Untouched GMP-Ready Purified Engineered Immune Cells to Treat Cancer

Abstract

Purpose: Engineering T cells with receptors to redirect the immune system against cancer has most recently been described as a scientific breakthrough. However, a main challenge remains the GMP-grade purification of immune cells selectively expressing the introduced receptor in order to reduce potential side effects due to poorly or nonengineered cells.Experimental Design: In order to test a novel purification strategy, we took advantage of a model gdT cell receptor (TCR), naturally interfering with endogenous TCR expression and designed the optimal retroviral expression cassette to achieve maximal interference with endogenous TCR chains. Following retroviral transduction, nonengineered and poorly engineered immune cells characterized by a high endogenous abTCR expression were efficiently depleted with GMP-grade anti-abTCR beads. Next, the engineered immune cells were validated for TCR expression, function against a panel of tumor cell lines and primary tumors and potential allo-reactivity. Engineered immune cells were further validated in two humanized mouse tumor models.Results: The untouched enrichment of engineered immune cells translated into highly purified receptor-engineered cells with strong antitumor reactivity both in vitro and in vivo. Importantly, this approach eliminated residual allo-reactivity of engineered immune cells. Our data demonstrate that even with long-term suboptimal interference with endogenous TCR chains such as in resting cells, allo-reactivity remained absent and tumor control preserved.Conclusions: We present a novel enrichment method for the production of untouched engineered immune cells, ready to be translated into a GMP-grade method and potentially applicable to all receptor-modified cells even if interference with endogenous TCR chains is far from complete.

TEG011 persistence averts extramedullary tumor growth without exerting off-target toxicity against healthy tissues in a humanized HLA-A*24:02 transgenic mice

“…Therefore, we proposed efficacy‐toxicity models for TEGs targeting joint spatial and conformational changes in CD277 (later referred as CD277J) 3 through a γ9δ2TCR (TEG001) by co‐incubating TEG001 with healthy and diseased tissues in an artificial 3D bone marrow niche 32 or in a mouse model where either healthy cord blood‐derived CD34 + progenitor or primary leukemia cells were engrafted 26 . These models partially overcome the absence of the natural ligand CD277J in mice 3,7,33 and allowed the initiation of a first‐in‐men study (NTR6541) 18,19,34 . With TEG011, we could utilize transgenic mice expressing human HLA‐A*24:02, 25 allowing thereby more extensive toxicity studies of TEG011 in different tissues as compared to TEG001 26,32 .…”

“…Depletion of nonengineered T cells was performed as previously described 19 . Briefly, TCR‐transduced T cells were incubated with a biotin‐labeled anti‐αβTCR antibody (clone BW242/412; Miltenyi Biotec, Leiden, The Netherlands) and incubated with an anti‐biotin antibody coupled to magnetic beads (anti‐biotin MicroBeads; Miltenyi Biotec).…”

“…To partially overcome these obstacles, we introduced the concept of TEGs: αβT cells engineered to express a defined γδTCR. TEGs allow the production of αβT cells transduced with highly tumor‐reactive γδTCR from both Vδ2 + 18‐20 and Vδ2 − 21‐23 subsets and thereby engineering strong tumor reactivity against a broad panel of malignancies. Within this context, we previously identified an allo‐HLA‐restricted and CD8α‐dependent Vγ5Vδ1TCR.…”

The next step toward GMP-grade production of engineered immune cells

“…5 We demonstrate that interference with endogenous abTCRs combined with GMP-grade anti-abTCR beads can provide highly purified untouched engineered immune cells without the additional need for selection markers. We used a tumor specific gdTCR 7,8 to naturally interfere with endogenous abTCRs, a readily translatable strategy.…”

“…In the context of allo-SCT, downregulation of endogenous receptors might be an additional important engineering step. 5 Regardless of the desired subset, processing cell fractions in a good-manufacturing-practices (GMP) certified environment is usually cumbersome and expensive due to the fact that sequential isolation steps using multiple GMP-grade antibodies are necessary.…”