Shiran Shapira

Recent advances in personalized colorectal cancer research.

Colorectal cancer is one of the most prevalent cancers and a leading cause ofcancer-related death. It is also curable if detected early. The prognosis for metastatic colorectal cancer remains poor and resistance to chemotherapy is still a major obstacle in effective treatment. While many patients do not clinically benefit from chemotherapy, others experience adverse reactions resulting in dose modifications or treatment withdrawal, thereby reducing treatment efficacy. Researchefforts attempt to identify reliable biomarkers which will guide clinicians in decision making, while matching suitable therapeutic regimens. We here review currently known molecular biomarkers used for the personalized treatment of colorectal cancer.

An Immunoconjugate of Anti-CD24 and Pseudomonas Exotoxin Selectively Kills Human Colorectal Tumors in Mice

BACKGROUND & AIMSnEffective and selective treatment options are needed for patients with colorectal cancer (CRC). The CD24 mucin-like glycoprotein is overexpressed in CRCs; monoclonal antibodies (mAbs) against CD24 inhibit tumor cell growth in vitro and in vivo. Based on the tumor-specific expression of CD24, we investigated the potential of anti-CD24 SWA11 mAb, to deliver a cytotoxic agent into CRC cells.nnnMETHODSnWe conjugated SWA11 to a Pseudomonas exotoxin derivative (PE38) via an Fc-binding ZZ domain from Staphylococcal protein A (which binds the Fc domain of mouse IgG2a immunoglobulins) to generate the immunotoxin SWA11-ZZ-PE38; IgG-ZZ-PE38 was used as control. Human HT-29 and COLO320 (CD24-positive) and HCT116 (CD24-negative) CRC cell lines were assayed for immunotoxin binding, cytotoxicity, viability, and apoptosis. Toxicity and antitumor efficacy were tested in mice.nnnRESULTSnThe immunotoxin preserved the affinity and specificity of SWA11, bound and selectively killed CD24-expressing CRC cells via apoptosis. IC(50) values ranged from 20 to 50 ng/mL-several orders of magnitude lower than that of the mAb alone. The immunotoxins were not toxic to mice at the maximum dose of 0.75 mg/kg. Growth of HT-29 xenograft tumors was significantly reduced in mice given SWA11-ZZ-PE38 (by 78%) compared to untreated mice.nnnCONCLUSIONSnAnti-CD24 SWA11 mAb can deliver a PE exotoxin derivative to CRC cells and cause them to undergo apoptosis, without toxicity to normal tissues. This immunotoxin might be developed as a therapeutic treatment for patients with CRC.

Removal of Hepatitis C Virus-Infected Cells by a Zymogenized Bacterial Toxin

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease and has become a global health threat. No HCV vaccine is currently available and treatment with antiviral therapy is associated with adverse side effects. Moreover, there is no preventive therapy for recurrent hepatitis C post liver transplantation. The NS3 serine protease is necessary for HCV replication and represents a prime target for developing anti HCV therapies. Recently we described a therapeutic approach for eradication of HCV infected cells that is based on protein delivery of two NS3 protease-activatable recombinant toxins we named “zymoxins”. These toxins were inactivated by fusion to rationally designed inhibitory peptides via NS3-cleavable linkers. Once delivered to cells where NS3 protease is present, the inhibitory peptide is removed resulting in re-activation of cytotoxic activity. The zymoxins we described suffered from two limitations: they required high levels of protease for activation and had basal activities in the un-activated form that resulted in a narrow potential therapeutic window. Here, we present a solution that overcame the major limitations of the “first generation zymoxins” by converting MazF ribonuclease, the toxic component of the E. coli chromosomal MazEF toxin-antitoxin system, into an NS3-activated zymoxin that is introduced to cells by means of gene delivery. We constructed an expression cassette that encodes for a single polypeptide that incorporates both the toxin and a fragment of its potent natural antidote, MazE, linked via an NS3-cleavable linker. While covalently paired to its inhibitor, the ribonuclease is well tolerated when expressed in naïve, healthy cells. In contrast, activating proteolysis that is induced by even low levels of NS3, results in an eradication of NS3 expressing model cells and HCV infected cells. Zymoxins may thus become a valuable tool in eradicating cells infected by intracellular pathogens that express intracellular proteases.

Targeted immunotherapy for colorectal cancer: monoclonal antibodies and immunotoxins

Colorectal cancer (CRC) is a major health concern worldwide. It is the third most frequently diagnosed cancer and the second leading cause of cancer death. There currently are a number of treatment options for CRC, however many of them have failed to demonstrate desired therapeutic benefit. Therefore, significant efforts are being directed towards the development of new biological therapies with improved efficacy. Immunotherapy is an emerging treatment modality for a variety of cancers. Several promising treatments have already been approved by the US FDA and are being tested in clinical trials. Antibodies have been proved to be useful in cancer therapy due to their ability to recognize tumor-associated antigens expressed at higher density on malignant cells in comparison with those that are normal. Antibodies can be used as a single therapy or in combination with other therapies. A large variety of monoclonal antibodies have been developed. However, only a very few are able to kill a sufficient number of malignant cells and cause tumor regression. Hence, it is often necessary to arm the antibody with a cytotoxic agent to enhance the efficacy of the anti-tumor activity. This review provides a brief overview of some of the current agents being employed in targeted immunotherapy for CRC.

The APC p.I1307K polymorphism is a significant risk factor for CRC in average risk Ashkenazi Jews.

BACKGROUNDnThe p.I1307K adenomatous polyposis coli (APC) gene variant, prevalent among Ashkenazi Jews, may increase the risk for colorectal neoplasia. We studied the clinical importance of screening for this polymorphism in 3305 Israelis undergoing colonoscopy.nnnPATIENTS AND METHODSnClinical data regarding potential risk factors for colorectal cancer (CRC) were collected from individuals undergoing colonoscopic examination at the Tel-Aviv medical center. The APC p.I1307K was detected using real-time PCR (polymerase chain reaction) from DNA extracted from peripheral mononuclear cells.nnnRESULTSnThe overall prevalence of the p.I1307K polymorphism was 8.0% (10.1% among Ashkenazi and 2.7% among Sephardic Jews, p<0.001). The overall adjusted odds ratio (OR) for colorectal neoplasia among carriers was 1.51 (95% confidence intervals (CI), 1.16-1.98). Among average risk Ashkenazi Jews, the adjusted OR was 1.75 (95% CI 1.26-2.45). A multiplicative interaction was identified between Ashkenazi ethnicity and APC p.I1307K carrier status (P(INTERACTION) = 0.055). The histopathological features of adenomas and carcinomas did not differ between carriers and non-carriers.nnnCONCLUSIONSnThe APC p.I1307K gene variant is an important risk factor for colorectal neoplasia in average risk Ashkenazi Jews. Carriers in this group should be considered for screening colonoscopy at the age of 40, to be repeated every 5 years, similar to recommendations in individuals with family history of colorectal cancer.

Novel approach to abuse the hyperactive K-Ras pathway for adenoviral gene therapy of colorectal cancer.

BACKGROUNDnFunctional activation of oncogenic K-Ras signaling pathway plays an important role in the early events of colorectal carcinogenesis (CRC). K-Ras proto-oncogene is involved in 35-40% of CRC cases. Mutations in the Ras gene trigger the transduction of proliferative and anti-apoptotic signals, even in the absence of extra cellular stimuli. The objective of the current study was to use a gene-targeting approach to kill human CRC cells selectively harboring mutated K-Ras.nnnRESULTSnA recombinant adenovirus that carries a lethal gene, PUMA, under the control of a Ras responsive promoter (Ad-Py4-SV40-PUMA) was used selectively to target CRC cells (HCT116, SW480, DLD1 and RIE-Ras) that possess a hyperactive Ras pathway while using HT29 and RIE cells as a control that harbors wild type Ras and exhibit very low Ras activity. Control vector, without the Ras responsive promoter elements was used to assess the specificity of our gene therapy approach. Both adenoviral vectors were assed in vitro and in xenograft model in vivo. Ad-Py4-SV40-PUMA showed high potency to induce ~50% apoptosis in vitro, to abolish completely tumor formation by infecting cells with the Ad-Py4-SV40-PUMA prior xenografting them in nude mice and high ability to suppress by ~35% tumor progression in vivo in already established tumors.nnnCONCLUSIONSnSelective targeting of CRC cells with the activated Ras pathway may be a novel and effective therapy in CRC. The high potency of this adenoviral vector may help to overcome an undetectable micro metastasis that is the major hurdle in challenging with CRC.

Delayed Wound Healing in Heat Stable Antigen (HSA/CD24)-Deficient Mice

Background Healthy individuals rarely have problems with wound healing. Most skin lesions heal rapidly and efficiently within one to two weeks. However, many medical and surgical complications can be attributed to deficiencies in wound repair. Open wounds have lost the barrier that protects tissues from bacterial invasion and allows the escape of vital fluids. Without expeditious healing, infections become more frequent. The CD24 gene encodes a heavily-glycosylated cell surface protein anchored to the membrane by phosphatidylinositol. CD24 plays an important role in the adaptive immune response and controls an important genetic checkpoint for homeostasis and autoimmune diseases in both mice and humans. We have previously shown that overexpression of CD24 results in increased proliferation and migration rates. Aim To examine the role of CD24 in the wound healing process. Methods An excisional model of wound healing was used and delayed wound healing was studied in genetically modified heat stable antigen (HSA/CD24)-deficient mice (HSA -/-) compared to wild-type (WT) mice. Results Large full-thickness skin wounds, excised on the back of mice, exhibited a significant delay in the formation of granulation tissue, and in wound closure when compared to their WTHSA +/+ littermates. Wounds were histologically analyzed and scored, based on the degree of cellular invasion, granulation tissue formation, vascularity, and re-epithelialization. Additionally, in stitched wounds, the HSA -/- mice failed to maintain their stitches; they did not hold and fell already 24 hours, revealing erythematous wound fields. Re-expression of HSA, delivered by lentivirus, restored the normal healing phenotype, within 24 hours post-injury, and even improved the healing in WT, and in BalbC mice. Conclusions Delayed wound-healing in the absence of HSA/CD24 suggests that CD24 plays an important role in this process. Increased expression of CD24, even in the normal state, may be used to enhance wound repair.

Chemoprevention for advanced CR neoplasia

Colorectal cancer (CRC) is a major health concern worldwide. In 2011 1,200,000 new cases are predicted and half of them are going to die from the disease. CRC carcinogenesis is a multi-step process that spans over 10-20 years, providing a window of opportunity for effective intervention. CRC can be prevented by life style modification and screening program. However, although these strategies are standard clinical practice, their impact is limited due to low adherence. The number of deaths due to CRC remains alarming high, and makes CRC prevention a paramount. Chemoprevention interferes with the carcinogenesis process by targeting key molecular pathways. It involves the use of a variety of natural or chemical compounds that can delay, prevent or even reverse the adenoma to carcinoma sequence. Numerous chemopreventive agents have been studied but the most efficient are the NSAID group of agents. Much of their efficacy and toxicity has been attributed to their potent inhibition of the cyclooxygenase (COX) enzymes. Chemoprevention has the potential to represent a cost-effective intervention, particularly when targeted at intermediate-risk populations, ages 61-70, following polypectomy. Chemoprevention in this setting is as very important as polyp recurrence in this population can be as high as 50%, even with surveillance colonoscopy every 1-3 years. The most challenging task is to find the proper place for these interventions in the entire effort of general wellbeing. Subjects are likely to be more adherent to prescribed regimens if cancer prevention may be combined with a cardiovascular and Alzheimer prophylaxis. Subjects with a normal colon or non advanced adenomas can be safely monitored with surveillance colonoscopy every 5-10 years. The ideal chemopreventive agent remains to be discovered with great emphasis on the need not to harm. Possibly, combinations of agents will maximize effectiveness while limiting drug toxicity. Finally, personalized approaches would include the ability to predict risk, as well as benefit for a specific individual based on specific SNPs or other genetic profiles.

Abstract 3805: A novel anti-CD24 monoclonal antibody, humanized and affinity maturated for targeting gastrointestinal cancers

Background: CD24 is a cell-surface heavily glycosylated GPI-anchored protein. We had previously shown that CD24 in an important player in the multistep process of GI carcinogenesis (Gastro 2006, Clin Can Res 2007, Can Res 2008). The creation of chimeric, humanized or fully human antibodies was a major breakthrough and led to a wave of US FDA-approved antibodies. Aim: To further improve the efficacy of the humanized anti-CD24 mAb by increasing its binding strength and thereby generating a novel therapy tool for GI malignancy Methods: From murine to humanized, unarmed and conjugated, small derivatives and full IgG antibodies were recombinantly engineered. The antibody genes were recovered, amplified and cloned into appropriate vectors. Then the vectors were introduced into a host (mammalian and E.coli) and adequate amounts of functional antibody were achieved. Sequence analysis of the CDR loops was the base for library designing. Affinity maturation was performed in two-steps selection (CDR walking) and by using phage display technique. The binding of the different derivatives were evaluated on full Glycan array in which more than 70 sugar moieties were printed. Results: In vivo antibody targeting and accumulation within a CD24 positive tumor and its excess clearance was clearly demonstrated using live imaging device (Maestro Cri device). High-affinity antibodies were selected and created from combinatorial phage-displayed antibody libraries that contain varying degrees of diversity at randomized positions. A chosen matured clone was isolated and showed higher binding strength (1.8×10-8), compared to the parental murine and humanized Abs. The matured antibody showed selective recognition and binding to the CD24 antigen which proves that the genetic manipulations carried out did not affect its properties. Its stability was enhanced following the maturation process,as well as its pharmacokinetics parameters which showed a long serum half-life.The matured antibody mediates ADCC (antibody-dependent cell cytotoxicity), 75% of target cell lysis was demonstrated. Combined treatment with standard chemotherapy and natural products, such as monoterpenes (terpinen-4-ol), showed significant reduction in cell viability (90% cell death). Binding of anti-CD24 Ab to glycan microarray could not be detected while high binding intensities were observed where the whole CD24 protein was printed, indicating that the antibodies bind to the core peptide and not to its sugar residues. Conclusion: Targeting CD24 may be a promising treatment for GI malignancies in combination with chemotherapy and natural agents. The resulted matured humanized anti-CD24 mAb proved to be more effective than the murine parental Ab. The long serum half-life is desirable as it would decrease the need for repetitive injections. Citation Format: Shiran Shapira, Dina Kazanov, Vered Padler Karavani, Itai Benhar, Nadir Arber. A novel anti-CD24 monoclonal antibody, humanized and affinity maturated for targeting gastrointestinal cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3805.

Abstract 5333: Novel Ab treatment tool for CRC

Background: We have shown that CD24, a cell surface molecule, is overexpressed early in the multistep process of colorectal neoplasia (Gastroenterology, 2006) hence, it can serve as a target for early detection and immunotherapy. The anti-CD24 murine SWA11 Mab (generous gift from Prof. Sthal, Zurich University) binds to CD24 and inhibits tumor cell growth, in vitro and in vivo, in a time- and dose-dependent manner (Cancer Research, 2008). However, murine Mabs have limitations because of their xenogeneic nature and due to the human anti-mouse antibody (HAMA) response. Aim: To produce an improved, chimeric form of the SWA11 Mab. Methods: Mammalian vector pMAZ-IgH for human γ1 heavy-chain and pMAZ-IgL for human κ light chain expression were designed. The variable region genes encoding the murine anti-CD24 SWA11 MAb were cloned for expression as chimeric γ1/kappa antibody into the mammalian pMAZ-IgH and pMAZ-IgL expression vectors and co-transfected into HEK293 cells. Stable clones were screened and selected for IgG production, analyzed by whole-cell and antigen-based ELISA, and Western blotting. Results: The new chimeric IgG derivative of the anti-CD24 SWA11 Mab possesses similar affinity and specificity (ELISA and Western blot), but has improved activity in inhibiting cell growth. The chimeric Mabs also have the ability to induce complement-dependent cytotoxicity, causing up to 60% cell lysis in the presence of complement. Conclusions: We had successfully generated a chimeric IgG derivative of the anti-CD24 SWA11 Mab that can serve as a novel therapeutic tool for cancer cells harboring CD24. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5333.