A therapeutic T cell receptor mimic antibody targets tumor-associated PRAME peptide/HLA-I antigens - PubMed (original) (raw)

. 2017 Jun 30;127(7):2705-2718.

doi: 10.1172/JCI92335. Epub 2017 Jun 19.

Tao Dao 1, Ron S Gejman 1 3, Casey A Jarvis 1, Andrew Scott 1 4, Leonid Dubrovsky 1, Melissa D Mathias 1, Tatyana Korontsvit 1, Victoriya Zakhaleva 1, Michael Curcio 1, Ronald C Hendrickson 1, Cheng Liu 5, David A Scheinberg 1 3

Affiliations

A therapeutic T cell receptor mimic antibody targets tumor-associated PRAME peptide/HLA-I antigens

Aaron Y Chang et al. J Clin Invest. 2017.

Erratum in

Abstract

Preferentially expressed antigen in melanoma (PRAME) is a cancer-testis antigen that is expressed in many cancers and leukemias. In healthy tissue, PRAME expression is limited to the testes and ovaries, making it a highly attractive cancer target. PRAME is an intracellular protein that cannot currently be drugged. After proteasomal processing, the PRAME300-309 peptide ALYVDSLFFL (ALY) is presented in the context of human leukocyte antigen HLA-A*02:01 molecules for recognition by the T cell receptor (TCR) of cytotoxic T cells. Here, we have described Pr20, a TCR mimic (TCRm) human IgG1 antibody that recognizes the cell-surface ALY peptide/HLA-A2 complex. Pr20 is an immunological tool and potential therapeutic agent. Pr20 bound to PRAME+HLA-A2+ cancers. An afucosylated Fc form (Pr20M) directed antibody-dependent cellular cytotoxicity against PRAME+HLA-A2+ leukemia cells and was therapeutically effective against mouse xenograft models of human leukemia. In some tumors, Pr20 binding markedly increased upon IFN-γ treatment, mediated by induction of the immunoproteasome catalytic subunit β5i. The immunoproteasome reduced internal destructive cleavages within the ALY epitope compared with the constitutive proteasome. The data provide rationale for developing TCRm antibodies as therapeutic agents for cancer, offer mechanistic insight on proteasomal regulation of tumor-associated peptide/HLA antigen complexes, and yield possible therapeutic solutions to target antigens with ultra-low surface presentation.

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Conflict of interest statement

Conflict of interest: D.A. Scheinberg has potential conflicts of interest, defined by the JCI by ownership in, income from, or research funds from Pfizer, Abbott, Progenics Pharmaceuticals, ContraFect Corp., Sellas Life Sciences, Sapience Therapeutics Inc., Allergan, Actinium Pharmaceuticals Inc., Eureka Therapeutics, and Intuitive Surgical Inc. D.A. Scheinberg, L.T. Dao, L. Dubrovsky, and C. Liu are inventors on intellectual property for which patents have been filed by MSKCC (WO2014143835 A1, WO2012135854 A2, and WO2016191246). C. Liu has ownership in Eureka Therapeutics.

Figures

Figure 1

Figure 1. Pr20 binds ALY/HLA-A2 complexes and PRAME+HLA-A2+ leukemia.

Pr20 was directly labeled by conjugation to the fluorophore APC. (A) TAP-deficient T2 cells were pulsed overnight with 50 μg/ml of ALY peptide or irrelevant control EW peptide or left unpulsed. Flow cytometry was used to determine P20 binding. (B) Each nonanchor residue in the ALY peptide was substituted for alanine, and peptides were pulsed onto T2. Pr20 binding was determined by flow cytometry relative to native ALY peptide–pulsed T2. Cell-surface HLA-A2 was also measured by flow cytometry to ensure altered peptides maintained the ability to bind and stabilize HLA-A2 compared with unpulsed T2. (C) PRAME mRNA expression was determined by qPCR, and samples that did not amplify after 40 cycles were considered negative. (D) The indicated cell lines were stained with Pr20 or an isotype control Ab, and binding was determined by flow cytometry. Surface HLA-A2 was also assessed compared with an isotype control. All data from A–D are representative of at least 3 experiments. (E) Whole blood populations from HLA-A2+ healthy donors were stained with Pr20 to determine possible crossreactivity. A representative gating strategy and Pr20 histogram compared with isotype control are shown, and data from all HLA-A2+ healthy donors (n = 5) are summarized. Staining was performed once independently for each healthy donor and an AML14 PRAME+HLA-A2+ leukemia–positive control was included in each assay to ensure assay reliability. SSC, side scatter; FSC, forward scatter.

Figure 2

Figure 2. Pr20M mediates Ab-dependent cellular cytotoxicity in vitro on PRAME+HLA-A2+ leukemias and lymphoma.

(A) ADCC assay was performed on hematopoietic cancers. 51Cr-labeled target leukemia or lymphoma cells were incubated with healthy donor PBMCs at an effector/target ratio of 50:1. Pr20M or an afucosylated isotype control Ab was added at the indicated concentration. Supernatant was collected after 6 hours, and scintillation counting was used to determine percentage of specific lysis. Data represent at least 3 experiments for each cell line except SKLY16 and MAC2A (done twice). (B) Healthy donor PBMCs were incubated overnight with 1 μg/ml of Pr20M or afucosylated isotype control. Flow cytometry was used to determine populations of B cells (CD19+CD3–), T cells (CD3+CD19–), monocytes (CD14+CD19–), and myeloid cells (CD33+CD19–). One representative analysis (n = 3) is shown, including a positive control to demonstrate that PBMCs in all assays were capable of depleting a PRAME+HLA-A2+ lymphoma (CD19– and transduced with GFP). Data from HLA-A2+ healthy donor PBMCs (n = 3) performed independently are summarized and plotted. Data analyzed by paired Wilcoxon signed-rank test.

Figure 3

Figure 3. Pr20M is therapeutically active against ALL and AML in vivo, and target epitope downregulation is not a mechanism of Pr20M resistance.

BV173 (ALL), SET2 (AML), and AML14 (AML) were transduced to express luciferase and GFP. NSG mice were engrafted though tail-vein injection, and on day 6 or 7, mice were randomized into groups and treated with 50 μg of Pr20M twice a week, left untreated (control for BV173 and SET2), or treated with an afucosylated isotype control Ab (AML14). Tumor burden was determined by BLI for BV173 (n = 5 mice) (A), SET2 (n = 5 mice) (B), and AML14 (n = 4 mice) (C) once a week throughout the experiment, and the BLI data are summarized below the images. The scales for days 7 and 14 for AML14 are lowered to indicate engraftment and early tumor growth. Total flux (photos/s) was normalized to each mouse’s total flux on day 6 or 7 immediately before initiation of Pr20M therapy and summarized with mean ± SEM. (D) Mice from the AML14 experiment were sacrificed on day 29, and bone marrow was harvested to determine tumor burden by flow cytometry for GFP+HLA-A2+ AML14 cells. Representative plots (n = 4 mice per group) are shown, and data are summarized. (E) MFI of AML14 for HLA-A2 and Pr20 was determined by flow cytometry. Because Pr20M-treated mice presumably had Pr20M already bound on tumor cells, staining was performed by an additional Pr20 stain on all samples followed by a secondary PE-conjugated anti-human Ab (n = 4 mice per group). Experiments were performed once per model. Differences were evaluated using the unpaired t tests on indicated times and samples. AML14 BLI data are representative of 3 similar experiments, while SET2 and BV173 BLI data are from 1 experiment. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 4

Figure 4. Melanomas and other solid tumors do not readily bind Pr20, but treatment with IFN-γ induces immunoproteasome expression and dramatically increases Pr20 binding.

(A) HLA-A2+ melanomas and a colon adenocarcinoma that expressed PRAME by qPCR (Table 1) were either left untreated (blue) or treated with 10 ng/ml of IFN-γ for 72 hours (red) and stained with Pr20 compared with untreated cells stained with an isotype control Ab (gray). HLA-A2 staining was performed in parallel. Data represent 3 independent experiments. (B) Melanomas were pretreated with 10 ng/ml IFN-γ for 72 hours or left untreated before 51Cr-ADCC assay was used to determine specific lysis by Pr20M. Samples were assayed in 3 technical replicates, and data are representative of 3 experiments per cell line. (C) PRAME expression after 72 hours of IFN-γ treatment was also measured by qPCR and Western blot analysis. qPCR data were analyzed by unpaired t test and are representative of 3 experiments with 3 technical replicates per experiment where mean ± SEM are plotted. Western blot data are representative of 3 experiments. (D) The expression of each immunoproteasome subunit was also determined after IFN-γ treatment by Western blot analysis. Blots were derived from replicate samples run on parallel gels with the GAPDH loading control shown from the β2i blot. Data are representative of 3 experiments.

Figure 5

Figure 5. Immunoproteasome catalytic subunit β5i is important for IFN-γ–mediated Pr20 binding in melanomas and other solid tumors.

β1i, β2i, and β5i were knocked out in the SK-Mel5 melanoma line using a CRISPR approach. A CRISPR construct against GFP was used as a vector control. (A, left panel) Cells were treated with 10 ng/ml IFN-γ for 72 hours, and Western blot analysis was used to demonstrate successful knockouts. Blots were derived from replicate samples run on parallel gels with the GAPDH loading control shown from the β2i blot. (B) Flow cytometry was used to determine Pr20 binding and surface HLA-A2 on the indicated knockouts (sgRNA against β1i, β2i, and β5i) untreated or treated with IFN-γ for 72 hours. (B, top panels). Data are normalized to MFI of untreated GFP sgRNA CRISPR control. (B, lower panels) β5i CRISPR knockout experiments were performed in the same manner on the UACC257 melanoma line. Successful knockout was determined by Western blot (A, right panel), and Pr20 binding and surface HLA-A2 were determined by flow cytometry (B, lower panels). (C) SK-Mel5 and UACC257 cells were left untreated or treated with 10 ng/ml IFN-γ for 72 hours in the presence or absence of 200 nM of the β5i inhibitor ONX-0914. Flow cytometry was used to determine MFI relative to untreated cells. All data are representative of 3 experiments with 3 technical replicates per experiment and mean ± SEM plotted. Statistical significance was determined by unpaired t test compared with control. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 6

Figure 6. The immunoproteasome catalyzes increased nondestructive cleavages on an ALY-precursor peptide.

A 20-mer ALY-elongated precursor peptide was incubated with purified constitutive proteasome or immunoproteasome for the indicated times. (A) All detectable fragments and their respective ion intensities were assigned to be nondestructive or destructive depending on whether the N- or C-terminal cleavages required to generate that fragment would have resulted in destruction of the ALY 10-mer. Ratios of ion intensity sums for nondestructive/destructive products are plotted. (B) Major cleavage sites along the precursor peptide after 1 hour were mapped by summing the ion intensities of each fragment resulting from a cleavage after the specific residue. Heat map with arbitrary units corresponding to ion intensities is shown, with 3 replicates illustrated in 3 bars for each proteasome preparation. Only fragments identified as at least 2 residues or more could be mapped, and thus cleavages before Q296 or after L313 were not accounted for. (C) Major differences in cleavage specificity between constitutive and immunoproteasome are schematized and mapped by red arrows. The green arrows denote the canonical proteasomal cleavage to generate the C-terminal end of the ALY 10-mer. Data are from 3 technical replicates per experimental condition with mean ± SEM plotted. Groups compared using multiple t tests. ***P < 0.001; ****P < 0.0001.

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