Adoptive transfer of Ag-specific T lymphocytes is an attractive form of immunotherapy for cancers. However, acquiring sufficient numbers of host-derived tumor-specific T lymphocytes by selection and expansion is challenging, as these cells may be rare or anergic. Using engineered T cells can overcome this difficulty. Such engineered cells can be generated using a chimeric Ag receptor based on common formats composed from Ag-recognition elements such as αβ-TCR genes with the desired specificity, or Ab variable domain fragments fused with T cell–signaling moieties. Combining these recognition elements are Abs that recognize peptide-MHC. Such TCR-like Abs mimic the fine specificity of TCRs and exhibit both the binding properties and kinetics of high-affinity Abs. In this study, we compared the functional properties of engineered T cells expressing a native low affinity αβ-TCR chains or high affinity TCR-like Ab–based CAR targeting the same specificity. We isolated high-affinity TCR-like Abs recognizing HLA-A2-WT1Db126 complexes and constructed CAR that was transduced into T cells. Comparative analysis revealed major differences in function and specificity of such CAR-T cells or native TCR toward the same antigenic complex. Whereas the native low-affinity αβ-TCR maintained potent cytotoxic activity and specificity, the high-affinity TCR-like Ab CAR exhibited reduced activity and loss of specificity. These results suggest an upper affinity threshold for TCR-based recognition to mediate effective functional outcomes of engineered T cells. The rational design of TCRs and TCR-based constructs may need to be optimized up to a given affinity threshold to achieve optimal T cell function.

Chimeric antigen receptors (CARs) are immunoreceptors that redirect T cells to selectively kill tumor cells. Given their clinical successes in hematologic malignancies, there is a strong aspiration to advance this immunotherapy for solid cancers; hence, molecular CAR design and careful target choice are crucial for their function. To evaluate the functional significance of the biophysical properties of CAR binding (i.e., affinity, avidity, and antigen density), we generated an experimental system in which these properties are controllable. We constructed and characterized a series of CARs, which target the melanoma tumor–associated antigen Tyr/HLA-A2, and in which the affinity of the single-chain Fv binding domains ranged in KD from 4 to 400 nmol/L. These CARs were transduced into T cells, and each CAR T-cell population was sorted by the level of receptor expression. Finally, the various CAR T cells were encountered with target cells that present different levels of the target antigen. We detected nonmonotonic behaviors of affinity and antigen density, and an interrelation between avidity and antigen density. Antitumor activity measurements in vitro and in vivo corroborated these observations. Our study contributes to the understanding of CAR T-cell function and regulation, having the potential to improve therapies by the rational design of CAR T cells. See related article on p. 946

Inherent and acquired multidrug resistance (MDR) is characterized by a simultaneous resistance to diverse anticancer drugs and is a major impediment towards curative chemotherapy of cancer. Hence one important goal is to develop strategies aimed at specific targeting of major anticancer drug efflux transporters of the ATP-binding cassette (ABC) superfamily including multidrug resistance protein 1 -MRP1 (ABCC1). To date, no monoclonal antibody has been isolated that can target an extracellular MRP1 epitope. Using a phage display approach, we have isolated a recombinant singlechain Fv (scFv) antibody that specifically reacts with the extracellular N-terminus of the human MRP1. Flow cytometric analysis revealed that this scFv fragment binds specifically to various viable human tumor cells that display variable Key words: multi drug resistance (MDR); scFv antibody; MRP1; phage displayCombination chemotherapy continues to play a major role in the treatment of various human malignancies. However, the efficacy of various chemotherapeutics has been limited by the frequent emergence of various anticancer drug resistance phenomena. The best-studied mechanisms of multidrug resistance (MDR) in malignant cells involve the overexpression of ATP-driven anticancer drug efflux pumps of the ABC superfamily. 1-3 These include the multidrug transporter, P-glycoprotein (Pgp), which extrudes various hydrophobic cytotoxic agents, as well as members of the multidrug resistance protein (MRP) family, currently comprising 9 exporters (MRP1 through MRP9). 3-5 MRP1 was discovered during a differential hybridization screen aimed at identifying mRNAs overexpressed in doxorubicin-resistant lung cancer cells that lack P-glycoprotein overexpression. 6 The 100 -200-fold MRP1 mRNA overexpression was a result of gene amplification. Analysis of the 1,531 amino acid sequence of MRP1 sequence identified this protein as a member of the ATP-binding cassette (ABC) superfamily of transporter proteins. 6 Importantly, transfection of the MRP1 cDNA conferred upon sensitive cells MDR to anthracyclines, Vinca alkaloids, etoposide, arsenical and antimonial oxyanions but not to cisplatinum and mitoxantrone. 7 These studies paved the way for the isolation of additional MRPs, MRP2 through MRP9, which have been shown to confer resistance to various toxic compounds that are either initially hydrophilic or that undergo intracellular conjugation to glutathione, sulfate and glucuronate. 5 Furthermore, another ABC transporter recently shown to mediate MDR in various tumor cells of epithelial origin is the breast cancer resistance protein (BCRP). 8 In order to study anticancer drug transporter proteins such as MRP1 in clinical specimens and to reveal their physiological functions, specific monoclonal antibodies (MAbs) were recently developed. 9 -11 Monoclonal antibodies against MDR proteins were used for 2 main purposes: a) detection and quantification of MRP and P-glycoprotein expression in drug-resistant cancer cells and b) reversal of MDR in cancer cells by abolishin...

Major histocompatibility complex class I molecules play a central role in the immune response against a variety of cells that have undergone malignant transformation by shaping the T-cell repertoire and presenting peptide antigens from endogeneous antigens to CD8+ cytotoxic T-cells. Diseased tumor or virus-infected cells are present on class I major histocompatibility complex molecule peptides that are derived from tumor-associated antigens or viral-derived proteins. Due to their unique specificity, such major histocompatibility complex-peptide complexes are a desirable target for novel approaches in immunotherapy. Targeted delivery of toxins or other cytotoxic drugs to cells which express specific major histocompatibility complex-peptide complexes that are involved in the immune response against cancer or viral infections would allow for a specific immunotherapeutic treatment of these diseases. It has recently been demonstrated that antibodies with the antigen-specific, major histocompatibility complex-restricted specificity of T-cells can be generated by taking advantage of the selection power of phage display technology. In addition to their tumor targeting capabilities, antibodies that mimic the fine specificity of T-cell receptors can serve as valuable research reagents that enable study of human class I peptide-major histocompatibility complex ligand presentation, as well as T-cell receptor peptide-major histocompatibility complex interactions. T-cell receptor-like antibody molecules may prove to be useful tools for studying major histocompatibility complex class I antigen presentation in health and disease as well as for therapeutic purposes in cancer, infectious diseases and autoimmune disorders.

Inherent and acquired MDR is characterized by simultaneous resistance to diverse anticancer drugs and continues to be a major impediment in the curative chemotherapy of cancer. The MDR1 gene product, Pgp, is an ATP-driven efflux pump, which extrudes a variety of dissimilar hydrophobic cytotoxic compounds from MDR cells. Pgp overexpression results in MDR of tumor cell lines in vitro as well as of a variety of human malignancies. Thus, one major goal is to develop strategies aimed at specifically disrupting Pgp drugefflux activity. To this end, we have developed a small recombinant antibody capable of potent reversal of MDR, by disrupting Pgp drug-efflux activity. Using a phage display approach, we isolated a small scFv recombinant antibody fragment that specifically reacts with the first extracellular loop of human Pgp. This scFv fragment binds specifically to various Pgp-overexpressing human MDR carcinoma cell lines, consequently disrupts Pgp drug-efflux function and thereby reverses the MDR phenotype. We have successfully disrupted anticancer drug-extrusion pump activity in MDR cells using a small recombinant scFv fragment. We propose that these novel small Fv-based recombinant antibody molecules may lead to the development of a new class of antibody fragment-based agents that specifically inhibit Pgp drug extrusion. Hence, these small recombinant antibody fragments may be applied in combination chemotherapy to overcome MDR in various human cancers. © 2004 Wiley-Liss, Inc. Key words: P-glycoprotein; multidrug resistance; single-chain FvCombination chemotherapy continues to play a major role in the treatment of various human malignancies. MDR, whereby tumor cells simultaneously possess intrinsic or acquired cross-resistance to diverse anticancer drugs, hampers the efficacy of the chemotherapeutic treatment of various human cancers. 1-3 Intense molecular investigations of the MDR phenomenon over the past 2 decades have resulted in the isolation and characterization of genes encoding for several plasma membrane glycoproteins associated with MDR. The best-studied mechanism of MDR in cancer cells is overexpression of an energy-dependent efflux pump, the multidrug transporter Pgp. 4 Pgp is a member of a large ABC superfamily of transport proteins. 5 Pgp-mediated MDR plays an important clinical role in the resistance of various tumor cells to chemotherapy, as judged by the high incidence of MDR1 expression in tumors of various types and the correlations between MDR1 expression and lack of response to chemotherapy in various human malignancies. 6,7 These findings prompted a major effort aimed at identifying strategies and agents capable of reversing Pgp-mediated MDR. 3 Inhibitors of the MDR phenotype in cancer cells may function in 2 main modes: they can either modify the expression (i.e., decrease it) or disrupt the drug-efflux function of the transporter proteins involved in MDR. 3 The search for "chemosensitizers" that disrupt the function of these drug exporters and thereby reverse MDR has grown in parallel with th...