SynNotch CAR circuits enhance solid tumor recognition and promote persistent antitumor activity in mouse models

Abstract

The first clinically approved engineered chimeric antigen receptor (CAR) T cell therapies are remarkably effective in a subset of hematological malignancies with few therapeutic options. Although these clinical successes have been exciting, CAR T cells have hit roadblocks in solid tumors that include the lack of highly tumor-specific antigens to target, opening up the possibility of life-threatening "on-target/off-tumor" toxicities, and problems with T cell entry into solid tumor and persistent activity in suppressive tumor microenvironments. Here, we improve the specificity and persistent antitumor activity of therapeutic T cells with synthetic Notch (synNotch) CAR circuits. We identify alkaline phosphatase placental-like 2 (ALPPL2) as a tumor-specific antigen expressed in a spectrum of solid tumors, including mesothelioma and ovarian cancer. ALPPL2 can act as a sole target for CAR therapy or be combined with tumor-associated antigens such as melanoma cell adhesion molecule (MCAM), mesothelin, or human epidermal growth factor receptor 2 (HER2) in synNotch CAR combinatorial antigen circuits. SynNotch CAR T cells display superior control of tumor burden when compared to T cells constitutively expressing a CAR targeting the same antigens in mouse models of human mesothelioma and ovarian cancer. This was achieved by preventing CAR-mediated tonic signaling through synNotch-controlled expression, allowing T cells to maintain a long-lived memory and non-exhausted phenotype. Collectively, we establish ALPPL2 as a clinically viable cell therapy target for multiple solid tumors and demonstrate the multifaceted therapeutic benefits of synNotch CAR T cells.

Figure 1.. ALPPL2 is a highly specific and broadly applicable tumor antigen for combinatorial antigen recognition of solid tumors with synNotch CAR T cells.

(A) ALPPL2 expression in FFPE sections of solid tumor tissues was measured by IHC. Positive staining (brown) was observed in mesothelioma, seminoma, gastric cancer, ovarian cancer, and pancreatic cancer. Scale bar: 100μm for mesothelioma, pancreatic cancer, and gastric cancer; 200μm for ovarian cancer and seminoma. (B) Representative images of ALPPL2 and MCAM co-expression in mesothelioma tissues are shown. Scale bar: 50μm. (C) ALPPL2 is a highly tumor-specific antigen found in a range of solid tumor types that display minimal expression in normal tissues. ALPPL2 can be targeted directly using an ALPPL2 CAR but can also function as a priming switch for CARs targeting other tumor-associated antigen through synNotch CAR circuits to minimize on-target/off-tumor toxicity. We identified ALPPL2/MCAM as an antigen signature with coverage across mesothelioma subtypes. Mesothelin is mainly restricted to epithelioid mesothelioma. In addition, ALPPL2/HER2 is a potential combinatorial antigen signature for ovarian cancer.

Figure 2.. SynNotch CAR circuit T cells exhibit multi-antigen specificity and paced elimination of mesothelioma.

(A) Primary human T cells were engineered with ALPPL2 sensing synNotch with genetic circuits encoding for either an MCAM (BBζ), mesothelin (MSLN, BBζ or 28ζ), or HER2 (BBζ) CAR. Antigen specific CAR expression in CD8⁺ T cells was determined by GFP expression after 48 hours of stimulation with K562 cells expressing MCAM, mesothelin, and HER2 ± ALPPL2 (representative of at least two independent experiments). (B) Killing kinetics of epithelioid (M28) and sarcomatoid (VAMT-1) mesothelioma tumor cells by CD8⁺ T cells expressing a MCAM CAR constitutively or through an ALPPL2 synNotch circuit. ALPPL2 synNotch regulating GFP expression acted as a control circuit (representative of at least two independent experiments). (C) Hours to 50% killing of M28 and VAMT-1, as compared to untransduced CD8⁺ T cells, were recorded for constitutive or ALPPL2 synNotch regulated expression of MCAM (BBζ) or MSLN (BBζ or 28ζ) CARs. n.a.; not applicable. Data are shown as mean±SD.

Figure 3.. ALPPL2 synNotch circuits are sensitive to a range of antigen densities across multiple tumor types.

(A) K562 cells constructed with doxycycline-inducible ALPPL2 expression show dose-dependent induction of ALPPL2 after 72 hours of doxycycline (Dox) treatment. CD8⁺ T cells engineered with an ALPPL2 (BBζ) CAR or ALPPL2 synNotch MCAM (BBζ) CAR circuit were challenged with K562 cells displaying a range of ALPPL2 expression and analyzed for antigen specific CAR expression, as determined by GFP expression, and T cell activation, as determined by CD69 expression (representative of at least two independent experiments). (B) ALPPL2⁺ K562 cells were constructed to display dose-dependent expression of either MCAM or HER2 upon treatment with doxycycline. MCAM^MED represents endogenous expression and HER2^HI represents cells expressing HER2 driven by the SFFV promoter. (C,D) CD8⁺ T cells engineered with either an MCAM (BBζ) CAR (C) or HER2 (BBζ) CAR (D) either under a constitutive SFFV promoter or ALPPL2 synNotch circuit were challenged with doxycycline-treated K562 target cells and assessed for CD25 expression after 48 hours. (E) ALPPL2⁺ A549 tumor cells were engineered to express either low or high densities of MCAM or HER2. (F) Cytotoxic kinetics for CD8⁺ T cells engineered with ALPPL2 synNotch circuits driving either a MCAM (BBζ) CAR or HER2 (BBζ) CAR challenged with A549 tumor cells expressing low or high densities of MCAM or HER2. Data is shown as mean±SD.

Figure 4.. SynNotch regulation of CAR expression maintains T cell stemness prior to therapeutic administration.

(A,B) Expansion of engineered CD4⁺ (A) or CD8⁺ (B) T cells expressing a MCAM (BBζ), mesothelin (MSLN, BBζ or 28ζ), or HER2 (BBζ) CAR either constitutively or through ALPPL2 synNotch circuit in two donors after removal of CD3/CD28 Dynabead stimulation. (C) Gating strategy for identifying T cells immunophenotypically resembling T stem cell memory-like (T_SCM), T central memory (T_CM), and T effector memory (T_EM) cells. SSC-A, side scatter area. (D) Composition of T_SCM, T_CM, and T_EM in non-antigen exposed CD8⁺ T cells from four different donors engineered to express a MCAM (BBζ) CAR either constitutively or through an ALPPL2 synNotch circuit 14 days post initial CD3/CD28 Dynabead stimulation. (E) Expression pattern analysis of CD39, LAG-3, PD-1, and TIM-3 in non-antigen exposed CD8⁺ T cells from three different donors engineered to express a MCAM (BBζ) CAR either constitutively or through ALPPL2 synNotch circuit 14 days post initial CD3/CD28 Dynabead stimulation. (F) Jurkat cells carrying AP-1, NFAT, or NF-κB response elements expressing an MCAM (BBζ) CAR constitutively or through an ALPPL2 synNotch circuit. The control circuit is ALPPL2 synNotch driving GFP expression (representative of two independent experiments). Statistics were calculated using two-way ANOVA with Šidák’s post-hoc test comparing CAR and Circuit (A,B) or unpaired (C) Student’s t-test. *P ≤0.05; **P ≤0.01; ***P ≤0.001; n.s.; not significant.

Figure 5.. SynNotch CAR circuits regulate T cell differentiation pre and post antigen stimulation in vitro.

(A) Experimental timeline of longitudinal T cell memory subset analysis for CD4⁺ or CD8⁺ T cells engineered to express either an MCAM (BBζ), mesothelin (MSLN, BBζ or 28ζ), or HER2 (BBζ) CAR constitutively or through ALPPL2 synNotch circuits without or after two stimulations with ALPPL2⁺ K562 cells expressing MCAM, mesothelin, and HER2. T_N/SCM, naïve/stem cell memory-like T cells (CD45RA⁺CD62L⁺); T_CM, central memory-like T cells (CD45RA⁻CD62L⁺); T_EM, effector memory-like T cells (CD45RA⁻CD62L⁻); T_EMRA, effector memory-like T cells re-expressing CD45RA (CD45RA⁺CD62L⁻). (B,C) Baseline memory phenotype of CD4⁺ (B) and CD8⁺ (C) T cells expressing an MCAM (BBζ), mesothelin (BBζ or 28ζ), or HER2 (BBζ) CAR constitutively or through ALPPL2 synNotch circuits 14 days post initial CD3/CD28 Dynabead stimulation. (D,E) Phenotypic evolvement for engineered CD4⁺ (D) and CD8⁺ (E) T cells expressing an MCAM (BBζ), mesothelin (BBζ or 28ζ), or HER2 (BBζ) CAR constitutively or through ALPPL2 synNotch circuits upon culture without (No Stim) or after two stimulations (Stim) of ALPPL2⁺ K562 cells expressing the cognate CAR antigens. Data are presented as mean±SEM and statistics were calculated using two-way ANOVA with Šidák’s post-hoc test. *P ≤0.05; **P ≤0.01; ***P ≤0.001 comparing T cells expressing CAR or Circuit.

Figure 6.. SynNotch CAR circuit T cells exhibit superior efficacy and persistence in vivo.

(A) NSG mice bearing contralateral subcutaneous tumors with ALPPL2 wild-type (WT) M28 tumors on the left side and with ALPPL2 knock-out (KO) M28 tumors on the right side received an adoptive transfer of a mix of CD4⁺ and CD8⁺ T cells engineered with an ALPPL2 synNotch→MCAM CAR circuit (n=5) or untransduced T cells (n=5). Tumor size was monitored over 35 days (representative of two independent experiments). (B) NSG mice bearing subcutaneous M28 tumors were injected i.v. with 1:1 ratio of CD4⁺:CD8⁺ T cells engineered with an ALPPL2 CAR (n=5), MCAM CAR (n=5), ALPPL2 synNotch regulating MCAM-CAR expression, or untransduced T cells (n=5). Tumor size was monitored over 49 days. (C) NSG mice bearing subcutaneous M28 tumors received an i.v. infusion of a 1:1 ratio of CD4⁺:CD8⁺ T cells engineered with either the MCAM CAR (n=10), the ALPPL2 synNotch→MCAM-CAR circuit (n=10), or untransduced T cells 21 days after tumor inoculation (n=5). Tumors, peripheral blood, and spleens were collected for phenotypic and numeric analysis of infused T cells 12 days post T cell infusion. (D) Number of tumor-infiltrating T cells (TILs) per mg tumor was determined by expression of human CD45 (hCD45) and CD4 or CD8. (E,F) Number of T cells per μL peripheral blood (E) and mg spleen tissue (F) was determined by expression of hCD45 (representative of two independent experiments). (G) Expression of PD-1 and CD39 in CD8⁺ TILs expressing a MCAM CAR either constitutively or through an ALPPL2 synNotch (representative of two independent experiments). Data are presented as mean±SEM. Statistics were calculated using either two-way ANOVA with Šidák’s (A) or Tukey’s post-hoc test (B), or unpaired Mann-Whitney’s t-test (D-G). *P ≤0.05; **P ≤0.01; ***P ≤0.001; n.s.; not significant.

Figure 7.. ALPPL2 synNotch CAR circuit T cells effectively target mesothelin and HER2 positive tumors and exhibit persistent activity upon rechallenge.

(A) NSG bearing subcutaneous M28 tumors were injected i.v. with 1:1 ratio of CD4⁺:CD8⁺ T cells engineered with an ALPPL2 synNotch regulating mesothelin (MSLN, BBζ or 28ζ) CAR expression (n=5 and n=3, respectively), or untransduced T cells (n=5) at 7 days post tumor implantation. Tumor size was monitored over 53 days. ALPPL2 synNotch - MSLN (28ζ) CAR is offset by +20 on the y-axis. (B) NSG mice bearing subcutaneous ALPPL2⁺ SK-OV-3 tumors were injected i.v. with 1:1 ratio of CD4⁺:CD8⁺ T cells engineered with an ALPPL2 CAR (n=5), HER2 CAR (n=5), an ALPPL2 synNotch regulating HER2 CAR expression (n=5), or untransduced T cells (n=5) at 7 days post tumor implantation. Tumor size was monitored over 40 days. (C) NSG mice bearing subcutaneous ALPPL2⁺ SK-OV-3 tumors on the left flank were injected i.v. with 1:1 ratio of CD4⁺:CD8⁺ T cells engineered with a HER2 CAR (n=6) or an ALPPL2 synNotch regulating HER2 CAR expression (n=4) at 7 days post tumor implantation. Control mice were injected with PBS (n=4). After 24 days, mice were contralaterally rechallenged with a subcutaneous injection of ALPPL2⁺HER2⁺ K562 tumor cells on the right flank. Tumor sizes were monitored over 51 days. (D) At 44 days post T cell infusion, presence of persistent human CD45⁺ (hCD45⁺) engineered T cells in the peripheral blood (PB) was assessed in tumor rechallenged mice by flow cytometry. Data are presented as mean±SEM. Statistics were calculated using two-way ANOVA with Tukey’s multiple comparison (C) or unpaired Mann-Whitney’s t-test (D). *P ≤0.05; **P ≤0.01; ***P ≤0.001.