TCR-like Antibody Research Articles

Circulating antigen presenting cells in chronically infected HBV patients do not constitutively cross-present HBV antigens (105.22)

Abstract Persistent exposure to antigen is thought to play a primary role in dysfunction of virus-specific T cells in chronic infection. Our study sought to determine whether antigen presenting cells (APCs) in the blood of chronic HBV patients constitutively cross-present circulating HBV antigen to virus-specific CD8 T cells. We isolated dendritic cells, CD14 or CD16 monocytes and B cells from HLA-A2+ healthy donors and chronic HBV patients with 10^8 IU HBV DNA/ml serum. APCs were co-incubated with TCR-redirected T cells specific for HLA-A2 restricted HBc18-27 and HBs183-91 epitopes and activation was monitored by staining for IFN-γ. APCs were also stained with TCR-like antibodies specific for the same peptide/HLA-A2 complexes. We did not observe any HBc18-27- or HBs183-91-specific T cell activation following incubation with sorted healthy donor APCs. Nor did we see T cell activation after co-culture with APCs isolated from chronic HBV patient PBMCs. Staining with TCR-like antibodies confirmed that there were no detectable HBc18-27- or HBs183-91-HLA-A2 complexes constitutively present on the cell surface. IFN-γ production and TCR-like antibody staining was observed with peptide loaded APCs. Our data suggests that APCs in the peripheral blood of chronically infected patients do not constitutively cross-present antigen to virus-specific CD8 T cells and therefore may not contribute to T cell dysfunction.

73 ADMINISTRATION OF THE ENTRY INHIBITOR MYRCLUDEX-B AFTER ESTABLISHMENT OF HEPATITIS B VIRUS INFECTION PREVENTS VIRAL SPREADING AMONG HUMAN HEPATOCYTES IN UPA MICE

with suppressed anti-viral innate and adaptive immune responses. To circumvent these immune defects and increase the efficacy of current interferon therapy against chronic HBV infection we created a novel tool that combines the exquisite specificity of HBV-specific CD8 T cells with the antiviral effect of cytokines in a format resistant to the hepatic suppression. Methods: We generated antibodies with specificity for the peptide/MHC of HBV surface (S 183–91/A201) and core (core 18–27/A201) antigens presented on HBV infected cells. These antibodies mimic the MHC/peptide complex recognition by the T cell receptor (TCR) of HBV-specific CD8 T cells, thus called TCRlike antibodies (TCRL). These antibodies were engineered to carry Interferon alpha moiety. Results: The TCRL antibodies demonstrated sensitivity similar to HBV-specific CD8 T cells from resolved hepatitis patients. They also recognized ex vivo HBV-infected hepatocytes from chronic HBV patients and the recognition was not affected by the presence of circulating HBV antigens. Importantly, IFN-alpha fusion did not alter the sensitivity of TCRL to cells expressing HBV antigens, while affinity of fused IFN-alpha to its own receptor was reduced. Interestingly, IFN-alpha activity was markedly enhanced on cells expressing HBV antigens. Pre-blocking of the MHC/peptide sites with TCRL abrogated the enhanced IFN-alpha activity of TCRLIFNa, suggesting that the enhanced IFNa activity is likely through a mechanism of increased local concentration of TCRL-IFNa as a result of anchoring to MHC/peptide complexes on target cells through TCRL. Conclusion: We demonstrate that TCRL can deliver IFN-alpha to HBV-specific target cells with greater potency than naked or pegylated interferon. Therefore modified TCRL can serve as a novel targeted therapeutic delivery platform to provide a treatment for HBV-infected patients with potentially reduced pleiotropic effects of interferon.

Targeting Hepatitis B Virus-Infected Cells with a T-Cell Receptor-Like Antibody

Virus-specific CD8 T cells are activated when their T-cell receptors (TCRs) recognize the specific viral peptide/major histocompatibility complex (MHC) class I (pMHC) complexes present on the surface of infected cells. Antibodies able to recognize the specific pMHC can mimic TCR specificity and both represent a valuable biological tool to visualize pMHC complexes on infected cells and serve as a delivery system for highly targeted therapies. To evaluate these possibilities, we created a monoclonal antibody able to specifically recognize a hepatitis B virus (HBV) envelope epitope (Env at positions 183 to 91 [Env183-91]) presented by the HLA-A201 molecule, and we tested its ability to recognize HBV-infected hepatocytes and to deliver a cargo to a specific target. We demonstrate that this antibody detects and visualizes the processed product of HBV proteins produced in naturally HBV-infected cells, is not inhibited by soluble HBV proteins present in patient sera, and mediates the intracellular delivery of a fluorescent molecule to target cells. Additionally, compared to CD8 T cells specific for the same HBV epitope, the TCR-like antibody has both a superior sensitivity and a specificity focused on distinct amino acids within the epitope. These data demonstrate that a T-cell receptor-like antibody can be used to determine the quantitative relationship between HBV replication and specific antigen presentation to CD8 T cells and serves as a novel therapeutic delivery platform for personalized health care for HBV-infected patients.

12 INCIDENCE AND DETERMINANTS OF SPONTANEOUS DECLINE OF HBV DNA TO UNDETECTABLE LEVEL IN PATIENTS WITH HIGH VIRAL LOAD

thus to distinguish HBV infected hepatocytes from non-infected ones. These antibodies were used to analyze the distribution and quantification of pMHC on HBV infected cells and to test their ability to deliver compounds to infected cells. Methods: Antibody secreting B cell hybridomas were generated from mice immunized with HBVc18–27/A201 and HBVe183–91/A201 pMHC monomers. The number of pMHC complexes on individual cells was enumerated by flow cytometry based quantification technique, while HBV antigen was quantified by Western blot and Quantity One Software. Confocal microscopy was used to detect intracellular delivery of fluorescent dye. Results: Culture supernatants from numerous hybridomas were screened for specificity. Five of them were able to produce antibodies that recognized T2 cells pulsed with core 18–27 (2 clones) or env 183–91 peptides (3 clones) but not T2 cells pulsed with irrelevant HLA-A0201 binding peptide. The two TCRlike antibodies could detect pMHC complexes on the surface of HepG2 cells with sensitivity lower than that of T cells (1nM versus 1pM) but they recognized the pMHC expressed naturally after endogenous processing on HBV infected cells. The binding of the antibodies was not inhibited by the presence of serum HBV antigens. The TCR-like antibodies were used to quantify pMHC complexes expressed on different cell types. On average, 1×105 core proteins produced one pMHC on B cells, while in hepatocytes, a single pMHC requires endogenous synthesis of approximately 1.5×106 proteins, directly demonstrating the limited antigen processing ability of hepatocytes. Furthermore, TCR-like antibodies could deliver the fluorescent dye to the endosomal compartment of HBV-infected hepatocytes. Conclusions: We have produced TCR-like antibodies recognizing HBV infected cells. These antibodies open the possibilities to obtain precise quantitative analysis of the peptide/HLA-class I complexes on HBV infected hepatocytes and provide the base for new therapeutic strategies to deliver drugs specifically to HBV infected cells.

Expression Hierarchy of T Cell Epitopes from Melanoma Differentiation Antigens: Unexpected High Level Presentation of Tyrosinase-HLA-A2 Complexes Revealed by Peptide-Specific, MHC-Restricted, TCR-Like Antibodies

Peptide Ags presented by class I MHC molecules on human melanomas and that are recognized by CD8(+) T cells are the subjects of many studies of antitumor immunity and represent attractive candidates for therapeutic approaches. However, no direct quantitative measurements exist to reveal their expression hierarchy on the cell surface. Using novel recombinant Abs which bind these Ags with a peptide-specific, MHC-restricted manner, we demonstrate a defined pattern of expression hierarchy of peptide-HLA-A2 complexes derived from three major differentiation Ags: gp100, Melan-A/Mart-1, and tyrosinase. Studying melanoma cell lines derived from multiple patients, we reveal a surprisingly high level of presentation of tyrosinase-derived complexes and moderate to very low expression of complexes derived from other Ags. No correlation between Ag presentation and mRNA expression was found; however, protein stability may play a major role. These results provide new insights into the characteristics of Ag presentation and are particularly important when such targets are being considered for immunotherapy. These results may shed new light on relationships between Ag presentation and immune response to cancer Ags.

Can oligomeric T-cell receptor be used as a tool to detect viral peptide epitopes on infected cells?

We have utilized soluble HIV Gag-specific T-cell receptor (TCR) D3 with low affinity and TCR-like antibody 25-D1.16 recognizing its natural peptide–MHC (pMHC) ligand with high affinity to determine how affinity and off-rate of the receptor-pMHC interactions affect the sensitivity of pMHC detection on the cell surface. We found that with soluble TCR cognate pMHCs can be detected only at relatively high cell surface densities when the TCR was oligomerized using either Streptavidin or quantum dot (QD) scaffolds. While the higher affinity probe led to a greater sensitivity of pMHC detection, monomers and oligomers of the probe showed essentially the same detection limit, which is restricted by the sensitivity of standard flow cytometry technique. We have also shown that imaging of QD/TCR specifically bound to cognate pMHC on the cell surface yielded a very bright fluorescent signal that can enhance the sensitivity of viral peptide detection on infected cells.

How a T Cell Receptor-like Antibody Recognizes Major Histocompatibility Complex-bound Peptide

We determined the crystal structures of the T cell receptor (TCR)-like antibody 25-D1.16 Fab fragment bound to a complex of SIINFEKL peptide from ovalbumin and the H-2K(b) molecule. Remarkably, this antibody directly "reads" the structure of the major histocompatibility complex (MHC)-bound peptide, employing the canonical diagonal binding mode utilized by most TCRs. This is in marked contrast with another TCR-like antibody, Hyb3, bound to melanoma peptide MAGE-A1 in association with HLA-A1 MHC class I. Hyb3 assumes a non-canonical orientation over its cognate peptide-MHC and appears to recognize a conformational epitope in which the MHC contribution is dominant. We conclude that TCR-like antibodies can recognize MHC-bound peptide via two different mechanisms: one is similar to that exploited by the preponderance of TCRs and the other requires a non-canonical antibody orientation over the peptide-MHC complex.

Antitumor Activity of Immunotoxins with T-Cell Receptor–like Specificity against Human Melanoma Xenografts

In this study, we have explored the use of Fab-toxin proteins (immunotoxin) to target antigen-specific MHC-peptide complexes of in vitro and in vivo cancer cells. A human phage display library was used to screen for T-cell receptor (TCR)-like antibodies that are highly specific for the peptide melanoma-associated antigen MART-1(26-35) presented by HLA-A201. We also used previously selected TCR-like antibodies specific for the peptide melanoma-associated antigen gp100(280-288) presented by HLA-A201. The recombinant immunotoxin constructs were generated by fusing the targeting Fab fragment to a truncated form of Pseudomonas exotoxin, PE38KDEL. These immunotoxins bound with high affinity to the EBV-transformed JY cell line pulsed with the aforementioned peptides and internalized within 30 min. A significant inhibition of protein synthesis, which resulted in cell death, was detected at 24 h. MART-1-specific and gp100-specific immunotoxins bound and killed HLA-A201 melanoma MART-1(+) and gp100(+) cell lines that were presented at natural levels but do not bind to HLA-A201(-) or to HLA-A201(+) MART-1(-) and gp100(-) cell lines. In severe combined immunodeficient mice, MART-1 and gp100 immunotoxins significantly and discriminately inhibited human melanoma growth. These results show that MHC class I/peptide complexes can serve as a specific target for passive immunotherapy of cancer.

Targeting TARP, a novel breast and prostate tumor-associated antigen, with T cell receptor-like human recombinant antibodies.

MHC class I molecules are important components of immune surveillance. There are no available methods to directly visualize and determine the quantity and distribution of MHC/peptide complexes on individual cells or to detect such complexes on antigen-presenting cells in tissues. MHC-restricted recombinant antibodies with the same specificity of T cell receptors (TCR) may become a valuable tool to address these questions. They may also serve as valuable targeting molecules that mimic the specificity of cytotoxic T cells. We isolated by phage display a panel of human recombinant Fab antibodies with peptide-specific, MHC-restricted TCR-like reactivity directed toward HLA-A2-restricted T cell epitopes derived from a novel antigen termed TCRgamma alternative reading frame protein (TARP) which is expressed on prostate and breast cancer cells. We have characterized one of these recombinant antibodies and demonstrated its capacity to directly detect specific HLA-A2/TARP T cell epitopes on antigen-presenting cells that have complexes formed by naturally occurring active intracellular processing of the antigen, as well as on the surface of tumor cells. Moreover, by genetic fusion we armed the TCR-like antibody with a potent toxin and demonstrated that it can serve as a targeting moiety killing tumor cells in a peptide-specific, MHC-restricted manner similar to cytotoxic T lymphocytes.

Antibody Specific for the Peptide·Major Histocompatibility Complex: IS IT T CELL RECEPTOR-LIKE?

Antibodies recognizing peptide bound to a major histocompatibility complex (MHC) protein usually have a higher affinity for the composite peptide.MHC (pMHC) ligand than T cell receptors (TCR) with the same specificity. Because the solvent-accessible peptide area constitutes only a small portion of the contacting pMHC surface, we hypothesized that the contribution of the MHC moiety to the TCR-pMHC complex stability is limited, ensuring a small increment of the binding energy delivered by the peptide to be distinguishable by the TCR or the peptide-specific antibody. This suggests that the gain in affinity of the antibody-pMHC interaction can be achieved through an increase in the on-rate without a significant change in the off-rate of the interaction. To test the hypothesis, we have analyzed the binding of an ovalbumin peptide (pOV8) and its variants associated with soluble H-2Kb protein to the 25-D1.16 monoclonal antibody and compared it with the binding of the same pMHC complexes to the OT-1 TCR. This comparison revealed a substantially higher on-rate of the antibody-pMHC interaction compared with the TCR-pMHC interaction. In contrast, both the antibody and the TCR-pMHC complexes exhibited comparably fast off-rates. Sequencing of the 25-D1.16 VH and VL genes showed that they have very few somatic mutations and those occur mainly in framework regions. We propose that the above features constitute a signature of the recognition of MHC-bound peptide antigens by TCR and TCR-like antibodies, which could explain why the latter are rarely produced in vivo.

Modification of a Tumor-Derived Peptide at an HLA-A2 Anchor Residue Can Alter the Conformation of the MHC-Peptide Complex: Probing with TCR-Like Recombinant Antibodies

A common assumption about peptide binding to the class I MHC complex is that each residue in the peptide binds independently. Based on this assumption, modifications in class I MHC anchor positions were used to improve the binding properties of low-affinity peptides (termed altered peptide ligands), especially in the case when tumor-associated peptides are used for immunotherapy. Using a new molecular tool in the form of recombinant Abs endowed with Ag-specific MHC-restricted specificity of T cells, we show that changes in the identity of anchor residues may have significant effects, such as altering the conformation of the peptide-MHC complex, and as a consequence, may affect the TCR-contacting residues. We herein demonstrate that the binding of TCR-like recombinant Abs, specific for the melanoma differentiation Ag gp100 T cell epitope G9-209, is entirely dependent on the identity of a single peptide anchor residue at position 2. An example is shown in which TCR-like Abs can recognize the specific complex only when a modified peptide, G9-209-2 M, with improved affinity to HLA-A2 was used, but not with the unmodified natural peptide. Importantly, these results demonstrate, using a novel molecular tool, that modifications at anchor residues can dramatically influence the conformation of the MHC peptide groove and thus may have a profound effect on TCR interactions. Moreover, these results may have important implications in designing modifications in peptides for cancer immunotherapy, because most such peptides studied are of low affinity.

Direct visualization of distinct T cell epitopes derived from a melanoma tumor-associated antigen by using human recombinant antibodies with MHC- restricted T cell receptor-like specificity.

Specificity in the cellular immune system is controlled and regulated by the T cell antigen receptor (TCR), which specifically recognizes peptide/major histocompatibility complex (MHC) molecules. In recent years many cancer-associated MHC-restricted peptides have been isolated and because of their highly restricted fine specificity, they are desirable targets for novel approaches in immunotherapy. Antibodies that would recognize tumor-associated MHC-peptide complexes with the same specificity as the TCR would be valuable reagents for studying antigen presentation by tumor cells, for visualizing MHC-peptide complexes on cells, and eventually for monitoring the expression of specific complexes during immunotherapy. To generate molecules with such a unique fine specificity, we selected a large nonimmune repertoire of phage Fab antibodies on recombinant HLA-A2 complexed with three common antigenic T cell, HLA-A2-restricted epitopes derived from the melanoma differentiation antigen gp100. We were able to isolate a surprisingly large panel of human recombinant Fab antibodies that exhibit a characteristic TCR-like binding specificity to each of the three gp100-derived epitopes, yet unlike TCRs, they did so with an affinity in the nanomolar range. These TCR-like antibodies recognize the native MHC-peptide complex expressed on the surface of antigen-presenting cells. Moreover, they can detect the specific MHC-peptide complexes on the surface of melanoma tumor cells. These results demonstrate the ability to isolate high-affinity human recombinant antibodies with the antigen-specific, MHC-restricted specificity of T cells, and this ability was demonstrated for three different epitopes of the same melanoma-derived antigen.