Antibody and T-cell receptors (TCRs) are the primary recognition molecules of the adaptive immune system. Antibodies have been extensively characterized and are being developed for a large number of therapeutic applications. This has been possible because of the ability to manufacture stable, soluble, monoclonal antibodies which retain the antigen specificity of B cells. Unlike antibodies, TCRs are not expressed in a soluble form, but are anchored to the T-cell surface by an insoluble trans-membrane domain. Characterization and development of TCRs has been hampered by the lack of suitable methods for producing them as soluble and stable proteins. Here we report the engineering of soluble human TCRs suitable for crystallization studies and potentially for in vivo therapeutic use.

Cancer cachexia is a syndrome of progressive wasting which has been suggested to be mediated by tumour-necrosis factor-alpha, interleukins 1 and 6, interferon-gamma and leukaemia-inhibitory factor. It has proved difficult to correlate levels of tumour-necrosis factor-alpha and interleukin-6 with cancer cachexia, and the weight loss induced by leukaemia-inhibitory factor may be due to toxicity. In the murine adenocarcinoma MAC16, cachexia is mediated by circulatory catabolic factors, which we have now isolated using an antibody cloned from splenocytes of mice transplanted with the MAC16 tumour, with a delayed cachexia. The material is a proteoglycan of relative molecular mass 24K which produces cachexia in vivo by inducing catabolism of skeletal muscle. The 24K material was also present in urine of cachectic cancer patients, but was absent from normal subjects, patients with weight loss due to trauma, and cancer patients with little or no weight loss. This suggests that cachexia in mice and humans may be produced by the same material.

T cell immunity can potentially eradicate malignant cells and lead to clinical remission in a minority of patients with cancer. In the majority of these individuals, however, there is a failure of the specific T cell receptor (TCR)–mediated immune recognition and activation process. Here we describe the engineering and characterization of new reagents termed immune-mobilizing monoclonal TCRs against cancer (ImmTACs). Four such ImmTACs, each comprising a distinct tumor-associated epitope-specific monoclonal TCR with picomolar affinity fused to a humanized cluster of differentiation 3 (CD3)-specific single-chain antibody fragment (scFv), effectively redirected T cells to kill cancer cells expressing extremely low surface epitope densities. Furthermore, these reagents potently suppressed tumor growth in vivo. Thus, ImmTACs overcome immune tolerance to cancer and represent a new approach to tumor immunotherapy.

Summary Urine from cancer patients with weight loss showed the presence of an antigen of Mr 24 000 detected with a monoclonal antibody formed by fusion of splenocytes from mice with cancer cachexia. The antigen was not present in the urine of normal subjects, patients with weight loss from conditions other than cancer or from cancer patients who were weight stable or with low weight loss (1 kg month-1). The antigen was present in the urine from subjects with carcinomas of the pancreas, breast, lung and ovary. The antigen was purified from urine using a combination of affinity chromatography with the mouse monoclonal antibody and reversed-phase high-performance liquid chromotography (HPLC). This procedure gave a 200 000-fold purification of the protein over that in the original urine extract and the material isolated was homogeneous, as determined by silver staining of gels. The N-terminal amino acid sequence showed no homology with any of the recognized cytokines. Administration of this material to mice caused a significant (P<0.005) reduction in body weight when compared with a control group receiving material purified in the same way from the urine of a normal subject. Weight loss occurred without a reduction in food and water intake and was prevented by prior administration of the mouse monoclonal antibody. Body composition analysis showed a decrease in both fat and non-fat carcass mass without a change in water content. The effects on body composition were reversed in mice treated with the monoclonal antibody. There was a decrease in protein synthesis and an increase in degradation in skeletal muscle. Protein degradation was associated with an increased prostaglandin E2 (PGE2) release. Both protein degradation and PGE2 release were significantly reduced in mice pretreated with the monoclonal antibody. These results show that the material of Mr 24 000 present in the urine of cachectic cancer patients is capable of producing a syndrome of cachexia in mice.

Natural T-cell responses generally lack the potency to eradicate cancer. Enhanced affinity T-cell receptors (TCRs) provide an ideal approach to target cancer cells, with emerging clinical data showing significant promise. Nevertheless, the risk of off target reactivity remains a key concern, as exemplified in a recent clinical report describing fatal cardiac toxicity, following administration of MAGE-A3 specific TCR-engineered T-cells, mediated through cross-reactivity with an unrelated epitope from the Titin protein presented on cardiac tissue. Here, we investigated the structural mechanism enabling TCR cross-recognition of MAGE-A3 and Titin, and applied the resulting data to rationally design mutants with improved antigen discrimination, providing a proof-of-concept strategy for altering the fine specificity of a TCR towards an intended target antigen. This study represents the first example of direct molecular mimicry leading to clinically relevant fatal toxicity, mediated by a modified enhanced affinity TCR designed for cancer immunotherapy. Furthermore, these data demonstrate that self-antigens that are expressed at high levels on healthy tissue should be treated with extreme caution when designing immuno-therapeutics.

A material of M r 24,000 has been isolated from a cachexia-inducing mouse tumor (MAC16) and shown to initiate protein degradation in isolated gastrocnemius muscle. Biological activity was destroyed by preincubation with peptide N-glycosidase F (PNGase F) and endo-␣-N-acetylgalactosaminidase (O-glycosidase) but not by neuraminidase or trypsin. Antibody reactivity was destroyed by treatment with periodate, indicating carbohydrate moieties to be the antigenic determinants. Antigenic activity was also reduced by treatment with PNGase F and O-glycosidase and was completely destroyed by treatment with chondroitinase ABC but was unaffected by treatment with either trypsin or chymotrypsin, confirming that the N-and O-linked sulfated oligosaccharide chains were both the antigenic and biological determinants. Biosynthetic labeling of MAC16 cells using a combination of [35 S]sulfate and [6-3 H]GlcN gave a single component of M r 24,000 containing both radiolabels. Similar material could not be isolated from a cell line (MAC13) originating from a tumor that does not cause cachexia in vivo. Digestion of 3 H/ 35 S material with PNGase F produced two fragments of M r 14,000 and 10,000 containing both radiolabels, and digestion with O-glycosidase produced three fragments of M r 14,000, 6,000, and 4,000, the first two contained both radiolabels and the third contained only 3 H. Digestion of the fragment of M r 14,000 released by PNGase F with O-glycosidase also gave fragments of M r 6,000 and 4,000. The products from both digestions were acidic as determined by anion exchange chromatography on DEAE-cellulose. The negative charge on the fragment of M r 4,000 was removed by treatment with alkaline phosphatase. This suggests that the charge originated from phosphate residues, and this has been confirmed by biosynthetic labeling of MAC16 cells with [ 32 P]orthophosphate, where radiolabel was incorporated into material of M r 24,000 and into the fragment of M r 4,000 after treatment with O-glycosidase. To determine the size of the polypeptide core MAC16 cells were biosynthetically labeled with L-[2,5-3 H]His which after chemical deglycosylation produced a major component of M r 4,000. These results suggest a model for the M r 24,000 material consisting of a central polypeptide chain of M r 4,000 and with phosphate residues that may be attached to the polypeptide or a short oligosaccharide chain containing GlcN, one O-linked sulfated oligosaccharide chain containing GlcN, and of M r 6,000 and one N-linked sulfated oligosaccharide chain of M r 10,000 also containing GlcN. Neither chain was cleaved into disaccharides with chondroitinase ABC, suggesting that the material is a sulfated glycoprotein.

PIF is associated with an accelerated rate of weight loss in patients with a tumour of the pancreatic head. This observation appears to be independent of the effect of an increased hepatic acute-phase protein response.

Naturally selected T-cell receptors (TCRs) are characterised by low binding affinities, typically in the range 1-100 microM. Crystal structures of syngeneic TCRs bound to peptide major histocompatibility complex (pMHC) antigens exhibit a conserved mode of binding characterised by a distinct diagonal binding geometry, with poor shape complementarity (SC) between receptor and ligand. Here, we report the structures of three in vitro affinity enhanced TCRs that recognise the pMHC tumour epitope NY-ESO(157-165) (SLLMWITQC). These crystal structures reveal that the docking mode for the high affinity TCRs is identical to that reported for the parental wild-type TCR, with only subtle changes in the mutated complementarity determining regions (CDRs) that form contacts with pMHC; both CDR2 and CDR3 mutations act synergistically to improve the overall affinity. Comparison of free and bound TCR structures for both wild-type and a CDR3 mutant reveal an induced fit mechanism arising from restructuring of CDR3 loops which allows better peptide binding. Overall, an increased interface area, improved SC and additional H-bonding interactions are observed, accounting for the increase in affinity. Most notably, there is a marked increase in the SC for the central methionine and tryptophan peptide motif over the native TCR.