Pre-clinical Evaluation of Chimeric Antigen Receptors Targeting CD70-Expressing Cancers

Mice, tumor lines and antibodies

C57BL/6J and NSG mice (Jackson Laboratory) were maintained per protocols in the NIH animal facility. Murine tumor lines B16 and B16/mCD70 (retrovirally transduced with murine CD70) were maintained in RPMI 1640 (Life Technologies) with 10% fetal bovine serum (FBS; Life Technologies). All mouse studies were approved by the National Cancer Institute Animal Care and Use Committee.

Tumor lines from RCC patients were established and maintained in DMEM (Life Technologies) including 10% FBS, 10% tryptose phosphate (Sigma), 1x insulin-transferrin-selenium (Life Technologies) and 1 × serum pyruvate (Life Technologies). Melanoma tumor lines were maintained in RPMI 1640 (Life Technologies) with 10% FBS. All cell lines included in the study were generated at Surgery Branch, NCI, and tested and identities confirmed by HLA genotyping. The cell lines were maintained in the cell culture only when they were needed in the experiments and usually kept in culture for approximately a month, and mycoplasma testing were done routinely using mycoplasma detection kit (Lonza). The cell lines were reassessed for HLA and antigen expression by flow cytometry and co-culture assays when they were thawed for each experiment.

Monoclonal antibodies (MoAbs) including FITC-labeled anti-mouse CD3, anti-mouse CD45.1 and anti-human CD8 Abs, PE-labeled anti-mouse and anti-human CD70 Abs, PE-cy7-labeled anti-human CD3 Ab, APC-labeled anti-mouse CD27 Ab, APC-cy7-labeled anti-mouse CD8, and purified anti-mouse CD3 and anti-mouse CD28 Abs were purchased from BD Pharmingen. APC-labeled anti-human CD27 Ab was purchased from eBiosciences.

Construction of human anti-CD70 CARs, retroviral production, retroviral transduction of human PBL, and in vitro reactivity of transduced cells

Genes encoding seven human anti-CD70 CARs, including cDNAs for full-length CD27 (flCD27) or truncated CD27 (trCD27; including extracellular and transmembrane portions of CD27, aa1-211) fused with CD28 and/or 41BB signaling domains and CD3-zeta were constructed and cloned into the pMSGV1 plasmid (Fig. 1A). Retroviral production and transduction were the same as described previously (19). Briefly, 293gp cells were transfected with 9 μg of anti-CD70 CARs and 4.5μg of plasmid RD114 using Lipofectamine 2000 (Life Technologies; 60μl). Two days later, the supernatants were harvested and used to transduce anti-CD3 stimulated PBL. PBL from allogeneic donors were stimulated with soluble OKT-3 (50ng/ml) and rhIL-2 (300 IU/ml) for 2 days before transduction was performed. The stimulated cells were added to 24-well plates initially coated with RetroNectin (Takara) and subsequently pre-coated with retrovirus by spinoculation (2000×g, 32°C, 2hrs) at 5 × 10⁵/ml. The plates were then centrifuged at 1000 × g for 10 min, and incubated overnight at 37°C in a 5% CO₂ incubator. This procedure was repeated the next day and cells were split as necessary to maintain cell density between 0.5 and 1 × 10⁶ cells/ml. Transduction efficiency was estimated by analyzing human CD27 expression on retrovirally-transduced cells and comparing this to mock transduced T-cells. To test their reactivity, retrovirally-transduced cells (1 × 10⁵) were co-cultured with 5 × 10⁴ human tumor lines with or without CD70 expression at 37°C, 5% CO₂ overnight. The supernatants were harvested and tested for IFN-γ secretion by ELISA (Thermo Fisher Scientific).

Mouse xenograft studies

NSG mice were injected subcutaneously with 1.5×10⁵ 2654R human renal cancer cells. Eighteen days after inoculation, when tumors were approximately 5 mm in diameter, mice received 6 × 10⁶ intravenous human T cells retrovirally transduced with anti-CD70 CARs or control T-cells, followed by intraperitoneal administration of 200,000 IU of rhIL-2 per day for 3 days. Each group included 5 randomly assigned tumor bearing mice, and all tumor measurements were performed by a blinded impartial observer.

Construction of murine anti-CD70 CAR, retroviral production, transduction of murine CD3 T cells, and in vitro functional analysis of transduced cells

cDNA encoding murine CD27 fused with murine CD3 zeta signaling domain (mCD27-zeta) was constructed in the pMSGV1 plasmid. To produce retrovirus, 293gp cells were transfected with 9 μg of pMSGV1-mCD27-zeta and 4.5μg of plasmid pCL-Eco (Addgene) using Lipofectamine 2000 (Life Technologies; 60μl). Two days later, the supernatants were harvested and used to transduce activated mouse T cells. Splenocytes from C57BL/6J mice were harvested and CD3⁺ T cells were isolated by negative selection using a mouse pan T cell isolation kit II (Miltenyi Biotec). Murine CD3 T cells were then stimulated with plate-bound anti-mouse CD3 (1μg/ml), and soluble anti-mouse CD28 (1μg/ml) and rhIL-2 (30 IU/ml) for 2 days before transduction was performed. Transduction of simulated cells was performed similarly as described above by spinoculation. Transduction efficiency was determining by analyzing mouse CD27 expression on retrovirally-transduced T cells, compared to cells just stimulated with anti-CD3, anti-CD28 and rhIL-2. To assess the reactivity of mCD27-zeta, retrovirally-transduced murine T cells (1×10⁵) were co-cultured with B16/mCD70 or B16 (5×10⁴) at 37°C, 5% CO₂ overnight and supernatant was harvested and tested for mouse IFN-γ secretion by ELISA (R&D systems).

Treatment efficacy of murine T cells retrovirally-transduced with murine anti-CD70 CAR in vivo

C57BL/6J mice were injected subcutaneously (s.c) with 0.5×10⁶ B16 or B16/mCD70 tumors. Ten days after inoculation, when tumors were approximately 5 mm in diameter, mice received up to 10⁷ intravenous (i.v) murine T cells retrovirally transduced with mCD27-zeta or stimulated, untransduced T-cells as a control, followed by intraperitoneal (i.p) administration of 200,000 IU of rhIL-2 per day for 3 days. Splenocytes from pmel-1 TCR transgenic mice, that are reactive to gp100 in both B16 and B16/mCD70 tumors, were activated in vitro in the presence of 1μM hgp100_25-33 peptide and 30IU/ml rhIL-2 for 7 days. As a positive adoptive cell therapy control, a total of 10⁶ activated pmel T cells were given to mice i.v. along with recombinant vaccinia virus encoding hgp100 (2× 10⁷ pfu) and the regimen of i.p. rhIL-2 as above. Where specified, sub-lethal total body irradiation (TBI; 500 cGy) was given to mice immediately prior to cell transfer. Mice were given from 10⁴ to 10⁷ retrovirally-transduced murine T cells when assessing minimal effective treatment dosage. All treatment groups were randomly assigned and all tumor measurements were performed by a blinded impartial observer.

In vivo persistence of transferred T cells transduced with murine anti-CD70 CAR and assessment of treatment-related toxicity

T cells from Ly5.1 congenic mice were stimulated with plate-bound anti-murine CD3 and anti-mouse CD28 Abs for 2 days, and retrovirally-transduced with mCD27-zeta. 4 days after transduction, T cells (10⁷) were injected i.v. to C57BL/6J mice, followed by i.p. administration of 200,000 IU of rhIL-2 per day for 3 days. Splenocytes were harvested from day 3 to day 12, and the presence of transferred cells was determined by expression of CD45.1-positive cells. Measurements of weight, absolute blood cell counts, blood chemistry, the number of splenocytes, serum cytokines were determined in both tumor-bearing and non-bearing mice.

Assessment of host immune responses after adoptive cell transfer

C57BL/6J mice were injected i.v. with murine T cells retrovirally-transduced with mCD27-zeta or untransduced control T-cells, followed by i.p. administration of 200,000 IU of rhIL-2 per day for 3 days. 32 days after transfer, mice were immunized with vaccinia virus encoding either OVA or gp100. Splenocytes from treated and control animals were harvested 7 days after immunization, and cultured in vitro in the presence of 1γM OVA_257-264 or gp100_25-33 peptides for 7 days, and then tested for IFN-γ production of by co-culturing T cells with LPS-stimulated lymphoblasts pulsed with either peptide.

Statistical analysis

Wilcoxon rank-sum test was used to compare tumor slopes between each treatment groups, and log-rank test was used to analyze survival.

The effectiveness of anti-human CD70 CAR in vitro and in vivo

Seven anti-human CD70 CAR retroviral vectors were constructed and transduced into anti-CD3 stimulated normal donor peripheral blood lymphocytes (PBL) to evaluate their expression and anti-tumor activities. As shown in Fig. 1A, full-length CD27 (flCD27) fused with CD3 zeta signaling domain (CD3-zeta) with or without CD28 and/or 41BB signaling domains were constructed, designated as flCD27-zeta, flCD27-CD28-zeta, flCD27-41BB-zeta and flCD27-CD28-41BB-zeta, as several studies demonstrated CD28 and 41BB signaling domains could augment anti-tumor reactivities and in vivo persistence (20-24). Similarly, three anti-human CD70 CARs using truncated CD27 (trCD27, aa 1-211; i.e. CD27 without its intracellular domain) were also constructed, shown as trCD27-CD28-zeta, trCD27-41BB-zeta, and trCD27-CD28-41BB-zeta. PBL retrovirally-transduced with each of the seven vectors showed various degrees of CD27 expression (Fig 1B). As might be expected, PBL successfully transduced with these CAR were devoid of CD3⁺CD70⁺ cells. flCD27-zeta appeared to be the best with ~92% of cells expressing CD27 in comparison with ~70% of CD27 expression on T cells transduced with trCD27-41BB-zeta, flCD27-41BB-zeta or flCD27-28-41BB-zeta (and 0.26% of stimulated but untransduced PBL). Since mock-transduced or untransduced T cells still expressed certain level of CD27, positivity of CD27 expression in these anti-CD70 CAR-expressed T cells was only counted when expression level was above the level of mock-transduced or untransduced T cells. This may lead to underestimate transduction efficiency of anti-CD70 CARs. Down-regulation of CD3 on these transduced T cells was also observed, and consistently seen with high transduction efficiency, suggesting that CD3 internalization may occur with introduced CD3 zeta chain signaling domain. Surprisingly, only 45%, 16% and 3% were CD27 positive when T cells were transduced with trCD27-28-zeta, trCD27-28-41BB-zeta and flCD27-28-zeta, respectively. To test antigen-dependent tumor recognition by these anti-human CD70 CARs, a panel of CD70-negative tumor lines (624mel and 938mel), and their stably transduced CD70 expressing counterparts (624/CD70 and 938/CD70), and RCC tumor lines naturally expressing high to low levels of CD70 (RCC HC, RCC RO, RCC DS, and RCC MW, respectively, Supplemental Fig. 1, and (25)) were included. All of the CARs, except for flCD27-28-zeta, demonstrated specific anti-CD70 reactivity, as they only recognized CD70-positive tumor lines, but not CD70-negative tumor lines (Fig.1C). The control vector did not show any reactivity against these tumors. Among the candidate CARs, trCD27-41BB-zeta appeared to possess the highest anti-tumor reactivity by IFN-γ production. In addition to flCD27-28-zeta, lower anti-CD70 reactivity was also observed in T cells transduced with trCD27-28-zeta, suggesting that signaling through CD28 may have adverse effects on these constructs. Based on transduction efficiency and anti-tumor reactivity of these CARs, we decided to compare the efficacy of flCD27-zeta and trCD27-41BB-zeta in vivo. The CD70 positive human renal tumor line, 2654R, was injected subcutaneously into NSG mice and once palpable, treated with human T cells transduced with either flCD27-zeta or trCD27-41BB-zeta. Curative effect was observed in both groups as shown in Fig. 2A and B. None of the control groups showed any delayed tumor growth. Our results suggest that either CAR could be effective in treating CD70-positive tumors in vivo. However, since normal tissues of NSG mice do not express human CD70, we could not assess the treatment-related toxicities using this model. Therefore, a mouse tumor model with tumors expressing murine CD70 was generated to address these issues.

Treatment Efficacy of murine anti-CD70 CAR

To generate a mouse model, an anti-murine CD70 CAR was constructed by fusing full-length murine CD27 with murine CD3-signaling domain (mCD27-zeta). Murine T cells expressed high levels of CD27 after retroviral transduction with the anti-murine CD70 CAR compared to mock-transduced or untransduced T-cells (Fig. 3A). Meanwhile, a stably transfected cell line, B16/mCD70, was generated by transducing B16 melanoma (a murine CD70 negative tumor line recognized by pmel T cells) (Fig. 3B) (26), with murine CD70. As shown in Fig. 3C, pmel T cells produced mouse IFN-γ when co-cultured with either parental B16 tumor, or B16/mCD70. However, mCD27-zeta could confer specific, anti-CD70 reactivity only against B16/mCD70. To test anti-tumor efficacy of mCD27-zeta in vivo, C57BL/6J mice were implanted with either B16 or B16/mCD70 subcutaneously, and then treated with mouse T cells retrovirally transduced with mCD27-zeta or other controls when tumors became palpable. While pmel T cells (given with concomitant vaccination and IL-2) could reduce tumor burdens in irradiated mice carrying either B16 or B16/mCD70, mCD27-zeta-transduced T cells (given only with IL-2) delayed tumor growth only in irradiated mice carrying B16/mCD70, in an antigen specific manner (Fig. 3D). Mice that were treated with untransduced T cells or left untreated did not show any delay of tumor growth. In addition, treatment efficacy was dose-dependent, with 1 ×10⁵ the lowest effective T cell number in irradiated mice (Fig. 3E). In non-irradiated mice, however, treatment effects could only be observed when 1 × 10⁷ cells were transferred. This may be attributable to superior persistence of transferred cells seen in irradiated mice compared to non-irradiated mice (Supplementary Fig. 2A). Furthermore, irradiated B16/mCD70-bearing mice treated with 1 ×10⁵ to 1 ×10⁷ CAR T-cells had significantly better survival than mice treated with no cells or 1 ×10⁷ mock transduced T-cells (Fig. 3F). Although curative effects could be achieved in mice that were treated with high cell doses (1 of 5 given either 1 ×10⁶ or 1× 10⁷ cells), the majority of mice showed tumor growth inhibition rather than durable regressions. To delineate possible mechanisms of immune escape, we analyzed mCD70 expression on tumors progressing in treated mice (Fig. 3G). While tumors from control animals remained mCD70 positive, tumors from mCD27-zeta treated mice completely lacked mCD70 expression, implicating antigen loss as a cause of immune escape. While efficacy data were interesting, this mouse model was primarily developed to analyze the in vivo toxicity of using a CD27-zeta CAR to target CD70.

Short-term treatment-related toxicities of anti-murine CD70 CAR

To assess treatment-related toxicities, C57BL/6J mice were implanted with either B16 or B16/mCD70, treated with mCD27-zeta transduced or control T cells, and body weight evaluated as an indicator of systemic cytokine toxicity. Irradiated B16/mCD70 mice receiving mCD27-zeta-transduced T cells showed significant lower body weight than those receiving untransduced T cells, pmel T cells or left untreated from day 6 to day 10, but recovered 2 weeks after the cell transfer (Fig. 4A). On the other hand, no significant differences were observed in non-irradiated recipients of T-cell transfers. Interestingly, lower body weight was also observed in irradiated, mCD27-zeta treated mice that were implanted with antigen-negative B16 tumors, which suggested that this transient toxicity results primarily from mCD27-zeta-transduced cells interacting with normal endogenous host cells.

To further define the toxicity of mCD27-zeta CAR cells independent of the effects of tumor growth, we conducted experiments in non-tumor bearing C57BL/6J mice. Body weight, peripheral white blood cell (WBC) and lymphocyte counts, splenocyte counts, blood chemistries, and serum cytokines were evaluated for two weeks after treatment. Similar to tumor bearing mice, irradiated mice that were treated with mCD27-zeta transduced T cells showed significant lower body weight at day 6 and 8 than those that were treated with mock-transduced or untransduced T cells, but recovered approximately 2 weeks after cell transfer (Supplementary Fig. 2B). This effect was not observed in non-irradiated mice. As would be predicted, whole body irradiation, with or without CAR T-cell transfer dramatically decreased absolute WBC and lymphocyte counts as well as splenocyte counts compared to all non-irradiated groups (Fig. 4B and Supplementary Fig. 2C). Although blood chemistry did not show any differences among groups, we could detect IFN-γ in mouse serum from day 3 to day 5 only in irradiated mice that were treated with mCD27-zeta CAR T-cells (Fig. 4C). However, no differences could be detected in the very low levels of CD70 expression on splenocytes among these groups (Fig. 4D). Taken together, our data suggest that transient cytokine toxicity, as indicated by weight loss and elevated serum IFN-γ levels, could be detected in mice undergoing whole body irradiation followed by mCD27-zeta CAR T-cell transfer, and this resulted primarily from reactivity against endogenous host cells.

Immune competence of mice treated with anti-murine CD70 CAR

Since successfully targeting CD70 could impact on some T-cells and APCs, we tested immune responses in treated mice. One month after cell transfer, treated mice were immunized with vaccinia-OVA or vaccinia-gp100. Splenocytes from treated mice were stimulated and tested for reactivity against OVA or gp100 peptides. As shown in Fig. 5A, 7 days after in vitro stimulation with OT-I_257-264, splenocytes from mice treated with mCD27-zeta or mock-transduced cells (with or without recipient pre-irradiation) were reactive to OT-I_257-264. Similar results were observed with mice immunized with vaccinia-gp100 (Fig. 5B), as T cells from mice immunized with vaccinia-gp100 were reactive to gp100_25-33, regardless which treatment the mice had received. Interestingly, T cells from mice given mCD27-zeta CAR T-cells with irradiation appeared to be more reactive than other groups. Nonetheless, our data show that mice treated with anti-murine CD70 CAR T-cells could still mount immune responses against exogenous antigens.