Tri-specific duoCAR-T cells can effectively treat antigen-heterogeneous cancers

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Tri-specific duoCAR-T cells can effectively treat antigen-heterogeneous cancers

Tri-specific duoCAR-T cells can effectively treat antigen-heterogeneous cancers. Science Sub-Journal: Tumor has nowhere to escape! Preclinical studies have shown that tri-specific duoCAR-T cells can effectively treat antigen-heterogeneous cancers.

With CD19-targeting chimeric antigen receptor (CAR) T cell (CAR-T) therapy (CAR19 T cell) and CD22-targeting CAR-T The emergence of cell therapy (CAR22 T cells) has significantly improved the treatment of B-cell leukemia and lymphoma.

However, recurrence after CAR-T cell therapy is still an obstacle, and as many as 50% of patients receiving CAR19 T cell therapy relapse within the first year after treatment, and a considerable number of these relapsed patients show loss of CD19 antigen.

Similarly, the recurrence of CAR22 T cells after the treatment of acute lymphoblastic leukemia (ALL) is related to the down-regulation of the CD22 protein of malignant tumor cells, so that CAR22 T cells can no longer recognize and kill them.

In the process of using CAR19 T cell therapy and then using CAR22 T cell therapy, the sequential loss of CD19 and CD22 tumor surface antigens has also been reported in diffuse large B-cell lymphoma.

Similarly, during treatment with the CD20 targeting antibody rituximab (Rituximab), loss of CD20 antigen has been reported in non-Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL) and follicular lymphoma.

In order to solve the problem of tumor antigen escape, researchers from Lentigen in the United States have previously developed a tandem bispecific CAR20-19 construct targeting CD19 and CD20 antigens, and it has shown promise in phase 1 clinical trials. Efficacy and tolerability.

Using multispecific CAR-T cells to target tumor cells is speculated to reduce the escape of tumor antigens and improve the therapeutic effect.

Previously, Lentigen researchers have confirmed in a humanized mouse xenograft model that duoCAR-T cells (dual CAR-T cells, that is, T cells expressing two CARs connected in series) are better than monoCAR in controlling HIV infection. -T cells (single CAR-T cells, that is, T cells expressing a single CAR) are more effective.

Now, in a new study, Lentigen researchers let lentiviral vectors expressing tandem CARs targeting CD19 and CD20 to transduce primary CD4+ T cells and CD8+ T cells. The expressed tandem CARs are combined with P2A self-cleaving peptides.

The monoCARs targeting CD22 antigen are operatively linked together, and tri-specific duoCAR-T cells were developed based on this, and the ability of this tri-specific duoCAR-T cells to solve the problem of antigen escape was evaluated.

Related research results were recently published in the journal Science Translational Medicine, with the title of the paper “Trispecific CD19-CD20-CD22–targeting duoCAR-T cells eliminate antigen-heterogeneous B cell tumors in preclinical models”.

These authors constructed 4 duoCAR constructs (called D1, D2, D3, and D4). The two open reading frames of each construct were separated by a P2A peptide. Their expression profiles on the surface of T cells are shown in the figure. 1 shown.

DuoCAR D1 consists of tandem single-chain variable region fragment (scFv) binding domains targeting B cell antigens CD19 and CD20, hinge and transmembrane domains derived from CD8, ICOS costimulatory domain, CD3 activation domain, and CD3 After the activation domain, the first generation of CAR gene targeting CD22, the hinge and transmembrane domain derived from CD8, and the CD3 activation domain are composed.

D2 is the same as D1, except that the OX40 domain is used instead of the ICOS costimulatory domain. D3 contains the same tandem CAR gene targeting CD19 and CD20 as the construct D2.

The tandem CAR gene is followed by a CD22 targeting CAR gene, the second-generation ICOS costimulatory domain and the CD3 activation domain.

D4 contains a tandem CAR gene targeting CD20 and CD19 and a CD27 costimulatory domain. It also contains a CAR base targeting CD22 and an ICOS costimulatory domain. In addition, each costimulatory domain is followed by a CD3 activation domain.

Tri-specific duoCAR-T cells can effectively treat antigen-heterogeneous cancers

Figure 1. Design, expression and characterization of the dicistronic DuoCAR D1, D2, D3 and D4. Picture from Science Translational Medicine, 2021, doi:10.1126/scitranslmed.abc6401.

Each duoCAR construct was transduced into primary human T cells, thereby obtaining duoCAR D1 T cells, duoCAR D2 T cells, duoCAR D3 T cells, and duoCAR D4 T cells, and proliferating in vitro for 8-10 days.

Based on four independent transduction experiments, the average expression of duoCAR D1 was 64%, D2 was 56%, D3 was 60%, and D4 was 45%. These authors can continue to produce duoCAR-T cells, and these duoCAR-T cells do not have any undesirable evidence of spontaneous activation, failure, or terminal differentiation.

Tri-specific duoCAR-T cells can effectively treat antigen-heterogeneous cancers

Figure 2. DuoCAR D1, D2, D3 and D4 have strong specific toxicity to tumor cells in vitro. Picture from Science Translational Medicine, 2021, doi:10.1126/scitranslmed.abc6401.

They used the tumor cell lines Raji (CD19+CD20+CD22+) and REH (CD19+CD20-CD22+) for in vitro tests and found that compared to monoCAR-T cells, duoCAR T cells lysed Raji cells and REH cells more effectively, but There is no duoCAR T cell lysis target antigen-negative cell line 293T (CD19-CD20-CD22-), which indicates that this tumor-killing effect is not antigen-independent (Figure 2).

In addition, they also found that these four duoCAR cells can specifically express any combination of the three surface antigens (CD19, CD20, and CD22) (such as expressing only one of them, only two of them, and expressing These three surface antigens) of tumor cells (Figure 5).

Tri-specific duoCAR-T cells can effectively treat antigen-heterogeneous cancers
Figure 5. In vitro cytotoxicity study of CAR-T cells to antigen engineering target cells. Picture from Science Translational Medicine, 2021, doi:10.1126/scitranslmed.abc6401.

They also collected the supernatant of duoCAR T cells when they were cultured together with the Raji cell line in vitro, and measured the soluble cytokines secreted by duoCAR T cells in the supernatant.

They found that duoCAR T cells produce cytokines IL-2, IFN-γ, TNF-α, GM-CSF and IL-9, in addition to IL-4 to a certain extent. As expected, T cells that were not transduced with CAR-carrying lentiviral vectors (hereinafter referred to as UTD T cells) did not produce cytokines, thus confirming that the production of such cytokines is dependent on CAR (Figure 3).

Tri-specific duoCAR-T cells can effectively treat antigen-heterogeneous cancers
Figure 3. Cytokines released by CD22+CAR-T when co-cultured with the CD19+CD20+ cell line Raji. Picture from Science Translational Medicine, 2021, doi:10.1126/scitranslmed.abc6401.

Then, they tested the efficacy of duoCAR-T cells in vivo. To this end, they tested duoCAR-T cells in the Raji NHL tumor xenograft mouse model and NALM-6 ALL mouse model. In these two mouse models, each model showed triple positive CD19+CD20+CD22+ Antigen expression phenotype.

In the Raji NHL tumor xenograft mouse model, the dose of duoCAR-T cells injected into each mouse was divided into high dose (5×106) and low dose (2×106). It was found that in the high-dose group, this The four duoCAR-T cells started to significantly and strongly inhibit Raji tumor growth on the 14th day, and in the low-dose group, these four duoCAR-T cells could all control Raji tumor burden.

In the NALM-6 ALL mouse model, these four duoCAR-T cells mediated significant tumor rejection (Figure 4).

Figure 4. DuoCAR-T cells eradicate B cell tumors in lymphoma and leukemia xenograft models. Picture from Science Translational Medicine, 2021, doi:10.1126/scitranslmed.abc6401.

In addition, in order to test the anti-tumor function of duoCAR-T cells against tumor cell mutants with missing antigens, NSG mice were implanted with 5×106 Raji cells (composed of Raji19KO cells, Raji20KO cells, Raji22KO cells and parental Raji cells, each Seed cells accounted for 25%).

This mixture of Raji cells represents an antigenic heterogeneous tumor. In this tumor, some tumor cells lose the expression of the target antigen to a certain extent.

For example, Raji19KO cells do not express CD19, Raji20KO cells do not express CD20, Raji22KO cells CD22 is not expressed, and the loss of this target antigen expression will prevent the corresponding CAR-T cell activation and anti-tumor immunity.

Seven days after injection of this Raji cell mixture, these mice received 5 x 106 duoCAR D1/D2/D3/D4 T cells and monoCAR-T cells as a control. On the 14th day, they found that these four duoCAR T cells strongly rejected Raji tumors with antigenic heterogeneity, and at the end of the study, these mice were still in remission. In contrast, monoCAR-T cells could not control the tumor burden in mice in the control group (Figure 6).

Figure 6. Anti-tumor activity and durability of duoCAR in vivo in an antigen heterogeneous Raji tumor model. Picture from Science Translational Medicine, 2021, doi:10.1126/scitranslmed.abc6401.

In addition, they also found that when combined with surface antigens on target cells, compared with the monoCAR design, duoCAR constructs can phosphorylate CD3 robustly and activate downstream signaling proteins Akt and MAPK p38, as well as the remote signaling mediator STAT5.

In addition, this duoCAR design retains the key activation characteristics of the homologous monoCAR, and the activation amplitude is minimal or not decreased.

Conclusion:

This study confirmed the high functionality of duoCAR-T cells in vitro and in vivo. This approach broadly addresses the pressing problem of antigen heterogeneity. In addition, these authors discovered an unexpected combination of the best costimulatory domains that did not include CD28 or 4-1BB domains, indicating that scientists have a strong interest in T cell signal transduction associated with high activity and long-term persistence.

Understanding continues to improve. Before the CAR-T method is considered to be truly optimized, activation (as indicated by CD3 phosphorylation), long-term persistence (as indicated by Akt activation), and response to physiological signals usually mediated by cytokines (such as STAT5 phosphorylation).

In addition, from the phosphorylation of p38 and Erk1/2, the reduction of some activation signals in duoCAR indicates that the maximum initial activation may not be related to disease control.

The duoCAR-T cell method developed in this research is improved on the basis of current CAR-T cell therapy, overcoming the clinical challenges of tumor heterogeneity and tumor antigen escape, while maintaining high anti-tumor efficacy and durability.

Tri-specific duoCAR-T cells can effectively treat antigen-heterogeneous cancers

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