Bispecific agents target endogenous murine T cells against human tumor xenografts.

A variety of immunological approaches to cancer treatment are currently being explored. These include strategies designed to enhance or redirect the activity of T cells against tumors. Bispecific antibodies comprise a class of agents capable of redirecting T cells by binding to a tumor antigen and the T-cell receptor (TCR). In vivo pre-clinical testing of bispecific antibodies against human tumors has to date been limited to the use of immunodeficient mice that receive the bispecific agent, activated human effector T cells, and human tumor cells. In this report, we show that TCR transgenic/RAG-1 knockout mice (TCR/RAG) serve as a unique model allowing endogenous T cells to be redirected against transplanted human tumors. The findings show that TCR/RAG mice (i) accepted transplants of human tumors, including the folate-receptor-positive tumor line KB; (ii) contained endogenous cytotoxic T lymphocytes that could be activated in vivo with an antigenic peptide recognized by the transgenic TCR; (iii) rejected human tumors after treatment with the activating peptide and bispecific agents that contained folic acid co-valently linked to an anti-TCR antibody. Successful rejection was achieved with folate conjugates of Fab or scFv fragments. Treatment with activating agents and bispecific conjugates resulted in the complete eradication of freshly transplanted tumors as well as significantly prolonging the survival of mice bearing established solid tumors. Our results highlight the importance of including T-cell-activating modalities in combination with bispecific antibodies. Additionally, we introduce a system that allows endogenous T cells to be redirected against human tumor xenografts and in which the T cells may be followed in vivo by use of a clonotypic marker.

Effects of complementarity determining region mutations on the affinity of an alpha/beta T cell receptor: measuring the energy associated with CD4/CD8 repertoire skewing.

It has been proposed that the generally low affinities of T cell receptors (TCRs) for their peptide-major histocompatibility complex (pMHC) ligands (Kd approximately 10(-4) to 10(-7) M) are the result of biological selection rather than an intrinsic affinity limitation imposed by the TCR framework. Using a soluble version of the 2C TCR, we have used complementarity determining region (CDR)-directed mutagenesis to investigate whether the affinity of this receptor for its allogeneic pMHC ligand can be improved upon. We report that several mutants at positions lying within CDR3alpha and CDR2beta showed increased affinities for pMHC compared with the wild-type receptor. Additionally, we have investigated whether Valpha mutations that have been implicated in the phenomenon of CD8(+) repertoire skewing achieve this skewing by means of generalized increases in affinity for MHC-I molecules. Two mutants (S27F and S51P), which each promote skewing toward a CD8(+) phenotype, exhibited significantly reduced affinity for pMHC-I, consistent with a quantitative-instructional model of CD4/CD8 lineage commitment. This model predicts that CD8 is downregulated on thymocytes that have TCR-ligand interactions above a minimal energy threshold. Together, the results (a) demonstrate that engineering higher affinity TCRs is feasible, and (b) provide TCR-pMHC energy values associated with CD4/CD8 repertoire skewing.

Targeting tumor cells with bispecific antibodies and T cells.

It has been known for some time that mammalian immune systems are capable of eliminating large tumor burdens. Redirecting the immune response of a patient to an established tumor has now become the focus of various therapeutic strategies. In this report, two projects toward this goal are described. The first project involves the development of a transgenic mouse model for T cell directed therapeutics. These mice express specific T cell receptor alpha and beta transgenes on a background in which the recombinational-activating-gene-1 (RAG) has been knocked out. The mice express cytotoxic T cells but not either T helper cells or B cells. Despite these deficiencies, the animals are capable of eliminating tumors that express the appropriate peptide/major histocompatibility complex ligand that is recognized by the alphabeta transgenic T cell receptor. Human tumors grow as transplants in these mice, thereby allowing various agents that redirect the endogenous T cells against human tumors to be tested. The second project involves a description of such agents: bispecific antibodies that simultaneously bind to an immune effector cell and a tumor cell. The bispecific antibody described here consists of folate attached to anti-T cell receptor antibodies, or their fragments. A single-chain Fv coupled with folate can redirect the lysis of human tumor cells that bear the high affinity folate receptor. Preliminary in vivo data showed that the folate/antibody conjugates were also capable of mediating rejection of the human tumor. This transgenic mouse model should now allow the evaluation and optimization of bispecific agents that can redirect a patient's own T cell response.

Alanine scanning mutagenesis of an alphabeta T cell receptor: mapping the energy of antigen recognition.

The T cell receptor (TCR) from the alloreactive T lymphocyte 2C recognizes a nonamer peptide QL9 complexed with the MHC class I molecule H2-Ld. Forty-two single-site alanine substitutions of the 2C TCR were analyzed for binding to QL9/Ld and anti-TCR antibodies. The results provided a detailed energy map of T cell antigen recognition and indicated that the pMHC and clonotypic antibody epitopes on the TCR were similar. Although residues in each Valpha and Vbeta CDR are important in binding pMHC, the most significant energy for the TCR/QL9/Ld interaction was contributed by CDRs 1 and 2 of both alpha and beta chains. The extent to which the individual energy contributions are directed at class I helices or peptide was also assessed.

Antigen recognition and allogeneic tumor rejection in CD8+ TCR transgenic/RAG(-/-) mice.

Three sources of help for the development of a CD8+ CTL response have been described: the CD4+ direct and indirect pathways and the CD8+ direct pathway. In an effort to understand the minimal requirements for the development of a CTL response in vivo, we have bred mice transgenic for the 2C TCR onto a RAG(-/-) background. The 2C T cells in this animal are exclusively CD8+ CTLs of a single specificity, and they exhibit altered thymic maturation compared with that of T cells from 2C TCR/RAG(+/+) mice. T cells from 2C TCR/RAG(-/-) mice can be activated to a high level in vivo by administration of a self-MHC-restricted antigenic peptide. The 2C TCR/RAG(-/-) mice are able to reject B7-negative allogeneic tumors bearing the appropriate peptide/MHC ligand p2C/Ld. These mice fail to reject syngeneic tumors, and their RAG(-/-) littermates lacking 2C T cells uniformly succumb to both allogeneic and syngeneic tumors. Moreover, blockade of B7 costimulatory molecules fails to prevent tumor rejection in the 2C TCR/RAG(-/-) mice, suggesting that allorejection is occurring independently of B7-mediated costimulation as well as in the absence of CD4+ T cells. CTLs isolated from the site of the tumor during the period of rejection express the activation marker CD25 and are able to mediate ex vivo cytolysis of tumor cells bearing the appropriate Ag. These results suggest that in this TCR transgenic model with a very high precursor frequency, CTL development can occur in the absence of B7:CD28 costimulation and without CD4+ help.

A residue in the center of peptide QL9 affects binding to both Ld and the T cell receptor.

The TCR from the alloreactive clone 2C recognizes p2C (LSPFPFDL)/Ld and QL9 (QLSPFPFDL)/Ld complexes with affinities of 2 x 10(6) and 10(7) M(-1). Recently, it was proposed that the Phe at position 4 of p2C is critical for recognition by the 2C TCR. To further characterize the role of this peptide position in binding to Ld and in recognition by the 2C TCR, we changed the corresponding peptide position in QL9 (position 5) to other amino acids. Binding affinities of these peptides for Ld and of the peptide/Ld complexes for a soluble single chain TCR were determined. Unexpectedly, it was shown that this peptide position has a significant effect on Ld binding (100-fold), with positively charged and hydrophobic residues having a beneficial effect and negatively charged residues having a detrimental effect. Measurements of the binding affinities of these peptide/Ld complexes for the 2C TCR showed that at 4 degrees C only a Tyr substitution at this position retained high affinity for the TCR. However, significant differences in TCR binding were observed among QL9 peptide variants at 4 degrees C compared with that at 37 degrees C. The influence of this peptide position on both Ld binding and TCR binding may suggest that the 2C TCR recognizes an Ld conformational determinant that is altered by interactions with the residue at position 5 of QL9. A strong correlation was also observed between peptide-Ld affinity and the ability of peptides to sensitize Ld target cells for lysis by CTL 2C. The results are considered in view of recent models on the relationship between T cell activity and TCR-peptide-MHC binding properties.

A strategy for the synthesis and screening of thiol-modified peptide variants recognized by T cells.

In this study we present a strategy for the identification of novel peptide conjugates which may be used to understand the molecular details of the recognition process or to potentially regulate T cell-mediated responses. The approach involves the incorporation of cysteine into a known peptide at a position of interest and subsequent chemical conjugation using thiol-specific agents. Conjugates derived from the nonapeptide QL9 that is recognized by CTL 2C had either enhanced or reduced activity compared to the original cys-peptides. Different classes of thiol-reactive agents (alkyl halides, alkylthiolsulfonates, and disulfides) were tested with increases in activity of over 100-fold. As with standard peptide analogs, the activity depended on the position of the cysteine within the peptide and the nature of the chemically linked functional group. Use of this approach in a cysteine 'scan' of all positions of the original peptide is cost effective and with the availability of many different thiol-specific functional groups will allow the screening of considerably larger libraries of chemically modified peptides than have been used to date. Additionally, these findings may provide insight into the pathogenesis of thiol agents involved in contact sensitivity reactions.