Nanobody Based Dual Specific CARs

Abstract

Recent clinical trials have shown that adoptive chimeric antigen receptor (CAR) T cell therapy is a very potent and possibly curative option in the treatment of B cell leukemias and lymphomas. However, targeting a single antigen may not be sufficient, and relapse due to the emergence of antigen negative leukemic cells may occur. A potential strategy to counter the outgrowth of antigen escape variants is to broaden the specificity of the CAR by incorporation of multiple antigen recognition domains in tandem. As a proof of concept, we here describe a bispecific CAR in which the single chain variable fragment (scFv) is replaced by a tandem of two single-antibody domains or nanobodies (nanoCAR). High membrane nanoCAR expression levels are observed in retrovirally transduced T cells. NanoCARs specific for CD20 and HER2 induce T cell activation, cytokine production and tumor lysis upon incubation with transgenic Jurkat cells expressing either antigen or both antigens simultaneously. The use of nanobody technology allows for the production of compact CARs with dual specificity and predefined affinity.

Keywords: CAR T cell; antigen escape; nanobody.

Figure 1

Nano chimeric antigen receptorS (CARs) are stably expressed on T cells at levels comparable to an scFv based CAR. (A) Scheme of the second generation nanoCAR constructs used to retrovirally transduce T cells. The antigen-binding domain is linked to the constant regions of the Fc tail of the human IgG1 antibody heavy chain, which is linked to the transmembrane and intracellular CD28 and intracellular CD3ζ chain. (B) Human peripheral blood T cells were activated and retrovirally transduced to express a nanoCAR. CAR expression was measured by flowcytometry on eGFP⁺ sorted cells after staining with a phycoerythrin (PE)-conjugated human anti-IgG antibody, which binds the spacer domain in the CAR. Non-transduced and scFv-CAR transduced T cells were used as controls. The figure is representative of five different healthy donors.

Figure 2

Characterization of the transgenic Jurkat lines expressing CD20, HER2, or both, that are used as target cells in the experiments shown in Figure 3. Jurkat cells were retrovirally transduced with the sequences coding for CD20 of for HER2 truncated at position 695 (HER2Δ695). The transgenic Jurkat cells were stained for CD20 and HER2 expression and analyzed by flowcytometry. As antigen negative control, non-transduced Jurkat cells (NTD) were used, MFIs are shown for each Jurkat clone.

Figure 3

Validation of the monospecific and tandem nanoCARs. (A) Cell lysis of Jurkat target cells expressing CD20, HER2, or both, after 4 h co-incubation with T cells expressing the monospecific or tandem nanoCAR in different effector-target ratios. Reported values are means of duplicate determinations with error bars indicating the standard deviation. Results are representative of two independent experiments, performed with three different donors. (B) Cytokine production of nanoCAR transduced T cells was analysed by intracellular staining after co-incubation with Jurkat cells. Mean percentages of interferon-γ (IFN-γ), interleukin-2 (IL-2) and tumor necrosis factor-α (TNF-α) positive cells are shown. Error bars represent standard deviations. The data is representative of two independent experiments, performed with three different donors.

Figure 4

It is not the affinity of the different CAR structures that affects their function, but rather, the CAR structure. (A) Affinity determination of the monospecific of bispecific nanobody constructs. Affinities are shown in molar concentration. (B) Lysis by mono-specific or tandem nanoCAR T cells against Jurkat target cells expressing one antigen. Data show the mean values of duplicates and are representative of three independent experiments on three different donors.