Ralph A. Willemsen

Circumventing tolerance to a human MDM2-derived tumor antigen by TCR gene transfer

We identified a tumor-associated cytotoxic T lymphocyte (CTL) epitope derived from the widely expressed human MDM2 oncoprotein and were able to bypass self-tolerance to this tumor antigen in HLA-A*0201 (A2.1) transgenic mice and by generating A2.1-negative, allo-A2.1–restricted human T lymphocytes. A broad range of malignant, as opposed to nontransformed cells, were killed by high-avidity transgenic mouse and allogeneic human CTLs specific for the A2.1-presented MDM2 epitope. Whereas the self-A2.1–restricted human T cell repertoire gave rise only to low-avidity CTLs unable to recognize the natural MDM2 peptide, human A2.1+ T lymphocytes were turned into efficient MDM2-specific CTLs upon expression of wild-type and partially humanized high-affinity T cell antigen receptor (TCR) genes derived from the transgenic mice. These results demonstrate that TCR gene transfer can be used to circumvent self-tolerance of autologous T lymphocytes to universal tumor antigens and thus provide the basis for a TCR gene transfer–based broad-spectrum immunotherapy of malignant disease.

Grafting primary human T lymphocytes with cancer-specific chimeric single chain and two chain TCR

Primary human activated T lymphocytes were genetically grafted with chimeric T cell receptors (TCR). Three domain single chain (sc-) TCR as well as two chain (tc-) TCR gene constructs were derived from the melanoma-specific cytotoxic human T cell (CTL) clone 82/30, and linked to the CD3-ζ signaling element. Chimeric TCR α and β receptor genes were structurally designed to prevent pairing with endogenous TCR α and β chains in order to prevent the generation of unpredictable immune specificities. After transduction of polyclonally activated human peripheral blood lymphocytes with retroviral vectors harboring the chimeric receptor genes, genetically engineered cells specifically recognized and responded to MAGE-A1POS/HLA-A1POS cells. Importantly, each type of transduced T lymphocytes that bound specifically to peptide/MHC complexes also showed specific anti-tumor reactivity as well as lymphokine production. Genetically engineered primary human T lymphocytes expressing chimeric sc- or tc-TCR therefore hold promise for disease-specific therapies.

Peptide fine specificity of anti-glycoprotein 100 CTL is preserved following transfer of engineered TCR alpha beta genes into primary human T lymphocytes

TCR with known antitumor reactivity can be genetically introduced into primary human T lymphocytes and provide promising tools for immunogene therapy of tumors. We molecularly characterized two distinct TCRs specific for the same HLA-A2-restricted peptide derived from the melanocyte differentiation Ag gp100, yet exhibiting different stringencies in peptide requirements. The existence of these two distinct gp100-specific TCRs allowed us to study the preservation of peptide fine specificity of native TCRαβ when engineered for TCR gene transfer into human T lymphocytes. Retroviral transduction of primary human T lymphocytes with either one of the two sets of TCRαβ constructs enabled T lymphocytes to specifically kill and produce TNF-α when triggered by native gp100pos/HLA-A2pos tumor target cells as well as gp100 peptide-loaded HLA-A2pos tumor cells. Peptide titration studies revealed that the cytolytic efficiencies of the T lymphocyte transductants were in the same range as those of the parental CTL clones. Moreover, primary human T lymphocytes expressing either one of the two engineered gp100-specific TCRs show cytolytic activities in response to a large panel of peptide mutants that are identical with those of the parental CTL. The finding that two gp100-specific TCR, derived from two different CTL, can be functionally introduced into primary human T lymphocytes without loss of the Ag reactivity and peptide fine specificity, holds great promise for the application of TCR gene transfer in cancer immunotherapy.

CGG repeat in the FMR1 gene: size matters

Willemsen R, Levenga J, Oostra BA. CGG repeat in the FMR1 gene: size matters.

A retroviral vector system ‘STITCH’ in combination with an optimized single chain antibody chimeric receptor gene structure allows efficient gene transduction and expression in human T lymphocytes

Genetic engineering of T lymphocytes for adoptive clinical immunotherapy calls for efficient gene transduction methods. Therefore, a transient retroviral gene transduction system ‘STITCH’ was developed comprising pSTITCH retroviral vector encoding the transgene, plasmids encoding Moloney murine leukemia virus gag/pol and gibbon ape leukemia virus envelope, and the human kidney cell line 293T as a packaging line. Cotransfection of retroviral vector and packaging plasmids in 293T cells results in the production of GALV env pseudotyped viral particles with a titer of 107 infectious units per milliliter. The ‘STITCH’ gene transduction system efficiently transduces genes into activated human T lymphocytes derived from healthy donors and cancer patients. The efficacy of gene transduction is donor-independent. A direct application of the ‘STITCH’ gene transduction system is the genetic engineering of activated human T lymphocytes to induce expression of antibody based chimeric receptors in their membrane. Introduction of these chimeric receptors into activated human T lymphocytes graft these cells with specificity for, for example, renal cell carcinoma. In order to study the effect of the chimeric receptor gene structure on the processes ultimately leading to functional membrane expression, we designed a number of different chimeric receptor gene structures and subsequently compared their membrane expression on 293T cells and activated human T lymphocytes. Distinct membrane expression densities were observed on 293T cells and human T lymphocytes for the different chimeric receptor gene constructs. Gene transduction of activated human T lymphocytes with four out of five chimeric receptor gene constructs resulted in functional expression of chimeric receptor as demonstrated by specific recognition and cytolysis of renal cell carcinoma.

Molecular design of the C alpha beta interface favors specific pairing of introduced TCR alpha beta in human T cells

A promising approach to adoptive transfer therapy of tumors is to reprogram autologous T lymphocytes by TCR gene transfer of defined Ag specificity. An obstacle, however, is the undesired pairing of introduced TCRα- and TCRβ-chains with the endogenous TCR chains. These events vary depending on the individual endogenous TCR and they not only may reduce the levels of cell surface-introduced TCR but also may generate hybrid TCR with unknown Ag specificities. We show that such hybrid heterodimers can be generated even by the pairing of human and mouse TCRα- and TCRβ-chains. To overcome this hurdle, we have identified a pair of amino acid residues in the crystal structure of a TCR that lie at the interface of associated TCR Cα and Cβ domains and are related to each other by both a complementary steric interaction analogous to a “knob-into-hole” configuration and the electrostatic environment. We mutated the two residues so as to invert the sense of this interaction analogous to a charged “hole-into-knob” configuration. We show that this inversion in the CαCβ interface promotes selective assembly of the introduced TCR while preserving its specificity and avidity for Ag ligand. Noteworthily, this TCR modification was equally efficient on both a Mu and a Hu TCR. Our data suggest that this approach is generally applicable to TCR independently of their Ag specificity and affinity, subset distribution, and species of origin. Thus, this strategy may optimize TCR gene transfer to efficiently and safely reprogram random T cells into tumor-reactive T cells.

TCR-Like Human Antibodies Expressed on Human CTLs Mediate Antibody Affinity-Dependent Cytolytic Activity

The permanent genetic programming via gene transfer of autologous T cells with cell surface receptors directed toward tumor-related Ags holds great promise for the development of more-specific tumor therapies. In this study we have explored the use of Abs directed to MHC-peptide complexes (or TCR-like Abs) to engraft CTLs with exquisite specificity for cancer cells. First, we affinity matured in vitro a previously selected TCR-like Ab, Fab-G8, which is highly specific for the peptide melanoma-associated Ag-A1 presented by the HLA-A1 molecule. A combination of L chain shuffling, H chain-targeted mutagenesis, and in vitro selection of phage display libraries yielded a Fab-G8 Ab derivative, Fab-Hyb3, with an 18-fold improved affinity yet identical peptide fine specificity. Fab-G8 and Fab-Hyb3 were expressed on primary human T lymphocytes as cell surface-anchored Fab, demonstrating that T cells expressing the high-affinity Fab-Hyb3 molecule eradicate tumor cells much more effectively. Furthermore, the gain in ligand-binding affinity resulted in a 2-log improvement in the detection of peptide/MHC complexes on melanoma-associated Ag-A1 peptide-loaded cells. In summary, an affinity-matured Ab specifically recognizing a cancer-related peptide/MHC complex was generated and used to improve the tumor cell killing capacity of human T cells. This strategy, based on engraftment of T cells with in vitro engineered Abs, is an attractive alternative to the laborious, and in many cases unsuccessful, generation of highly potent tumor-specific T lymphocytes.

The FMR1 gene and fragile X‐associated tremor/ataxia syndrome

The CGG‐repeat present in the 5′UTR of the FMR1 gene is unstable upon transmission to the next generation. The repeat is up to 55 CGGs long in the normal population. In fragile X patients, a repeat length exceeding 200 CGGs (full mutation: FM) generally leads to methylation of the repeat and the promoter region, which is accompanied by silencing of the FMR1 gene. The gene product FMRP is involved in regulation of transport and translation of certain mRNA in the dendrite, thereby affecting synaptic plasticity. This is central to learning and memory processes. The absence of FMRP seen in FM is the cause of the mental retardation seen in fragile X patients. The premutation (PM) is defined as 55–200 CGGs. Female PM carriers are at risk of developing primary ovarian insufficiency. Recently it was discovered that elderly PM carriers might develop a progressive neurodegenerative disorder called fragile X‐associated tremor/ataxia syndrome. Although arising from the mutations in the same gene, distinct mechanisms lead to fragile X syndrome (absence of FMRP) and FXTAS (toxic RNA gain of function). The pathogenic mechanisms thought to underlie these disorders are discussed, with a specific emphasis on FXTAS. This review gives insight on the implications of all possible repeat length categories seen in fragile X families.

Human TCR That Incorporate CD3ζ Induce Highly Preferred Pairing between TCRα and β Chains following Gene Transfer

TCR gene therapy is adversely affected by newly formed TCRαβ heterodimers comprising exogenous and endogenous TCR chains that dilute expression of transgenic TCRαβ dimers and are potentially self-reactive. We have addressed TCR mispairing by using a modified two-chain TCR that encompasses total human CD3ζ with specificities for three different Ags. Transfer of either TCRα:CD3ζ or β:CD3ζ genes alone does not result in surface expression, whereas transfer of both modified TCR chains results in high surface expression, binding of peptide-MHC complexes and Ag-specific T cell functions. Genetic introduction of TCRαβ:ζ does not compromise surface expression and functions of an endogenous TCRαβ. Flow cytometry fluorescence resonance energy transfer and biochemical analyses demonstrate that TCRαβ:CD3ζ is the first strategy that results in highly preferred pairing between CD3ζ-modified TCRα and β chains as well as absence of TCR mispairing between TCR:CD3ζ and nonmodified TCR chains. Intracellular assembly and surface expression of TCR:CD3ζ chains is independent of endogenous CD3γ, δ, and ε. Taken together, our data support the use of TCRαβ:CD3ζ to prevent TCR mispairing, which may provide an adequate strategy to enhance efficacy and safety of TCR gene transfer.

A phage display selected Fab fragment with MHC class i-restricted specificity for MAGE-A1 allows for retargeting of primary human T lymphocytes

The clinical benefit of adoptive transfer of MHC-restricted cytotoxic T lymphocytes(CTL) for the treatment of cancer is hampered by the low success rate to generate antitumor CTLs. To bypass the need for tumor-specific CTL, we developed a strategy that allows for grafting of human T lymphocytes with MHC-restricted antigen specificity using in vitro selected human Fab fragments fused to the Fc(ɛ)RI-γ signaling molecule. Retroviral introduction of a Fab-based chimeric receptor specific for MAGE-A1/HLA-A1 into primary human T lymphocytes resulted in binding of relevant peptide/MHC complexes. Transduced T lymphocytes responded to native MAGE-A1/HLA-A1POS target cells by specific cytokine production and cytolysis. Therefore, peptide/MHC-specific Fab fragments represent new alternatives to TCR to confer human T lymphocytes with tumor specificity, which provides a promising rationale for developing immunogene therapies. Gene Therapy (2001) 8, 000-000.