Ideally, efficient tumour-specific effector and memory T cells can be induced by therapeutic vaccination. Nevertheless, in certain cases, active immunization is difficult due to the lack of an effective endogenous T-cell repertoire against the tumour antigens targeted.
T cells can recognize peptides derived from all parts of the cell, including nuclear proteins, which greatly expands the number of potential targets in the tumour cells and offers a large number of new therapeutic opportunities.
Adoptive cell therapy (ACT) involves the administration of large number of highly selected cells with high avidity for tumour antigens.
T cells can be programmed and activated ex vivo to exhibit anti-tumour functions. These cells occur naturally in cancer patients, but are inhibited by numerous immunosuppressive mechanisms in vivo. Adoptive transfer of tumour-specific T cells from tumour infiltrating lymphocytes (TIL) expanded in vitro has already been shown to induce objective cancer regression in patients with metastatic melanoma. A limitation of this approach is the requirement for pre-existing tumour-reactive cells that can be expanded ex vivo and TILs can only be reliably grown from patients with a very limited number of cancer types and mainly melanoma.
In the absence of an adequate level of endogenous tumour-reactive T-cell response, recent clinical studies have shown that it is feasible to compensate for this by engineering a tumour-specific T-cell repertoire by the transfer of genes encoding TCRs or chimeric antigen receptors (CARs). Both of these methods are developed in-house (see below).
Antibodies are produced by B cells and released into blood and body fluids to block pathogens. They are highly specific and show a high affinity to their antigen (e.g. a part of a protein). By genetic modification, the antigen recognition part of the antibody, the variable part (Fv), can be minimized to a small unit (single chain Fv, scFv) that conserves the specificity and the affinity of the original antibody. Twenty five years ago, it was reported that the fusion of a scFv to the signaling domain of the TcR (CD3ζ) could stimulate the expressing T cell upon antigen binding (Eshar et al, 1993). This type of constructs was further named CAR, for chimeric antigen receptor. Since then, the hunt for extracellular cancer markers has exploded and several targets were defined, mainly for B-cell malignancies. CD19, CD20, and CD22 CARs are nowadays in development or already in advanced clinical trials with promising outcomes.
The Radium Hospital possesses a large collection of hybridomas, each producing monoclonal antibodies. Some of these antibodies have already been used in clinical trials, but never tested as CARs. We therefore aim at designing CARs targeted against unexploited antigens.
We design novel CARs that target B-cell malignancies. CD37 is a tetraspanin that is widely expressed on the surface of mature B cells. Due to its high level expression across all subtypes of B-cell non-Hodgkin lymphoma (B-cell NHL), CD37 is one of many potential antibody targets for B-cell malignancies. Since adoptive immunotherapy using CAR gene-modified T-cells has generated impressive clinical responses in B-cell malignancies, we designed a novel second-generation CAR that redirects T-cell specificity towards CD37. Initial testing showed that CD37-directed human peripheral blood CAR T cells potently killed CD37+ B-cell NHL cell lines such as SU-DHL-4, BL-41, Mino and U-2932. We compared the anti-lymphoma activity of CD37-specific CAR T cells with that of CD19-specific CAR T cells and found no difference except for U-2932 cells which were more susceptible to CD37-specific CAR T cells, in concordance with high CD37 expression and low CD19 expression in these cells. The cell line U-2932 was originally derived from a patient with ABC-type of diffuse large B-cell lymphoma (DLBCL), who suffered from many relapses after multiple chemo- and radiotherapy regimens. Assessment of expression of CD19 and CD37 as well as other B-cell antigens such as CD20, CD22 and CD23 demonstrated a dramatic variation in their expression across the B-cell NHL cell lines. Genetic heterogeneity is common in B-cell NHLs and loss of or reduced expression of CD19 has already been reported in some B-cell NHLs. Moreover, extensive chemo- and radiotherapy regimens could potentially contribute to the loss of or reduced CD19 expression. Therefore, we are now in the process of screening B-cell NHL patient tumors for loss of or reduced expression of particular B-cell antigens, including CD19, CD20 and CD37. This type of screening can help us to identify the patient groups that are likely to benefit from CD19-, CD20- or CD37-targeted therapies. In summary, our findings suggest that CD37-directed CAR T cells can be used as an alternative to CD19-targeted CAR T cells, especially when CD19 expression is lost or reduced in patients’ tumor cells.
- TCR CAR (H. Köksal):
Effector T cells equipped with engineered antigen receptors specific for cancer targets have proven to be very efficient. Two methods have emerged: the Chimeric Antigen Receptors (CARs) and T-cell Receptor (TCR) redirection. Although very potent, CAR recognition is limited to membrane antigens which represent around 1% of the total proteins expressed, whereas TCRs have the advantage of targeting any peptide resulting from cellular protein degradation. However, TCRs depend on heavy signalling machinery only present in T cells which restricts the type of eligible therapeutic cells. Hence, an introduced therapeutic TCR will compete with the endogenous TCR for the signalling proteins and carries the potential risk of mixed dimer formation giving rise to a new TCR with unpredictable specificity. We have fused a soluble TCR construct to a CAR-signalling tail and named the final product TCR-CAR. In this project we prove that, if expressed, the TCR-CAR conserved the specificity and the functionality of the original TCR. In addition, we demonstrate that TCR-CAR redirection was not restricted to T cells. Indeed, after transduction, the NK cell line NK-92 became TCR positive and reacted against pMHC target. This opens therapeutic avenues combing the killing efficiency of NK cells with the diversified target recognition of TCRs.
- IGK CAR (H. Köksal):
Chimeric antigen receptor (CAR) based immunotherapy is coming under the spotlight in the cancer treatment. This is mainly due to the success of CAR T cells targeting B-lymphocyte antigen CD19, which has led to astonishing results in clinical trials. Considering that all B cells express CD19 antigen, CAR-T cells eliminate all B cells, including non-malignant B cells. Therefore, the patients suffer from impaired humoral immune response, specifically B-cell aplasia and hypogammaglobulinemia, which might increase susceptibility to severe infections. Another problem is related to the target itself. Accumulation of data demonstrates the possibility of immune escape by down regulation of CD19 or alternative splicing variant which becomes resistant to standard CD19 CAR. There is therefore a need for alternative targets. Taking into account that most B-cell lymphomas and chronic lymphocytic leukemia cells have a clonally restricted expression of Immunoglobulin (Ig) light chains, either Ig-kappa or Ig-lambda, Ig-kappa+ tumor cells can be targeted while sparing normal Ig-lambda+ B-cells. Hence, Ig-kappa CAR T cells could provide lower on-target toxicity than CD19 CAR T cells and would be expected to improve the life quality of the patients.
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