Functional expression in mast cells of chimeric receptors with antibody specificity

Cell Biochemistry and Biophysics - Chimeric genes composed of a single-chain Fv domain (scFv) of an antibody linked with receptor chains normally present in cells of hematopoietic origin were...     

T cells expressing VHH-directed oligoclonal chimeric HER2 antigen receptors: Towards tumor-directed oligoclonal T cell therapy

Background

Adoptive cell therapy with engineered T cells expressing chimeric antigen receptors (CARs) originated from antibodies is a promising strategy in cancer immunotherapy. Several unsuccessful trials, however, highlight the need for alternative conventional binding domains and the better combination of costimulatory endodomains for CAR construction to improve the effector functions of the engineered T cells. Camelid single-domain antibodies (VHHs), which are the smallest single domain antibodies, can endow great targeting ability to CAR-engineered T cells.

Methods

We have developed a method to generate genetically engineered Jurkat T cells armed with a CAR comprising the anti-HER2 VHH as targeting moiety. From an immune camel library, five VHH clones were selected as a set of oligoclonal anti-HER2 VHHs that exhibited diverse binding abilities and joined them to CD28-CD3ζ and CD28-OX40-CD3ζ signaling endodomains. Jurkat T cells expression of VHH-CARs and cell functions were evaluated.

Results

The oligoclonal engineered T cells showed higher proliferation, cytokine secretion and cytotoxicity than each individual VHH-CAR-engineered Jurkat T cells.

Conclusions

The combination of superior targeting ability of oligoclonal VHHs with the third generation CAR can substantially improve the function of engineered T cells.

General significance

Antigen-specific directed oligoclonal T cells are alternatively promising, but safer systems, to combat tumor cells.     

Genetic engineering of T cells with chimeric antigen receptors for hematological malignancy immunotherapy

The host immune system plays an instrumental role in the surveillance and elimination of tumors by recognizing and destroying cancer cells. In recent decades, studies have mainly focused on adoptive immunotherapy using engineered T cells for the treatment of malignant diseases. Through gene engraftment of the patient’s own T cells with chimeric antigen receptor (CAR), they can recognize tumor specific antigens effectively and eradicate selectively targeted cells in an MHC-independent fashion. To date, CAR-T cell therapy has shown great clinical utility in patients with B-cell leukemias. Owing to different CAR designs and tumor complex microenvironments, genetically redirected T cells may generate diverse biological properties and thereby impact their long-term clinical performance and outcome. Meanwhile some unexpected toxicities that result from CAR-T cell application have been examined and limited the curative effects. Diverse important parameters are closely related with adoptively transferred cell behaviors, including CAR-T cells homing, CAR constitutive signaling, T cell differentiation and exhaustion. Thus, understanding CARs molecular design to improve infused cell efficacy and safety is crucial to clinicians and patients who are considering this novel cancer therapeutics. In this review, the developments in CAR-T cell therapy and the limitations and perspectives in optimizing this technology towards clinical application are discussed.     

Human T cell lines differing in phenotype and specificity are reactive with the same anti-idiotypic antibody

3D6, a monoclonal antibody selected for reactivity with the T cell antigen receptor on the T leukemic cell line HPB-ALL, was found to react with 3 to 13% of peripheral blood T lymphocytes of 10 out of 15 normal donors. Peripheral T cells of two donors were stimulated with allogeneic cells, and the 3D6+ cells were enriched by rosetting 3D6-coated cells with goat anti-mouse-coupled human red blood cells and were expanded in interleukin 2-containing medium. In this way, 90 to 100% 3D6+ cell lines were obtained that were cytotoxic for the allogeneic stimulator cells. 3D6 antibody could block antigen-specific cytotoxicity, as well as induce nonspecific cytotoxicity toward target cells that could not be killed in the absence of the 3D6 antibody. The 3D6+ cell populations contained T4+, as well as T8+ cells, indicating that 3D6 antibody defined a T cell receptor population that might harbor various antigenic specificities. One 3D6+ cell line was separated into T4+ T8- and T4- T8+ populations. 3D6 reactive T cell receptors isolated from HPB-ALL and normal cell lines were analyzed biochemically by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing, and V8 protease peptide mapping. Isoelectric focusing analysis provided additional evidence for the idea that 3D6 antibody detected a number of structurally distinct T cell receptors, because the T cell receptor alpha-chain was homogeneous in charge after desialation on the clonal tumor line HPB-ALL, but remained heterogeneous in charge on the 3D6+ normal cell lines. No great differences in charge were found between T cell receptors isolated from T4+ and T8+ 3D6+ lines, but their isoelectric focusing patterns were not identical. V8 protease peptide mapping revealed structural differences between the T cell receptor alpha-chain isolated from HPB-ALL on one hand and from the normal 3D6+ lines on the other, whereas the beta-chains did not differ greatly in primary structure according to this analysis. In addition, the peptide mapping suggested differences in primary structure between T cell receptors present on the T4+ population vs those present on the T8+ populations.     

Cloning and high level expression of a chimeric antibody with specificity for human carcinoembryonic antigen

A mouse/human chimeric antibody has been constructed by using variable light and variable heavy regions from a murine hybridoma specific for human carcinoembryonic antigen (CEA) (CEM231.6.7). These V regions were combined with kappa and gamma-1 constant region genes cloned from human lymphocytes. The chimeric constructs were sequentially electroporated into murine non-Ig-producing myeloma (P3.653) and hybridoma (SP2/0) cell. Significant differences were seen in expression levels between the two cell types. High levels of expression (24 to 32 micrograms/ml/10(6) cells) were seen with several of the anti-CEA SP2/0 transfectomas but not with the P3.653 cells. The SP2/0 transfectoma lines were adapted to serum-free, chemically defined media and grown in large scale fermentation cultures where they continued to secrete high levels of antibody. The chimeric antibodies remain reactive against human CEA with affinity constants comparable to that of the parental hybridoma antibody. High level expression will make practical the production of chimeric antibodies for in vivo therapeutic and diagnostic purposes.     

A chimeric mouse-human antibody that retains specificity for HIV gp120 and mediates the lysis of HIV-infected cells

Murine mAb BAT123, which was made against the envelope glycoprotein gp120 of HTLV-IIIB strain of HIV type 1 (HIV-1), is capable of neutralizing HTLV-IIIB in vitro. It also inhibits the fusion between uninfected CD4+ cells and HIV-1-infected cells to form syncytia. As a step to explore the potential utility of the anti-HIV antibody in vivo, we have constructed a mouse-human chimeric antibody by rDNA techniques. The chimeric antibody, which bears the variable domains of mouse antibody BAT123 and constant domains Cr1 and C kappa of human Ig retains the Ag specificity of BAT123 as determined by its reactivity with HIV-1-infected H9 cells, gp120 in Western blot analysis, and the oligopeptide recognized by BAT123. The antiviral activities of the chimeric antibody in neutralizing HIV-1 infection as well as inhibiting the syncytia formation are also found identical to those of the parent murine antibody. Moreover, in the presence of human blood mononuclear cells, the chimeric antibody but not BAT123 (mouse IgG1) induces antibody-dependent cellular cytotoxicity. The findings point to the potential usefulness of the chimeric antibody in treating patients infected with HIV-1.     

Electron microscopic investigation of cell surface antigens using a multivalent hybrid antibody with double specificity

Multivalent hybrid antibody (MHA) complexes with double specificity were prepared by combining two different antibodies, one specific against ferritin (Fer), the other against horseradish peroxidase using protein A from Staphylococcus aureus (SpA). Electron microscopy of mouse spleen lymphocytes and thymocytes (the latter coated with mouse anti-Thy-1 antibody) reacted with anti-HRP/SpA/anti-mIg complex and HRP showed the pattern of surface Ig receptors visualized by the specific peroxidase labelling. When IgG anti-Fer/SpA/IgG-anti-mIg complex was applied to the same cellular stuff, better resolution and higher accuracy were obtained although the distribution was similar. Surface Thy-1 alloantigen and Fe receptors (charged with hIgG) were simultaneously detected on the thymocyte surface by using in the same way a mixture of anti-Fer/SpA/anti-Thy-1 and anti-HRP/SpA/anti-Fab. The concomitant ultrastructural visualization of Thy-1 (with Fer) and Fc (with HRP) on mouse thymocytes clearly showed that their distribution was mainly independent and that the amount of Thy-1 antigen prevailed. These data show that electron microscopy with a mixture of MHA may be a useful technique for the concomitant location of two antigenic determinants on the cell surface.     

Encephalitogenic T cell clones with variant receptor specificity

We explored antigenic differences between guinea pig (GP)-basic protein (BP), rat (Rt)-BP, and respective peptides from the encephalitogenic region for Lewis rats by comparing the fine specificity of T lymphocyte lines and clones selected from animals primed with these Ag. Encephalitogenic T cell lines specific for GP-BP or Rt-BP predictably recognized the corresponding 72-89 and to a lesser degree the 72-84 (S55S) amino acid sequence. T cell lines selected from rats primed with GP-S55S responded preferentially to GP-S55S compared to other peptides. A T cell line raised to Rt-S55S, however, initially recognized the S55S and S72-89 peptides but were nearly unresponsive to the intact GP-BP or Rt-BP. T cell clones selected from the Rt-S55S line at that point had two distinct patterns of response: clones that recognized both of the BP and the S55S peptides adoptively transferred delayed-type hypersensitivity and experimental autoimmune encephalomyelitis. These clones also recognized residues 69-81 (S67) but not peptide S75-89. In contrast, T cell clones that responded only to synthetic peptides GP-S55S and Rt-S55S but not to the parent BP adoptively transferred delayed-type hypersensitivity but not disease in Lewis rats. The same clones failed to respond to either the S67 or the S75-89 sequences. These results demonstrate that the encephalitogenic Rt-S55S sequence houses a minimum of two T cell epitopes with differing specificities and functions. One epitope is immuno-dominant and resembles the encephalitogenic region of the intact BP molecule. The second non-encephalitogenic epitope is restricted to the S55S sequences and is not shared by the parent BP, the S67, or the S75-89 sequences. Both types of Rt-S55S-specific clones differ in fine specificity from encephalitogenic clones selected from GP-BP immunized rats, thus indicating that uniformity of T cell recognition of the encephalitogenic epitope is not an absolute condition for T cells to be encephalitogenic.     

Relationship between T cell receptors and antigen-binding factors. I. Specificity of functional T cell receptors on mouse T cell hybridomas that produce antigen-binding T cell factors

Upon antigenic stimulation with OVA-pulsed syngeneic macrophages, the mouse T cell hybridoma 231F1 produced glycosylation inhibiting factor (GIF) having affinity for OVA and IgE-suppressive factors, whereas another T cell hybridoma, 12H5, cells produced OVA-binding glycosylation enhancing factor (GEF) and IgE-potentiating factor. The OVA-binding GIF from the 231F1 cells is an Ag-specific Ts cell factor, whereas OVA-binding GEF from the 12H5 cells is an Ag-specific augmenting factor. Both hybridomas express CD3 complex and functional TCR-alpha beta. Cross-linking of TCR-alpha beta or CD3 molecules on the hybridomas by anti-TCR-alpha beta mAb or anti-CD3 mAb and protein A resulted in the formation of the same factors as those obtained by the stimulation of the cells with OVA-pulsed syngeneic macrophages. It was also found that both the 231F1 cells and 12H5 cells formed IgE-binding factors upon incubation with H-2d and H-2b APC, respectively, with a synthetic peptide corresponding to residues 307-317 in the OVA molecules (P307-317). Six other synthetic peptides, including those containing the major immunogenic epitope, i.e., P323-339, failed to stimulate the hybridomas in the presence of APC. Indeed, all of the 10 T cell hybridoma clones, which could produce either OVA-binding GIF or OVA-binding GEF, responded to P307-317 and APC for the formation of IgE-binding factors. In contrast, GIF/GEF derived from six other hybridoma clones, whose TCR recognized P323-339 in the context of a MHC product, failed to bind to OVA-coupled Sepharose. The results indicate the correlation between the fine specificity of TCR and the affinity of GIF/GEF to the nominal Ag. The amino acid sequence of P307-317 suggested that TCR on the cell sources of Ag-binding factors are specific for an external structure of the Ag molecules.     

Endowing human pancreatic ribonuclease with toxicity for cancer cells

Onconase is an amphibian protein that is now in Phase III clinical trials as a cancer chemotherapeutic. Human pancreatic ribonuclease (RNase 1) is homologous to Onconase but is not cytotoxic. Here, ERDD RNase 1, which is the L86E/N88R/G89D/R91D variant of RNase 1, is shown to have conformational stability and ribonucleolytic activity similar to that of the wild-type enzyme but > 10(3)-fold less affinity for the endogenous cytosolic ribonuclease inhibitor protein. Most significantly, ERDD RNase 1 is toxic to human leukemia cells. The addition of a non-native disulfide bond to ERDD RNase 1 not only increases the conformational stability of the enzyme but also increases its cytotoxicity such that its IC(50) value is only 8-fold greater than that of Onconase. Thus, only a few amino acid substitutions are necessary to make a human protein toxic to human cancer cells. This finding has significant implications for human cancer chemotherapy.     

Suppression of antibody synthesis by CD4+ T cell clones and normal T cells stimulated with monoclonal anti-CD3 antibody

CD4+ve Th1 clones, as well as normal splenic T cells, were found to suppress LPS-driven antibody secretion in a non-Ag-specific and non-MHC-restricted manner when the T cells were activated with the anti-CD3 mAb, 145-2C11. Suppression was observed with both primed and naive B cells, as well as with purified hapten-specific B cells, a result that suggests a direct effect of anti-CD3-activated T cells on B cell differentiation. Th1 clones activated by cognate Ag also suppressed LPS-driven antibody secretion. Furthermore, suppression of LPS-driven antibody secretion could be achieved across a cell-impermeable porous membrane when T cells were activated with anti-CD3. Suppression by Th1 clones and by normal T cells could not be attributed to a concomitant decrease in B cell proliferation or to a shift in the kinetics or isotype of the antibody response. These data demonstrate that CD4+ve Th1 clones, as well as normal T cells, can effect suppression of polyclonal antibody formation.     

Construction, expression, and biologic activity of murine/human chimeric antibodies with specificity for the human alpha/beta T cell receptor.

Murine/human chimeric antibodies with specificity for the human TCR-alpha/beta have been produced by genetic engineering. The L and H chain V region exons encoding the murine mAb BMA 031 were isolated and inserted into mammalian expression vectors containing the human kappa and gamma 1 or gamma 4 C region exons. The chimeric genes were transfected into murine Sp2/O hybridoma cells by electroporation and transfectomas secreting chimeric antibody were isolated. Secretion levels ranged from 1 to 7 pg/cell/24 h. The chimeric antibodies bound specifically to T cells and competed effectively with the parental murine mAb for binding to these sites. The ability to promote antibody-dependent cell-mediated cytolysis was significantly enhanced in the chimeric antibodies as compared with murine BMA 031. C-dependent cytolysis, however, was not detectable with any of the antibodies. Chimeric BMA 031 is a clinically relevant, genetically engineered antibody with potential uses in transplantation, graft-vs-host disease, autoimmune diseases and other T cell-related disorders.     

A genetically engineered murine/human chimeric antibody retains specificity for human tumor-associated antigen

Chimeric immunoglobulin genes were constructed by fusing murine variable region exons to human constant region exons. The ultimate goal was to produce an antibody capable of escaping surveillance by the human immune system while retaining the tumor specificity of a murine monoclonal. The murine variable regions were isolated from the functionally expressed kappa and gamma 1 immunoglobulin genes of the murine hybridoma cell line B6.2, the secreted monoclonal antibody of which reacts with a surface antigen from human breast, lung, and colon carcinomas. The kappa and gamma 1 chain fusion genes were co-introduced into non-antibody producing murine myeloma cells by electroporation. Transfectants that produced murine/human chimeric antibody were obtained at high frequency as indicated by immunoblots probed with an antisera specific for human immunoglobulin. Enzyme-linked immunoabsorbent assay analysis demonstrated that this chimeric antibody was secreted from the myeloma cells and retained the ability to bind selectively to membrane prepared from human tumor cells. The chimeric immunoglobulin was also shown by indirect fluorescence microscopy to bind to intact human carcinoma cells with specificity expected of B6.2. The ability of chimeric antibody to recognize human tumor-associated antigen makes feasible a novel approach to cancer immunotherapy.     

T cell regulation of antibody responses: an I-A-specific, autoreactive T cell collaborates with antigen-specific helper T cells to promote IgG responses

In earlier studies we showed that hapten-specific inducer T cell clones specifically induce B cells from immunized donors to secrete IgM antibodies. However, IgG responses were not observed, suggesting that an additional signal(s) was required. In this report, we show that an autoreactive T cell clone produces a factor(s) that collaborates with antigen-specific inducer T cells to promote specific IgG responses. This factor is not restricted by antigen or MHC determinants and promotes IgG production both in vivo and in vitro. These findings suggest that autoreactive cells may play an important role in the regulation of isotype expression.     

Virus specificity of human influenza virus-immune cytotoxic T cells.

The virus specificity of human in vitro cytotoxic T cell responses to influenza virus was studied with the use of peripheral blood mononuclear leukocytes from normal adult volunteers. Previous natural exposure of these donors to a variety of type A influenza viruses was documented by HI antibody titers. Cells sensitized in vitro with A/HK or A/PR8 were cytotoxic for autologous target cells infected with A/HK, A/PR8, or A/JAP 305 type A influenza viruses, but not for B/HK-infected or uninfected cells. B/HK-sensitized effector cells lysed target cells infected with B/HK but not targets infected with type A viruses. A/HK- and A/PR8-immune effector populations were shown to recognize cross-reactive antigens on A/HK- and A/PR8-infected target cells by cold target competition. Influenza-immune effector cells were cytotoxic for virus-infected autologous targets but much less so for virus-infected allogeneic targets. This self-restriction suggested that the cytotoxicity was largely T cell-mediated and was confirmed by cell separation analysis. Thus, the human secondary cytotoxic T cell response in vitro to influenza viruses is predominantly directed against cross-reactive determinants on cells infected with serologically distinct type A influenza viruses.     

Characterization of a monoclonal antibody which detects all murine alpha beta T cell receptors

Research on the specificities, functions, and maturation of T cells would be greatly aided by a collection of monoclonal antibodies which distinguishes different types of TCR. With this end in mind hamsters were immunized and tested for production of pan-reactive anti-mouse alpha beta TCR antibodies. In this report we describe the properties and uses of a mAb, H57-597, produced from one of these animals. The mAb reacts with surface receptors on all alpha beta TCR-bearing cells and does not react with receptors on gamma delta+ T cells. In an immobilized form, this antibody can directly activate T cells bearing alpha beta TCR. It can be used in immunoprecipitation reactions to precipitate receptor from the appropriate cell types. In combination with anti-CD3, the antibody can be used in cytofluorographic analyses to measure numbers of CD3+, alpha beta+, and CD3+, gamma delta+ cells in the thymus and periphery.     

Redirecting therapeutic T cells against myelin-specific T lymphocytes using a humanized myelin basic protein-HLA-DR2-zeta chimeric receptor

Therapies that Ag-specifically target pathologic T lymphocytes responsible for multiple sclerosis (MS) and other autoimmune diseases would be expected to have improved therapeutic indices compared with Ag-nonspecific therapies. We have developed a cellular immunotherapy that uses chimeric receptors to selectively redirect therapeutic T cells against myelin basic protein (MBP)-specific T lymphocytes implicated in MS. We generated two heterodimeric receptors that genetically link the human MBP84-102 epitope to HLA-DR2 and either incorporate or lack a TCRzeta signaling domain. The Ag-MHC domain serves as a bait, binding the TCR of MBP-specific target cells. The zeta signaling region stimulates the therapeutic cell after cognate T cell engagement. Both receptors were well expressed on primary T cells or T hybridomas using a tricistronic (alpha, beta, green fluorescent protein) retroviral expression system. MBP-DR2-zeta-, but not MBP-DR2, modified CTL were specifically stimulated by cognate MBP-specific T cells, proliferating, producing cytokine, and killing the MBP-specific target cells. The receptor-modified therapeutic cells were active in vivo as well, eliminating Ag-specific T cells in a humanized mouse model system. Finally, the chimeric receptor-modified CTL ameliorated or blocked experimental allergic encephalomyelitis (EAE) disease mediated by MBP84-102/DR2-specific T lymphocytes. These results provide support for the further development of redirected therapeutic T cells able to counteract pathologic, self-specific T lymphocytes, and specifically validate humanized MBP-DR2-zeta chimeric receptors as a potential therapeutic in MS.     

Down-regulation of T cell receptors on self-reactive T cells as a novel mechanism for extrathymic tolerance induction.

By generating two types of transgenic mice we have investigated how extrathymic events can contribute to self tolerance. The major histocompatibility complex class I gene Kb was expressed under the control of the glial fibrillary acidic protein promoter in cells of neuroectodermal origin outside the thymus. These mice were tolerant to Kb. When crossed to transgenic mice expressing a Kb-specific T cell receptor (TCR), clonotype+, CD8+CD4- mature T cells could be detected in normal numbers in the thymus of the double-transgenic mice but were strongly reduced in spleen and lymph nodes in comparison with TCR single-transgenic mice. After isolation of clonotype negative splenic T cells and activation in vitro, reappearance of the clonotype+, CD8+CD4- cells was observed. These results indicate that down-regulation of TCR and CD8 molecules on the antigen-specific T cells is a novel mechanism, by which peripheral tolerance to this antigen can occur.