Preclinical Development of Bivalent Chimeric Antigen Receptors Targeting Both CD19 and CD22

Abstract

Despite high remission rates following CAR-T cell therapy in B-ALL, relapse due to loss of the targeted antigen is increasingly recognized as a mechanism of immune escape. We hypothesized that simultaneous targeting of CD19 and CD22 may reduce the likelihood of antigen loss, thus improving sustained remission rates. A systematic approach to the generation of CAR constructs incorporating two target-binding domains led to several novel CD19/CD22 bivalent CAR constructs. Importantly, we demonstrate the challenges associated with the construction of a bivalent CAR format that preserves bifunctionality against both CD19 and CD22. Using the most active bivalent CAR constructs, we found similar transduction efficiency compared to that of either CD19 or CD22 single CARs alone. When expressed on human T cells, the optimized CD19/CD22 CAR construct induced comparable interferon γ and interleukin-2 in vitro compared to single CARs against dual-antigen-expressing as well as single-antigen-expressing cell lines. Finally, the T cells expressing CD19/CD22 CAR eradicated ALL cell line xenografts and patient-derived xenografts (PDX), including a PDX generated from a patient with CD19^- relapse following CD19-directed CAR therapy. The CD19/CD22 bivalent CAR provides an opportunity to test whether simultaneous targeting may reduce risk of antigen loss.

Keywords: adoptive cell therapy; chimeric antigen receptor; leukemia.

Figure 1

Heterogeneous Expression of CD19 and CD22 on B-ALL (A) CD19 and CD22 expression on primary patient samples was measured using a flow-cytometric quantitative assay prior to CAR T cell therapy. ****p < 0.0001. (B) CD19 and Cd22 expression on parental Nalm6 and following CRISPR-Cas9-mediated deletion of CD19 and CD22.

Figure 2

Simultaneous Targeting on Both CD19 and CD22 Surface Antigens with Co-infusion of Two Mono-targeting CARs or Co-transduced T Cell Products Result in Slowing Down of Tumor Progression in the Relapse Leukemia Model (A) NSG mice were challenged with a mixture of 2.5−E5 each of NALM6, NALM6-CD19neg, and NALM6-CD22neg leukemia lines on day 0. Mice in the sequential treatment group received 3E−6 CAR⁺ T cells on day 3 and 3E−6 CAR⁺ T cells on day 9. Mice in the co-injection group received a total of 6E−6 CAR⁺ T cells with 3E−6 CD19 CAR⁺ and 3E−6 CD22 CAR⁺ T cells on day 3. Mice in the co-infusion group received 3E−6 CD19 CAR⁺ and 3E−6 CD22 CAR⁺ T cells. Mice in the co-transduction group received 6E−6 CAR-expressing T cells (see Figure S1 for distribution of CAR-expressing cells). Mice in the CD19 or CD22 group received 3E−6 CD19 CAR⁺ or CD22 CAR⁺ T cells, respectively. Quantification of luminescence is shown on the right. (B). Representative flow-cytometric plots demonstrating the CD19 and CD22 surface expression of the leukemia before injection or at takedown at the times indicated post-CAR treatment. (C) Graphic plot presentation of the leukemia phenotype post-CAR treatment. ****p < 0.0001. ADT, adoptive transfer day (day at which CAR T cells were given). See also Figure S1.

Figure 3

Development of the Bivalent Tandem CARs (A) Schematic of TanCAR structures. (B) Flow-cytometric plot demonstrating the surface binding of CD22Fc and CD19 idiotypes. (C) Cytokine production by CD19-CAR-, CD22-CAR-, TanCAR1-, and TanCAR4-expressing T cells co-incubated with K562, K562-CD19, K562-CD22, and K562-CD19CD22 cell lines. (D) Comparison of in vivo efficacy of TanCAR1 and TanCAR4 CAR T cells. NSG mice were challenged with 1E−6 luciferase-expressing NALM6 leukemia cells on day 0. On day 3, mice were i.v. injected with 3E−6 tandem-CAR-expressing T cells. Quantification of luminescence is shown on the right. ****p < 0.0001. ns, not significant.

Figure 4

Development of the Bivalent Loop CARs (A) Schematic of Loop CAR structures. (B) Flow-cytometric plot demonstrating the surface binding of CD22Fc and CD19 idiotype. (C) Cytokine production of CD19 CAR, CD22 CAR, and LoopCAR1-5 with K562, K562-CD19, K562-CD22, and K562-CD19CD22 target cell lines. (D) Cytokine production of CD19 CAR, CD22 CAR, LoopCAR4, and LoopCAR6 with K562, K562-CD19, K562-CD22, and K562-CD19CD22 target cell lines. ****p < 0.0001. (E and F) Killing of a 10:1 ratio of NALM6:NALM6-CD19neg (E) and NALM6:NALM6-CD22neg (F) cells by CD19-CAR-, CD22-CAR-, and LoopCAR6-expressing T cells. GFU, green fluorescent units.

Figure 5

LoopCAR6 Demonstrates Comparable Efficacy with CD19 CAR but Diminished Potency compared to CD22 CAR against CD19⁻ NALM6 In Vivo (A) NSG mice were challenged with 1E−6 luciferase-expressing NALM6 leukemia cells on day 0. On day 3, mice were i.v. injected with 3E−6 CAR-expressing T cells. (B) NSG mice were challenged with 1E−6 luciferase-expressing NALM6 leukemia cells on day 0. On day 3, mice were i.v. injected with titrated doses of 9E−6, 3E−6, and 1E−6 LoopCAR6-expressing T cells. (C) NSG mice were challenged with a mixture of 5E−5 NALM6-CD19neg and 5E−5 NALM6-CD22neg luciferase-expressing leukemia cells on day 0. On day 3, mice were treated with 3E−6 CAR-expressing T cells. (D) NSG mice were challenged with 1E−6 luciferase-expressing leukemia cells, as indicated in the table on day 0. On day 3, mice were treated with 6E−6 CAR-expressing T cells. Quantification of luminescence is shown below each image. ****p < 0.0001.

Figure 6

LoopCAR6 Eradicates CD19⁺CD22⁺ and CD19⁻ Patient-Derived Xenografts (A) Flow-cytometric plot showing the surface expression of CD19 and CD22 on HMB15 ALL PDXs. (B) NSG mice were challenged with 1E−6 of HMB15 cells on day 0. Mice received treatment with 8E−6 of CD19, CD22, or LoopCAR6 CAR⁺ T cells on day 7. (C) Flow-cytometric plot showing the surface expression of CD19 and CD22 on HMB28 ALL PDXs. (D) NSG mice were challenged with 1E−6 of HMB28 cells on day 0. Mice received treatment with 8E−6 CD19, CD22, or LoopCAR6 CAR⁺ T cells on day 7. Quantification of luminescence is shown on the right. ***p < 0.001; ****p < 0.0001.