained higher levels of phosphorylated AKT, and displayed enhanced sensitivity to the proliferative and anti-apoptotic effects of γ-chain cytokines compared to CTLs derived from TN/SCM. Higher frequencies of CTLs derived from TCM retained CD28 expression and upon activation secreted higher levels of IL-2. In NOD/Scid IL-2RγCnull mice, CD8+ TCM derived CTLs engrafted to higher frequencies in response to human IL-15 and mounted robust proliferative responses to an immunostimulatory vaccine. Similarly, CD8+ TCM derived CD19CAR+ CTLs exhibited superior antitumor potency following adoptive transfer compared to their CD8+ TN/SCM derived counterparts. These studies support the use of TCM enriched cell products for adoptive therapy of cancer.

Lenalidomide enhances the function of CS1 chimeric antigen receptor redirected T cells against multiple myeloma

Purpose: Multiple myeloma remains an incurable malignancy of plasma cells despite considerable advances in treatment. The purpose of the study was to develop novel chimeric antigen receptors (CAR) for the treatment of multiple myeloma and explore combinatorial therapy using CAR T cells and immunomodulatory drugs such as lenalidomide for increasing treatment efficacy. Experimental Design: We redirected central memory T cells to express second-generation CAR-specific for CS1 and adoptively transferred them into multiple myeloma tumor-bearing mice to test their anti-multiple myeloma activity. CS1 CAR T cells were transduced and expanded in the presence of lenalidomide in vitro. The phenotype and effector function of CS1 CAR T cells treated with and without lenalidomide were compared. Finally, CS1 CAR T cells and lenalidomide were administered to treat multiple myeloma–bearing mice as combinatorial therapy. Results: CS1 CAR T cells exhibited efficient antitumor activity when adoptively transferred into mice. Mechanistic studies indicated that the addition of lenalidomide during CS1 CAR T-cell expansion in vitro enhanced the immune functions of CS1 CAR T cells, including cytotoxicity, memory maintenance, Th1 cytokine production, and immune synapse formation. Furthermore, lenalidomide enhanced the antitumor activity and persistence of adoptively transferred CS1 CAR T cells in vivo. Conclusions: The study demonstrates that lenalidomide improves the anti-multiple myeloma properties of CS1-directed CAR T cells and provides a basis for a planned clinical trial using the combination of lenalidomide with engineered T cells against CS1 in relapsed myeloma. Clin Cancer Res; 24(1); 106–19. ©2017 AACR.

640. Ex Vivo AKT Inhibition Promotes the Generation of Potent CD19CAR T Cells for Adoptive Immunotherapy

Insufficient persistence and effector function of chimeric antigen receptor (CAR) re-directed T cells in vivo has been a challenge for adoptive T cell therapy. Generation of long-lived potent CAR T cells is an increasing demand in the field. AKT activation triggered by convergent extracellular signals evokes a transcription program that enhances effector functions. However, sustained AKT activation severely impairs T cell memory and protective immunity because AKT drives differentiation of effectors, diminishing T cell potential to survive and differentiate into memory cells. We now investigate whether inhibition of AKT signaling can prevent terminal differentiation of T cells that are genetically modified to express CD19-specific chimeric antigen receptors (CD19CAR), as well as increase the number of memory CD19CAR T cells, which would enhance the antitumor activity following adoptive therapy. CD8+ T cells from healthy donors were isolated, activated with CD3/CD28 beads, and transduced with a lentiviral vector encoding a second-generation CD19CAR containing a CD28 co-stimulatory domain, which carries mutations at two sites (L235E; N297Q) within the CH2 region on the IgG4-Fc spacers to block diminshed potency and persistence due to Fc receptor binding. The lentiviral vector also expressed a truncated human epidermal growth factor receptor (huEGFRt) for selection and ablation purposes. IL-2 (50U/mL) and AKT inhibitor (1uM/mL) were supplemented every other day. Transduced CD19CAR T cells without AKT inhibitor treatment were used as controls. The engineered CD19CAR T cells were expanded in vivo for 21 days before in vitro and in vivo assays. We found that AKT inhibitor did not compromise the CD19CAR T cell proliferation and survival in vitro; comparable CD19CAR T cell expansion was observed after culturing in the presence or absence of AKT inhibitor. Functionally, AKT inhibitor did not dampen the effector function of CD19CAR T cells as indicated by equivalent levels of interferon gamma production and CD107a expression upon CD19 antigen stimulation. Memory-like phenotype such as CD62L and CD28 expression on CAR T cells is associated with better antitumor activity in vivo. We therefore characterized the CD19CAR T cells after ex vivo expansion. We found that 40% of AKT-inhibited CD19CAR T cells expressed CD62L and co-expressed CD28. In contrast, only 10% of control untreated CD19CAR T cells expressed CD62L and they were CD28 negative, indicating that AKT-inhibited CD19CAR T cells may have superior anti-tumor activity following adoptive transfer. To test the potency of the AKT inhibitor treated CAR T cells, 0.5×106 CD19+ acute lymphoid leukemic cells (SupB15) that were engineered to express firely lucifierase were inoculated intravenously into NOD/Scid IL-2RgammaCnull (NSG) mice. Five days post tumor engraftment, 2×106 CD8+ CD19CAR T cells were intravenously injected into tumor bearing mice. Control mice received either no T cells, non-transduced T cells (Mock), or CD19CAR T cells that were not treated with AKT inhibitor during in vitro expansion. Tumor signals post T cells infusion were monitored by biophotonic imaging. In contrast to the untreated CD19CAR T cells, which exhibited lower and transient anti-tumor activity, AKT-inhibited CD19CAR T cells completely eradicated the CD19+ tumor in all mice (Figure 1) 21 days post CD19CAR T cell infusion. In conclusion, inhibition of AKT signaling during the ex vivo priming and expansion gives rise to a CD19CAR T cell population that possesses superior antitumor activity. These findings suggest that therapeutic modulation of AKT might be a strategy to augment antitumor immunity for adoptive CAR T cell therapy, which could easily be transitioned into the clinic with the availability of pharmaceutical grade AKT inhibitor.