CAR T cells targeting α_vβ₃ integrin are effective against advanced cancer in preclinical models

1 INTRODUCTION

Integrins are heterodimeric cell surface receptors expressed by all nucleated cells and involved in adhesion and signaling processes between cells and their microenvironment. They are formed by noncovalent association of one α- and one β-subunit.1 Upon binding of their respective ligands, integrins recruit large signaling complexes to their cytoplasmic domain and transmit information about the cell's functional state to the microenvironment by modulating the binding affinity to their ligands through conformational changes.2 Because integrins influence tumor cell migration, invasion, proliferation, and survival, they are being pursued as molecular and immunologic targets in cancer therapy.3 Integrin α_vβ₃, also known as vitronectin receptor, is one of the best-studied integrins in cancer research. It primarily interacts with ligand proteins containing an RGD-motif and can increase tumor cell survival.4, 5 Several mechanisms have been identified that link expression of integrin α_vβ₃ on tumor cells to increased metastatic spread.6, 7 First, the activated form of integrin α_vβ₃ enables binding to platelets, which protects tumor cells from shear stress and enhances their adhesion to blood vessels.8, 9 Second, recruitment and activation of the tyrosine kinase c-Src by the cytoplasmic tail of the β₃ subunit of integrin α_vβ₃ promotes anchorage-independent cell survival.10 Furthermore, integrin α_vβ₃ promotes the epithelial-mesenchymal transition of tumor cells by direct interaction with the TGF-β receptor II and binding of TGF-β. Exposure to TGF-β also leads to increased integrin α_vβ₃ expression.11, 12 Expression of α_vβ₃ integrin has been demonstrated in several tumor entities such as melanoma and glioblastoma, as well as breast, pancreatic, and prostate cancer.7, 13-16 In melanoma, transition from benign radial growth to malignant vertical growth correlates with de novo expression of α_vβ₃ integrin.17 In addition to tumor cells, integrin α_vβ₃ is also expressed on cells that are essential components of the tumor environment including cancer-associated fibroblasts (CAFs), tumor-associated macrophages, and angiogenic endothelial cells.18-20

There have been prior attempts of exploiting α_vβ₃ integrin as a therapeutic target. This includes immunotherapy with monoclonal antibodies (mABs) that inhibit ligand binding to α_vβ₃ integrin_, which has been reported to be safe but only of limited efficacy.21 Here, we report on the development of α_vβ₃-specific chimeric antigen receptor (CAR) T cells and their antitumor function in preclinical models. CARs are synthetic receptors that most commonly employ the variable heavy (VH) and variable light (VL) chains of a mAB for antigen targeting. To construct α_vβ₃ integrin-specific CARs, we utilized the VH and VL chains of a super-humanized mAB LM609 (hLM609), which we developed in previous work.22 We have recently demonstrated that binding domain affinity and extracellular spacer domain design affect tumor cell recognition and CAR T-cell function,23, 24 and are modulating both variables to derive an α_vβ₃-CAR with optimal antitumor reactivity. Through these iterations, we have obtained an α_vβ₃-CAR that confers potent reactivity against α_vβ₃-expressing hematologic and nonhematologic tumor cells in vitro and eliminates metastatic melanoma in a murine xenograft model in vivo.

2 MATERIALS AND METHODS

3 RESULTS

3.1 α_vβ₃ integrin is expressed on hematologic and nonhematologic tumor cell lines

We analyzed expression of α_vβ₃ integrin by flow cytometry on a panel of hematologic and nonhematologic tumor cell lines using a mAb directed against the α_vβ₃ integrin complex. We detected high-level α_vβ₃ integrin surface expression on the melanoma cell lines A-375, M14, Malme-3M, UACC-62 and UACC-257, and the triple-negative breast cancer cell line MDA-MB-231 (xMFI = MFI mAb/MFI isotype: 7.3-19.5); and intermediate-level expression on the T-cell acute lymphoblastic leukemia cell line Jurkat (xMFI: 3.6) (Figure 1A). The chronic myelogenous leukemia cell line K562 and the mantle cell lymphoma cell line JeKo-1, as well as the lung carcinoma cell line A-549 and the melanoma cell line LOX-IMVI showed only a minor increase in MFI compared to isotype control (xMFI: <2). To determine if this was due to low-level expression of α_vβ₃ integrin complex or rather unspecific binding, we analyzed expression of the α_v and β₃ subunits on these cell lines individually (Figure 1B, S1). We detected high-level expression of α_v on LOX-IMVI, A-549, and K562, but not on the surface of JeKo-1 cells. The β₃ subunit was not expressed on LOX-IMVI and A-549 cells and only detectable at extremely low levels on K562 and JeKo-1 cells. For comparison, on K562 cells that we transduced with a lentiviral vector encoding the β₃ integrin subunit (K562_β₃), we detected uniform, high-level expression of α_vβ₃ (xMFI: 13.3) (Figure 1A).

We also analyzed normal T cells, B cells, and monocytes from peripheral blood, and mobilized hematopoietic stem cells (HSCs) and did not detect relevant expression of α_vβ₃ integrin complex (xMFI: <2) (Figure S2, S3A). Taken together, these data affirm the prior notion, that integrin α_vβ₃ is expressed on malignant cells in hematologic and nonhematologic cancers, including prevalent high-level expression in melanoma cell lines. We selected the K562 and A-549 (negative); A-375, UACC-62, UACC-257 and MDA-MB-231 (positive), and K562_β₃ cell lines (positive control) as target cells to analyze the specificity and antitumor potency of T cells that we gene-engineered to express an α_vβ₃-specific CAR.

3.2 Expression of α_vβ₃-CARs in human T cells

We designed a set of four α_vβ₃-CAR constructs comprising: i. a scFv targeting domain derived from a super-humanized variant of anti-α_vβ₃ integrin-specific mAb LM609 with either high (hLM609v7; K_d = 3 nmol/L) or low (hLM609v11; K_d = 160 nmol/L) monovalent affinity22; ii. an IgG4-Fc derived spacer of either Hinge only (short spacer, 12 amino acids) or of Hinge-CH2-CH3 (long spacer, 229 amino acids).23 Each of the constructs contained a CD28_CD3zζ signaling domain and was coexpressed with a truncated epidermal growth factor receptor (EGFRt) as transduction marker (Figure 2A).25 We transduced each of the constructs into CD8⁺ and CD4⁺ T cells and enriched CAR⁺ T cells using the EGFRt marker (Figure 2B). At the end of the manufacturing process and prior to functional testing, each of the CD8⁺ and CD4⁺ α_vβ₃-CAR T-cell lines was negative for α_vβ₃ integrin–expression by flow cytometry (Figure 2C).

3.3 α_vβ₃-CAR T cells elicit potent antitumor functions in vitro

We sought to determine the antitumor function of T cells expressing our α_vβ₃-CAR constructs. First, we analyzed the cytolytic activity of CD8⁺ α_vβ₃-CAR T cells and found that each of the four CAR constructs conferred specific recognition and elimination of A-375, UACC-62, UACC-257, MDA-MB-231, and K562_β₃ target cells (Figure 3A). Overall, the four α_vβ₃-CAR constructs with high versus low affinity and short versus long spacer were similarly potent and not significantly different in their ability to induce cytolysis of target cells. K562_β₃ cells were more susceptible to cytolysis compared to the melanoma cell lines and MDA-MB-231, especially at low effector-to-target cell ratios. We detected low-level recognition of native K562 cells, potentially due to very low-level expression of α_vβ₃ integrin complex, as suggested in our expression analysis. Accordingly, we did not detect cytolysis of A-549 cells (Figure 3A). We also evaluated recognition of normal HSCs, and consistent with their α_vβ₃-negative phenotype, viability was not affected by coculture with α_vβ₃-CAR T cells (Figure S3B).

We confirmed that CD8⁺ and CD4⁺ α_vβ₃-CAR T cells produced IFN-ɣ and IL-2 after stimulation with α_vβ₃-positive tumor cells (Figure 3B,C). The α_vβ₃-CAR with high affinity and short spacer domain (hLM609v7/short) induced the strongest IFN-ɣ production, particularly in CD4⁺ T cells (Figure 3C). The α_vβ₃-CARs with short spacer domain induced more IL-2 production compared to the constructs with long spacer. In CD4⁺ T cells, the hLM609v7/short α_vβ₃-CAR induced the strongest production of IL-2 (Figure 3B,C). Overall, we observed stronger cytokine production by CD4⁺ compared to CD8⁺ CAR T cells. We also evaluated the proliferation of α_vβ₃-CAR T cells after stimulation with α_vβ₃-positive tumor cells (Figure 3D,E). Again, there was stronger proliferation of CD4⁺ compared to CD8⁺ CAR T cells, and the hLM609v7/short α_vβ₃-CAR was superior in inducing proliferation compared to the other receptor designs. Notably, there was some background proliferation of T cells equipped with the hLM609v7/long α_vβ₃-CAR, potentially due to tonic signaling.

In summary, the data show that α_vβ₃-CAR T cells confer specific and potent antitumor functions against α_vβ₃-expressing target cells in vitro. The data also show that the α_vβ₃-CAR construct with higher affinity and short spacer domain conferred the strongest effector functions, particularly cytokine secretion and proliferation, in line with our prior observation that these parameters affects the antitumor function of CAR T cells.23 On the basis of our analysis, we selected the α_vβ₃-CAR constructs with short spacer domain for evaluation in an in vivo model.

3.4 α_vβ₃-CAR T cells are effective against metastatic melanoma in vivo

We established xenografts of metastatic melanoma in immunodeficient NSG mice using the aggressive A-375 cell line. We injected NSG mice with 1 × 10⁶ firefly luciferase-labeled A-375 cells i.v. on day 0 and treated them with a single dose of 5 × 10⁶ T cells i.v. after 7 days (CD8:CD4 ratio = 1:1). Mice were either treated with α_vβ₃-CAR T cells (hLM609v7/short) or untransduced control T cells derived from the same donor. We performed serial bioluminescence imaging and observed rapid tumor regression in all mice treated with α_vβ₃-CAR T cells (n = 3) and rapid tumor progression in all control mice (n = 3) (Figure 4A,B). All CAR T-cell-treated mice were alive until the end of the observation period on day 65 (Figure 4C); only one mouse had a slowly progressing, singular tumor lesion in the lower back (Figure 4A,B).

In the next set of experiments, we included a group of NSG/A-375 mice that received T cells expressing the hLM609v11/short α_vβ₃-CAR to compare the higher and lower affinity α_vβ₃-CAR constructs. On day 3 and day 7 after adoptive T-cell transfer, we analyzed peripheral blood samples and confirmed T-cell engraftment in all treatment groups (Figure 5A). Between day 3 and day 7, the percentage of α_vβ₃-CAR T cells increased, in particular in the group of mice that had received the lower affinity hLM609v11/short α_vβ₃-CAR. In serum samples, we measured elevated concentrations of IFN-ɣ and GM-CSF in mice that received α_vβ₃-CAR T cells, but not in mice that had received control T cells (Figure 5B). Interestingly, also the cytokine concentrations were higher in mice that had been treated with the lower affinity hLM609v11/short compared to the higher affinity hLM609v7/short α_vβ₃-CAR. Treatment with α_vβ₃-CAR T cells conferred a potent antitumor effect. On day 3 after T-cell transfer (ie, day 10 after tumor inoculation), we already detected lower levels of bioluminescence signal in all of the mice that had received CAR T cells, whereas there was increasing bioluminescence signal in all mice in the control groups that had either received untransduced T cells or no treatment (Figure 5C). By day 28, the bioluminescence signal had reached background levels in 4 of 6 mice in the hLM609v7/short, and 5 of 6 mice in the hLM609v11/short α_vβ₃-CAR T-cell groups, respectively, indicating that all tumor lesions had been eradicated (Figure 5C,D). Out of the 12 mice treated with α_vβ₃-CAR T cells, 3 did not reach the background bioluminescence signal and had a recurrent tumor at the same site as one of the first lesions. Three of the six mice in the hLM609v7/short treatment group were lost to follow-up for reasons unrelated to tumor progression. Kaplan-Meier analysis showed that by day 44, all of the mice in the control groups had to be sacrificed due to tumor progression (Figure 5E). In contrast, all of the mice that completed follow-up were alive in the hLM609v7 α_vβ₃-CAR (3 out of 3), and all of the mice were alive in the hLM609v11 α_vβ₃-CAR cohort (6 out of 6). Bone marrow analysis at the end of the experiment confirmed that CAR T cells and untransduced T cells had persisted for the entire duration of the experiment (Figure 5F).

Taken together, these data show that α_vβ₃-CAR T cells confer potent antitumor efficacy in vivo. Several lines of evidence, including stronger CAR T-cell expansion and cytokine secretion, and a higher rate of complete responses indicate that α_vβ₃-CAR T cells with the lower affinity binding domain hLM609v11 conferred stronger antitumor reactivity than α_vβ₃-CAR T cells with the higher affinity hLM609v7 binding domain in vivo, which reverses the hierarchy established in our in vitro analysis.

4 CONCLUSIONS

Adoptive immunotherapy with gene-engineered CAR T cells has curative potential against advanced hematologic malignancies.27 At present, significant efforts are being invested to extend the clinical success that has been obtained with CD19-specific CAR T cells in B-cell leukemia and lymphoma, to prevalent nonhematologic tumors, which requires the identification and validation of novel target antigens. Here, we demonstrate that CAR T cells specific for α_vβ₃ integrin exhibit potent antitumor reactivity, including effective tumor cell lysis, as well as cytokine production and proliferation after stimulation with α_vβ₃-expressing cancer cells in vitro. Furthermore, we demonstrate that α_vβ₃-CAR T cells are capable of completely eradicating established A-375 melanoma in a xenograft model in mice.

Our data show that α_vβ₃-CAR constructs with hLM609-derived binding domains confer superior antitumor function when equipped with a short IgG-Fc Hinge rather than a long IgG-Fc Hinge-CH2-CH3 spacer domain. This is consistent with our previous observation with CD19- and ROR1-specific CARs, showing that the length of the extracellular spacer domain affects tumor cell recognition and CAR T-cell function, likely because of the spatial interaction of CAR and tumor antigen, and the localization of the targeted epitope.23, 24 The paradigm that emerged from our previous studies is that the “long” spacer domain is optimal for targeting epitopes that are proximal to the tumor cell membrane, whereas a “short” spacer domain is optimal for targeting membrane-distal epitopes. The mAB LM609 recognizes a membrane-distal epitope of integrin α_vβ₃28 and accordingly, our data that α_vβ₃-CAR constructs with “short” spacer domain confer the strongest antitumor functions, are in line with this paradigm.

We show that α_vβ₃-CAR T cells are able to eradicate metastatic A-375 melanoma in an NSG mouse xenograft model. The A-375 melanoma model is commonly used to evaluate novel anticancer agents and notably, the efficacy that we observed with α_vβ₃-CAR T cells in our study is superior to the efficacy that has recently been reported for several alternative investigational treatments, including a combination treatment of oncolytic adenovirus and dacarbazine chemotherapy, and itraconazole, that both achieved tumor reduction but not tumor clearance like in our study.29, 30

Interestingly, we observed stronger CAR T-cell expansion and cytokine secretion in mice that had been treated with the lower affinity hLM609v11 α_vβ₃-CAR, indicating the lower affinity receptor had induced stronger T-cell activation in vivo. In contrast, in our in vitro analysis, the higher affinity hLM609v7 α_vβ₃-CAR had induced stronger cytokine secretion and proliferation. Other investigators have reported similar findings with CAR T cells targeting ErbB2.31 A potential explanation is that the lower affinity hLM609v11 binding domain has a three-times faster off-rate than the higher affinity variant hLM609v7 (16 × 10⁻⁴ s⁻¹ vs 5.4 × 10⁻⁴ s⁻¹ measured for the corresponding monovalent Fab) and therefore, hLM609v11 CAR T cells are more likely to sequentially interact with tumor cells and thus, may receive a higher net activation signal. Experiments with additional affinity variants of hLM609 are warranted in order to define the “sweet spot” of hLM609 affinity that permits maximum antitumor function of the α_vβ₃-CAR.

In this study, we used super-humanized LM609 VH and VL variants to derive α_vβ₃-CAR targeting domains with minimal immunogenicity. Indeed, we and others have demonstrated in previous work that immune responses directed against immunogenic epitopes located in murine VH/VL CAR targeting domain can lead to rejection of CAR T cells, which may limit or abrogate the antitumor effect.32, 33 Notably, the super-humanized hLM609v7 and hLM609v11 variants employed in our α_vβ₃-CARs retained only the third complementarity-determining region (CDR) of the parental mouse mAB LM609 VH and VL chains. All other CDRs and framework regions were replaced by human amino acid sequences.22

A previous study has reported on the construction of an α_vβ₃-CAR that instead of a mAB-derived scFv, utilized a modified echistatin peptide, a disintegrin as targeting moiety, which binds to both human and mouse α_vβ₃ integrin.34 These α_vβ₃-specific CAR T cells were tested in a syngeneic mouse model against B16 melanoma and inhibited tumor growth, but did not achieve tumor eradication. Furthermore, this study showed that echistatin-based α_vβ₃-CAR T cells damage tumor vasculature, due to α_vβ₃-expression on tumor endothelium, which was evident from hematomas in the tumor tissue. Encouragingly, the adoptive transfer of echistatin-based α_vβ₃-CAR T cells did not cause any evident toxicity to normal tissues.34 Expression of α_vβ₃ integrin has been reported on several healthy tissues including angiogenic endothelial cells, osteoclasts, vascular smooth muscle cells, platelets, macrophages, and HSCs.4, 35-37 The expression density and conformation of α_vβ₃ may be modulated, for example, under inflammatory conditions.1-3

Notably, clinical studies with the two humanized LM609 mABs vitaxin and abegrin did not report any severe adverse events and did not report toxicity to these normal tissues and cell subsets.38-41 While encouraging for the clinical translation of adoptive therapy with α_vβ₃-CAR T cells, the safety data obtained with mABs have to be considered with caution because of the substantially higher potency of CAR-T cells. In a first-in-human study with α_vβ₃-CAR T cells, the lower affinity hLM609v11 scFv may be a preferable targeting domain because of data obtained with other CAR constructs indicating that higher affinity may come at the expense of reduced selectivity, and that higher affinity scFvs are more likely to confer recognition of normal tissues that express the targeted antigen with low density.31, 42, 43 In a clinical study, it is advisable to equip CAR T cells with a depletion marker or suicide gene to provide a safety switch in case of toxicity.44 The α_vβ₃-CAR T cells presented in our study are equipped with an EGFRt marker that we have shown can be used to deplete CAR T cells by administering the anti-EGFR mAB cetuximab.25

Our data suggest that adoptive therapy with α_vβ₃-CAR T cells has the potential to confer greater therapeutic efficacy compared to immunotherapy with anti-α_vβ₃ mABs. The anti-α_vβ₃ mABs that were employed in previous clinical studies exerted their mechanism of action by sterically hindering the binding of RGD-ligands to α_vβ₃ integrin to inhibit tumor vascularization.28 We demonstrate that α_vβ₃-CAR T cells exert a distinct set of effector mechanisms including direct cytolytic activity against α_vβ₃-positive tumor cells. In addition, we anticipate that α_vβ₃-CAR T cells will also alter the tumor microenvironment and limit tumor angiogenesis by destruction of α_vβ₃-expressing cancer-associated fibroblasts and endothelial cells in tumor-associated blood vessels. Furthermore, the expression of α_vβ₃ on tumor vasculature should facilitate the migration of α_vβ₃-CAR T cells to and invasion into primary and metastatic tumor lesions. Indeed, in prior work, other investigators have equipped primary lymphocytes with a membrane-bound fusion-molecule that targets integrin α_vβ₃ through the disintegrin kistrin and observed increased lymphocyte infiltration of tumor sites.45

A recent observation is the strong resistance of α_vβ₃-expressing cancer cells to cisplatin and doxorubicin, the standard-of-care chemotherapy agents for several solid tumor malignancies.46 This observation suggests that adoptive immunotherapy with α_vβ₃-CAR T cells may be used either concomitantly or sequentially to chemotherapy in order to delete chemotherapy-resistant tumor cells and improve therapeutic outcomes.

Collectively, our data introduce α_vβ₃ integrin as a novel target for CAR T cells with potential utility in several prevalent solid tumors. Our data affirm our previous notion that binding domain affinity and spacer length can be calibrated to augment the antitumor reactivity of CARs. Because α_vβ₃ integrin is expressed on primary and metastatic tumor cells, endothelial cells of tumor vasculature and tumor stroma, α_vβ₃-CAR T cells bear significant therapeutic potential in a clinical setting.

CONFLICT OF INTEREST

M.H. is coinventor on a patent application (PCT/US2013/055862) related to CAR spacer design that has been filed by the Fred Hutchinson Cancer Research Center (Seattle, WA) and licensed by JUNO Therapeutics, Inc. C.R. is coinventor of U.S. Patent 7,087,409 that claims the humanized LM609 variants and is owned by The Scripps Research Institute (La Jolla, CA).

ETHICS STATEMENT

The authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to and the appropriate ethical review committee approval has been received.

CLINICAL IMPLICATIONS

Adoptive immunotherapy with CAR T cells has been shown to be effective in advanced hematologic malignancies. Here, we demonstrate that α_vβ₃-expressing tumors can be targeted with α_vβ₃-specific CAR-T cells. Integrin α_vβ₃ is expressed on several prevalent epithelial, mesenchymal, and neuroectodermal cancers, including melanoma, glioblastoma, breast, pancreatic, and prostate cancer. CAR T cells exert a unique mode of action that can overcome resistance to conventional chemoradiotherapy and antibody-mediated immunotherapy. Intriguingly, α_vβ₃ integrin is also present on tumor vasculature and tumor stroma cells, for example, cancer-associated fibroblasts suggesting, that α_vβ₃-CAR T cells would be able to effectively migrate to and invade a solid tumor mass. Our data encourage the initiation of clinical studies to assess the safety and efficacy of α_vβ₃-CAR T-cell therapy.