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Antibody Therapy Section

Mitchell Ho, Ph.D.

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Gastroenterology cover: GPC3-targeted CAR T cells for liver cancer therapy

GPC3-targeted CAR T cells for liver cancer therapy

Published Date

Background and Aims:  Glypican 3 (GPC3) is an oncofetal antigen involved in Wnt-dependent cell proliferation that is highly expressed in hepatocellular carcinoma (HCC). We investigated whether the functions of chimeric antigen receptors (CARs) that target GPC3 are affected by their antibody- binding properties. 

Methods:  We collected peripheral blood mononuclear cells from healthy donors and patients with HCC and used them to create CAR T cells, based on the humanized YP7 (hYP7) and HN3 antibodies, which have high affinities for the C-lobe and N-lobe of GPC3, respectively. NOD/SCID/IL- 2Rgcnull (NSG) mice were given intraperitoneal injections of luciferase-expressing (Luc) Hep3B or HepG2 cells and after xenograft tumors formed, mice were given injections of saline or untransduced T cells (mock control), or CAR (HN3) T cells or CAR (hYP7) T cells. In other NOD/SCID/IL-2Rgcnull (NSG) mice, HepG2-Luc or Hep3B-Luc cells were injected into liver, and after orthotopic tumors formed, mice were given 1 injection of CAR (hYP7) T cells or CD19 CAR T cells (control). We developed droplet digital polymerase chain reaction and genome sequencing methods to analyze persistent CAR T cells in mice. 

Results:  Injections of CAR (hYP7) T cells eliminated tumors in 66% of mice by week 3, whereas CAR (HN3) T cells did not reduce tumor burden. Mice given CAR (hYP7) T cells remained tumor free after re-challenge with additional Hep3B cells. The CAR T cells induced perforin- and granzyme-mediated apoptosis and reduced levels of active b-catenin in HCC cells. Mice injected with CAR (hYP7) T cells had persistent expansion of T cells and subsets of polyfunctional CAR T cells via antigen- induced selection. These T cells were observed in the tumor microenvironment and spleen for up to 7 weeks after CAR T- cell administration. Integration sites in pre-infusion CAR (HN3) and CAR (hYP7) T cells were randomly distributed, whereas integration into NUPL1 was detected in 3.9% of CAR (hYP7) T cells 5 weeks after injection into tumor-bearing mice and 18.1% of CAR (hYP7) T cells at week 7. There was no common site of integration in CAR (HN3) or CD19 CAR T cells from tumor-bearing mice. 

Conclusions:  In mice with xenograft or orthoptic liver tumors, CAR (hYP7) T cells eliminate GPC3- positive HCC cells, possibly by inducing perforin- and granzyme-mediated apoptosis or reducing Wnt signaling in tumor cells. GPC3-targeted CAR T cells might be developed for treatment of patients with HCC.

Citation

Persistent Polyfunctional Chimeric Antigen Receptor T Cells That Target Glypican 3 Eliminate Orthotopic Hepatocellular Carcinomas in Mice. Li D, Li N, Zhang YF, Fu H, Feng M, Schneider D, Su L, Wu X, Zhou J, Mackay S, Kramer J, Duan Z, Yang H, Kolluri A, Hummer AM, Torres MB, Zhu H, Hall MD, Luo X, Chen J, Wang Q, Abate-Daga D, Dropulic B, Hewitt SM, Orentas RJ, Greten TF, Ho M. Gastroenterology. 2020 Jun;158(8):2250-2265.e20.

Hepatology cover: GPC3-targeted CAR T cells for liver cancer therapy

Engineered immunotoxin targeting GPC3 promotes liver cancer regression in mice

Published Date

Background and Aims:  Treatment of hepatocellular carcinomas using our glypican‐3 (GPC3)‐targeting human nanobody (HN3) immunotoxins causes potent tumor regression by blocking protein synthesis and down‐regulating the Wnt signaling pathway. However, immunogenicity and a short serum half‐life may limit the ability of immunotoxins to transition to the clinic.

Approach and Results:  To address these concerns, we engineered HN3‐based immunotoxins to contain various deimmunized Pseudomonas exotoxin (PE) domains. This included HN3‐T20, which was modified to remove T‐cell epitopes and contains a PE domain II truncation. We compared them to our previously reported B‐cell deimmunized immunotoxin (HN3‐mPE24) and our original HN3‐immunotoxin with a wild‐type PE domain (HN3‐PE38). All of our immunotoxins displayed high affinity to human GPC3, with HN3‐T20 having a KD value of 7.4 nM. HN3‐T20 retained 73% enzymatic activity when compared with the wild‐type immunotoxin in an adenosine diphosphate–ribosylation assay. Interestingly, a real‐time cell growth inhibition assay demonstrated that a single dose of HN3‐T20 at 62.5 ng/mL (1.6 nM) was capable of inhibiting nearly all cell proliferation during the 10‐day experiment. To enhance HN3‐T20’s serum retention, we tested the effect of adding a streptococcal albumin‐binding domain (ABD) and a llama single‐domain antibody fragment specific for mouse and human serum albumin. For the detection of immunotoxin in mouse serum, we developed a highly sensitive enzyme‐linked immunosorbent assay and found that HN3‐ABD‐T20 had a 45‐fold higher serum half‐life than HN3‐T20 (326 minutes vs. 7.3 minutes); consequently, addition of an ABD resulted in HN3‐ABD‐T20–mediated tumor regression at 1 mg/kg.

Conclusion:  These data indicate that ABD‐containing deimmunized HN3‐T20 immunotoxins are high‐potency therapeutics ready to be evaluated in clinical trials for the treatment of liver cancer.

Citation

Engineered Anti-GPC3 Immunotoxin, HN3-ABD-T20, Produces Regression in Mouse Liver Cancer Xenografts Through Prolonged Serum Retention. Fleming BD, Urban DJ, Hall MD, Longerich T, Greten TF, Pastan I, Ho M. Hepatology. 2020 May;71(5):1696-1711.

Antibody Therapeutics cover: A Shark Single Domain Antibody Library

A Shark Single Domain Antibody Library

Published Date

The laboratory of Mitchell Ho at the National Cancer Institute has established a large phage-displayed VNAR single domain antibody library from nurse sharks. The shark single domain library provides a new platform for selecting therapeutic antibodies. For additional information, see Antibody Therapeutics 2019 Jan;2(1):1-11.

Citation

 Construction and next-generation sequencing analysis of a large phage-displayed VNAR single-domain antibody library from six naïve nurse sharks. Feng M, Bian H, Wu X, Fu T, Fu Y, Hong J, Fleming BD, Flajnik MF, Ho M. Antibody Therapeutics 2019 Jan;2(1):1-11.

Antibody Therapeutics cover: single domain antibody (shark VNAR) versus conventional antibody (Fv)

Searching for Magic Bullets

Published Date

Single domain antibodies have emerged as a new class of therapeutic molecules in antibody engineering. A single domain antibody (shark VNAR), in contrast to a conventional antibody (Fv), is capable of binding a buried functional site consisting of the enzyme’s substrate binding pocket.  For additional information, see Antibody Therapeutics 2018 Jun;1(1):1-5.

Citation

Inaugural Editorial: Searching for Magic Bullets. Ho M.  Antibody Therapeutics 2018 Jun;1(1):1-5.

mAbs cover: A novel anti-GPC3 monoclonal antibody (YP7)

A novel anti-GPC3 monoclonal antibody (YP7)

Published Date

Glypican-3 (GPC3) is an emerging therapeutic target in hepatoma. A novel anti-GPC3 monoclonal antibody (YP7) has been generated through a combination of peptide immunization and high-throughput flow cytometry screening. YP7 binds cell-surface-associated GPC3 with high affinity and exhibits significant hepatoma xenograft growth inhibition in nude mice. The new antibody may have potential for hepatoma therapy. For additional information, see MAbs. 2012 Sep-Oct;4(5):592-9.

Citation

High-affinity monoclonal antibodies to cell surface tumor antigen glypican-3 generated through a combination of peptide immunization and flow cytometry screening. MAbs. 2012 Sep-Oct;4(5):592-9.

Alumni

Heungnam Kim, Ph.D.
2008-2011
Postdoctoral Fellow
Liping Yu
2009
Graduate Student