Reinder L. H. Bolhuis

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.

Lymphokine-activated killer cell activity:Characteristics of effector cells and their progenitors in blood and spleen

Leukocytes in blood and spleen can be activated by interleukin 2 (IL-2) to become cytotoxic to certain tumor cell lines in vitro. Recent evidence suggests that such lymphokine-activated killer (LAK) cells can bring about the regression of solid tumors in animals and patients, under certain circumstances. Here, Ronald Herberman and colleagues from eight international laboratories, review what is known of the characteristics of LAK cell activity and conclude that most of it can be attributed to natural killer cells stimulated by IL-2.

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.

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.

Functional balance between T cell chimeric receptor density and tumor associated antigen density: CTL mediated cytolysis and lymphokine production

Genetically engineered expression of tumor-specific single chain antibody chimeric receptors (ch-Rec) on human T lymphocytes endow these cells with the parental monoclonal antibody (mAb) dictated tumor specificity and may be useful for clinical immuno-genetherapy. Therefore it was of importance to assess how the densities of tumor-specific receptors and tumor associated antigens (TAA), respectively, affect primary human T lymphocyte functions in relation to target cell susceptibilities to lysis. We therefore studied the functional balance between ch-Rec densities on human T lymphocytes and TAA on tumor cells. The gene construct encoding a ch-Rec derived from (1) a renal carcinoma cell (RCC) specific mouse mAb (G250), and (2) the human signal transducing Fc(ε)RI γ-chain was used. To obtain ch-RecHIGH-POS and ch-RecLOW-POS T lymphocytes, two distinct retroviral vectors were used to introduce the gene constructs into primary human T lymphocytes. Levels of ch-Rec-redirected T lymphocyte mediated tumor cell lysis, as well as lymphokine production were determined using RCC lines as target/stimulator cells, which express either no or increasing densities of the TAA. A functional and dynamic balance between ch-Rec densities on cytotoxic T lymphocytes (CTLs) on the one hand and TAA densities on RCCs on the other, was found. In short, ch-RecHIGH-POS CTLs are triggered by TAAHIGH-POS as well as TAALOW-POS RCCs to lyse tumor cells and produce (IFN-γ and TNF-α) lymphokine. In contrast, ch-RecLOW-POS T lymphocytes are only triggered for cytolysis and lymphokine production by relatively TAAHIGH-POS RCCs. In conclusion, (1) the activation of T lymphocyte responses is co-determined by the expression levels of the ch-Rec on T lymphocytes and the TAA on tumor cells and (2) at relatively high T lymphocyte ch-Rec expression levels the CTLs lyse tumor cells with a wide range of TAA densities.

‘Nonspecific’ MHC-unrestricted killer cells and their receptors

The receptors involved in apparently nonspecific, MHC- unrestricted effector cell-target cell interaction and lysis continue to raise controversy. They bind to distinct ligands on their target cells and activate diverse cellular functions such as gene expression, lymphokine production, proliferation and/or cytolytic activity by the effector cells. Several distinct receptors may mediate MHC-unrestricted cytotoxicity. Here, Peter Hersey and Reinder Bolhuis review evidence that the four main receptors involved in triggering this form of lytic activity are the CD2 molecule (the sheep red cell receptor), CD3-associated αβ chain T-cell receptor (TCR), the γδ chain TCR-CD3 complex and the CD16 molecule (the IgG0Fc receptor). The apparent non-specificity specificity of killing is a reflection of the widespread expression of natural ligands for these receptors on target 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.

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.

An entirely humanized CD3 ζ‐chain signaling receptor that directs peripheral blood t cells to specific lysis of carcinoembryonic antigen–positive tumor cells

Recombinant T‐cell receptors with antibody‐like specificity for tumor‐associated antigens are successfully used to direct the cytolytic activity of T cells toward tumor cells. Clinical application, however, needs to comply with the low immunogenicity of the recombinant receptor, efficient gene transfer into peripheral blood T cells, and enrichment of receptor‐grafted cells. Here, we address these issues and describe an entirely humanized immune receptor for use in adoptive immunotherapy of colorectal carcinoma. The receptor consists of a single‐chain antibody (scFv) binding domain specific for carcinoembryonic antigen (CEA), the IgG hinge and CH2/CH3 (Fc) joining region, and the transmembrane and intracellular CD3 ζ signaling chain. To express the receptor in peripheral blood T cells, both GALV envelope and MuLV 4070A pseudotyped retrovirus turned out to be equally efficient, with transduction efficiencies of about 5% to 40%, depending on the lymphocyte donor. Furthermore, receptor‐grafted T cells could be 2‐ to 6‐fold enriched by magnetic activated cell sorting, utilizing an antibody directed to the extracellular IgG domain of the receptor. Upon co‐culture with CEA+ tumor cells, receptor‐grafted T cells are specifically and efficiently activated to cytolysis and IFN‐γ secretion, demonstrating their feasibility for the adoptive immunotherapy of CEA+ carcinomas. Int. J. Cancer 88:115–120, 2000.

T Cell Retargeting with MHC Class I-Restricted Antibodies: The CD28 Costimulatory Domain Enhances Antigen-Specific Cytotoxicity and Cytokine Production

T cells require both primary and costimulatory signals for optimal activation. The primary Ag-specific signal is delivered by engagement of the TCR. The second Ag-independent costimulatory signal is mediated by engagement of the T cell surface costimulatory molecule CD28 with its target cell ligand B7. However, many tumor cells do not express these costimulatory molecules. We previously constructed phage display derived FAB, G8, and Hyb3, Ab-based receptors with identical specificity but distinct affinities for HLA-A1/MAGE-A1, i.e., “TCR-like” specificity. These chimeric receptors comprised the FcεRI-γ signaling element. We analyzed whether linking the CD28 costimulation structure to it (γ + CD28) could affect the levels of MHC-restricted cytolysis and/or cytokine production. Human scFv-G8POS T lymphocytes comprising the γ + CD28 vs the γ signaling element alone produced substantially more IL-2, TNF-α, and IFN-γ in response to HLA-A1/MAGE-A1POS melanoma cells. Also a drastic increase in cytolytic capacity of scFv-G8POS T cells, equipped with γ + CD28 vs the γ-chain alone was observed.