Zhili Zheng

High Efficiency TCR Gene Transfer into Primary Human Lymphocytes Affords Avid Recognition of Melanoma Tumor Antigen Glycoprotein 100 and Does Not Alter the Recognition of Autologous Melanoma Antigens

The α- and β-chains of the TCR from a highly avid anti-gp100 CTL clone were isolated and used to construct retroviral vectors that can mediate high efficiency gene transfer into primary human lymphocytes. Expression of this TCR gene was confirmed by Western blot analysis, immunocytometric analysis, and HLA Ag tetramer staining. Gene transfer efficiencies of >50% into primary lymphocytes were obtained without selection for transduced cells using a method of prebinding retroviral vectors to cell culture vessels before the addition of lymphocytes. The biological activity of transduced cells was confirmed by cytokine production following coculture with stimulator cells pulsed with gp100 peptides, but not with unrelated peptides. The ability of this anti-gp100 TCR gene to transfer high avidity Ag recognition to engineered lymphocytes was confirmed in comparison with highly avid antimelanoma lymphocytes by the high levels of cytokine production (>200,000 pg/ml IFN-γ), by recognition of low levels of peptide (<200 pM), and by HLA class I-restricted recognition and lysis of melanoma tumor cell lines. CD4+ T cells engineered with this anti-gp100 TCR gene were Ag reactive, suggesting CD8-independent activity of the expressed TCR. Finally, nonmelanoma-reactive tumor-infiltrating lymphocyte cultures developed antimelanoma activity following anti-gp100 TCR gene transfer. In addition, tumor-infiltrating lymphocytes with reactivity against non-gp100 melanoma Ags acquired gp100 reactivity and did not lose the recognition of autologous melanoma Ags following gp100 TCR gene transfer. These results suggest that lymphocytes genetically engineered to express anti-gp100 TCR may be of value in the adoptive immunotherapy of patients with melanoma.

A Herceptin-Based Chimeric Antigen Receptor with Modified Signaling Domains Leads to Enhanced Survival of Transduced T Lymphocytes and Antitumor Activity

To generate chimeric Ag receptors (CARs) for the adoptive immunotherapy of cancer patients with ErbB2-expressing tumors, a single-chain Ab derived from the humanized mAb 4D5 Herceptin (trastuzumab) was initially linked to T cell signaling domains derived from CD28 and the CD3ζ to generate a CAR against ErbB2. Human PBLs expressing the 4D5 CAR demonstrated Ag-specific activities against ErbB2+ tumors. However, a gradual loss of transgene expression was noted for PBLs transduced with this 4D5 CAR. When the CD3ζ signaling domain of the CAR was truncated or mutated, loss of CAR expression was not observed, suggesting that the CD3ζ signaling caused the transgene decrease, which was supported by the finding that T cells expressing 4D5 CARs with CD3ζ ITAM mutations were less prone to apoptosis. By adding 4-1BB cytoplasmic domains to the CD28-CD3ζ signaling moieties, we found increased transgene persistence in 4D5 CAR-transduced PBLs. Furthermore, constructs with 4-1BB sequences demonstrated increased cytokine secretion and lytic activity in 4D5 CAR-transduced T cells. More importantly, PBLs expressing this new version of the 4D5 CAR could not only efficiently lyse the autologous fresh tumor digests, but they could strongly suppress tumor growth in a xenogenic mouse model.

Recognition of glioma stem cells by genetically modified T cells targeting EGFRvIII and development of adoptive cell therapy for glioma.

No curative treatment exists for glioblastoma, with median survival times of less than 2 years from diagnosis. As an approach to develop immune-based therapies for glioblastoma, we sought to target antigens expressed in glioma stem cells (GSCs). GSCs have multiple properties that make them significantly more representative of glioma tumors than established glioma cell lines. Epidermal growth factor receptor variant III (EGFRvIII) is the result of a novel tumor-specific gene rearrangement that produces a unique protein expressed in approximately 30% of gliomas, and is an ideal target for immunotherapy. Using PCR primers spanning the EGFRvIII-specific deletion, we found that this tumor-specific gene is expressed in three of three GCS lines. Based on the sequence information of seven EGFRvIII-specific monoclonal antibodies (mAbs), we assembled chimeric antigen receptors (CARs) and evaluated the ability of CAR-engineered T cells to recognize EGFRvIII. Three of these anti-EGFRvIII CAR-engineered T cells produced the effector cytokine, interferon-γ, and lysed antigen-expressing target cells. We concentrated development on a CAR produced from human mAb 139, which specifically recognized GSC lines and glioma cell lines expressing mutant EGFRvIII, but not wild-type EGFR and did not recognize any normal human cell tested. Using the 139-based CAR, T cells from glioblastoma patients could be genetically engineered to recognize EGFRvIII-expressing tumors and could be expanded ex vivo to large numbers, and maintained their antitumor activity. Based on these observations, a γ-retroviral vector expressing this EGFRvIII CAR was produced for clinical application.

Immunogenicity of somatic mutations in human gastrointestinal cancers

Low mutation rate okay for T cells Cancers that tend to have high numbers of mutations, such as melanoma and smoking-induced lung cancer, respond well to immunotherapies, whereas those with fewer mutations, such as pancreatic cancer, do not. Tran et al. searched for tumor mutation–reactive T cells in 10 patients with metastatic gastrointestinal cancers, which have relatively low mutation burdens, and discovered that 9 out of 10 harbored such cells. T cells from one patient recognized a mutation common to many types of cancers. Engineering T cells to express this particular mutation-reactive T cell receptor may extend adoptive cell immunotherapy to a larger pool of patients than previously anticipated. Science, this issue p. 1387 Individuals with cancers that have low mutation frequencies often harbor mutation-reactive T cells. It is unknown whether the human immune system frequently mounts a T cell response against mutations expressed by common epithelial cancers. Using a next-generation sequencing approach combined with high-throughput immunologic screening, we demonstrated that tumor-infiltrating lymphocytes (TILs) from 9 out of 10 patients with metastatic gastrointestinal cancers contained CD4+ and/or CD8+ T cells that recognized one to three neo-epitopes derived from somatic mutations expressed by the patient’s own tumor. There were no immunogenic epitopes shared between these patients. However, we identified in one patient a human leukocyte antigen–C*08:02–restricted T cell receptor from CD8+ TILs that targeted the KRASG12D hotspot driver mutation found in many human cancers. Thus, a high frequency of patients with common gastrointestinal cancers harbor immunogenic mutations that can potentially be exploited for the development of highly personalized immunotherapies.

Single and Dual Amino Acid Substitutions in TCR CDRs Can Enhance Antigen-Specific T Cell Functions

Single and dual amino acid substitution variants were generated in the TCR CDRs of three TCRs that recognize tumor-associated Ags. Substitutions that enhance the reactivity of TCR gene-modified T cells to the cognate Ag complex were identified using a rapid RNA-based transfection system. The screening of a panel of variants of the 1G4 TCR, that recognizes a peptide corresponding to amino acid residues 157–165 of the human cancer testis Ag NY-ESO-1 (SLLMWITQC) in the context of the HLA-A*02 class I allele, resulted in the identification of single and dual CDR3α and CDR2β amino acid substitutions that dramatically enhanced the specific recognition of NY-ESO-1+/HLA-A*02+ tumor cell lines by TCR gene-modified CD4+ T cells. Within this group of improved TCRs, a dual substitution in the 1G4 TCR CDR3α chain was identified that enhanced Ag-specific reactivity in gene-modified CD4+ and CD8+ T cells. Separate experiments on two distinct TCRs that recognize the MART-1 27–35 (AAGIGILTV) peptide/HLA-A*02 Ag complex characterized single amino acid substitutions in both TCRs that enhanced CD4+ T cell Ag-specific reactivity. These results indicate that simple TCR substitution variants that enhance T cell function can be identified by rapid transfection and assay techniques, providing the means for generating potent Ag complex-specific TCR genes for use in the study of T cell interactions and in T cell adoptive immunotherapy.

Primary Human Lymphocytes Transduced with NY-ESO-1 Antigen-Specific TCR Genes Recognize and Kill Diverse Human Tumor Cell Lines

cDNAs encoding TCR α- and β-chains specific for HLA-A2-restricted cancer-testis Ag NY-ESO-1 were cloned using a 5′RACE method from RNA isolated from a CTL generated by in vitro stimulation of PBMC with modified NY-ESO-1-specific peptide (p157–165, 9V). Functionality of the cloned TCR was confirmed by RNA electroporation of primary PBL. cDNA for these α- and β-chains were used to construct a murine stem cell virus-based retroviral vector, and high titer packaging cell lines were generated. Gene transfer efficiency in primary T lymphocytes of up to 60% was obtained without selection using a method of precoating retroviral vectors onto culture plates. Both CD4+ and CD8+ T cells could be transduced at the same efficiency. High avidity Ag recognition was demonstrated by coculture of transduced lymphocytes with target cells pulsed with low levels of peptide (<20 pM). TCR-transduced CD4 T cells, when cocultured with NY-ESO-1 peptide pulsed T2 cells, could produce IFN-γ, GM-CSF, IL-4, and IL-10, suggesting CD8-independent, HLA-A2-restricted TCR activation. The transduced lymphocytes could efficiently recognize and kill HLA-A2- and NY-ESO-1-positive melanoma cell lines in a 4-h 51Cr release assay. Finally, transduced T cells could efficiently recognize NY-ESO-1-positive nonmelanoma tumor cell lines. These results strongly support the idea that redirection of normal T cell specificity by TCR gene transfer can have potential applications in tumor adoptive immunotherapy.

High-Affinity TCRs Generated by Phage Display Provide CD4+ T Cells with the Ability to Recognize and Kill Tumor Cell Lines

We examined the activity of human T cells engineered to express variants of a single TCR (1G4) specific for the cancer/testis Ag NY-ESO-1, generated by bacteriophage display with a wide range of affinities (from 4 μM to 26 pM). CD8+ T cells expressing intermediate- and high-affinity 1G4 TCR variants bound NY-ESO-1/HLA-A2 tetramers with high avidity and Ag specificity, but increased affinity was associated with a loss of target cell specificity of the TCR gene-modified cells. T cells expressing the highest affinity TCR (KD value of 26 pM) completely lost Ag specificity. The TCRs with affinities in the midrange, KD 5 and 85 nM, showed specificity only when CD8 was absent or blocked, while the variant TCRs with affinities in the intermediate range—with KD values of 450 nM and 4 μM—demonstrated Ag-specific recognition. Although the biological activity of these two relatively low-affinity TCRs was comparable to wild-type reactivity in CD8+ T cells, introduction of these TCR dramatically increased the reactivity of CD4+ T cells to tumor cell lines.

Development of optimal bicistronic lentiviral vectors facilitates high-level TCR gene expression and robust tumor cell recognition

In human gene therapy applications, lentiviral vectors may have advantages over γ-retroviral vectors in several areas, including the ability to transduce nondividing cells, resistance to gene silencing and a potentially safer integration site profile. However, unlike γ-retroviral vectors it has been problematic to drive the expression of multiple genes efficiently and coordinately with approaches such as internal ribosome entry sites or dual promoters. Using different 2A peptides, lentiviral vectors expressing two-gene T-cell receptors directed against the melanoma differentiation antigens gp100 and MART-1 were constructed. We demonstrated that addition of amino-acid spacer sequences (GSG or SGSG) before the 2A sequence is a prerequisite for efficient synthesis of biologically active T-cell receptors and that addition of a furin cleavage site followed by a V5 peptide tag yielded optimal T-cell receptor gene expression. Furthermore, we determined that the furin cleavage site was recognized in lymphocytes and accounted for removal of residual 2A peptides at the post-translational level with an efficiency of 20–30%, which could not be increased by addition of multiple furin cleavage sites. The novel bicistronic lentiviral vector developed herein afforded robust anti-melanoma activities to engineered peripheral blood lymphocytes, including cytokine secretion, cell proliferation and lytic activity. Such optimal vectors may have immediate applications in cancer gene therapy.

Improving Adoptive T Cell Therapy by Targeting and Controlling IL-12 Expression to the Tumor Environment

Interleukin-12 (IL-12) is an important immunostimulatory cytokine, yet its clinical application has been limited by the systemic toxicity associated with its administration. In this work, we developed a strategy to selectively deliver IL-12 to the tumor environment using genetically engineered lymphocytes. However, peripheral blood lymphocytes (PBLs) transduced with a γ-retroviral vector, which constitutively expressed IL-12, failed to expand in culture due to apoptosis. To circumvent this problem, a vector was designed where IL-12 expression was directed by a composite promoter-containing binding motifs for nuclear factor of activated T-cells (NFAT.hIL12.PA2). The NFAT-responsive promoter was activated to drive IL-12 expression upon the recognition of tumor-specific antigen mediated by a T cell receptor (TCR) that was engineered into the same lymphocytes. We tested the efficacy of the inducible IL-12 vector in vivo in a murine melanoma model. Adoptive transfer of pmel-1 T cells genetically engineered with NFAT-murineIL12 (NFAT.mIL12.PA2) significantly enhanced regression of large established B16 melanoma. Notably, this targeted and controlled IL-12 treatment was without toxicity. Taken together, our results suggest that using the NFAT.hIL12.PA2 vector might be a promising approach to enhance adoptive cancer immunotherapy.

Recognition of Fresh Human Tumor by Human Peripheral Blood Lymphocytes Transduced with a Bicistronic Retroviral Vector Encoding a Murine Anti-p53 TCR

The p53 protein is markedly up-regulated in a high proportion of human malignancies. Using an HLA-A2 transgenic mouse model, it was possible to isolate high-avidity murine CTLs that recognize class I-restricted human p53 epitopes. We isolated the α- and β-chain of a TCR from a highly avid murine CTL clone that recognized the human p53264–272 epitope. These genes were cloned into a retroviral vector that mediated high efficiency gene transfer into primary human lymphocytes. Efficiencies of >90% for gene transfer into lymphocytes were obtained without selection for transduced cells. The p53 TCR-transduced lymphocytes were able to specifically recognize with high-avidity, peptide-pulsed APCs as well as HLA-A2.1+ cells transfected with either wild-type or mutant p53 protein. p53 TCR-transduced cells demonstrated recognition and killing of a broad spectrum of human tumor cell lines as well as recognition of fresh human tumor cells. Interestingly, both CD8+ and CD4+ subsets were capable of recognizing and killing target cells, stressing the potential application of such a CD8-independent TCR molecule that can mediate both helper and cytotoxic responses. These results suggest that lymphocytes genetically engineered to express anti-p53 TCR may be of value for the adoptive immunotherapy of patients with a variety of common malignancies.