Paul Y. Liu

Anti-CD3 x anti-epidermal growth factor receptor (EGFR) bispecific antibody redirects T-cell cytolytic activity to EGFR-positive cancers in vitro and in an animal model.

Purpose: Targeting epidermal growth factor receptor (EGFR) overexpressed by many epithelial-derived cancer cells with anti-EGFR monoclonal antibodies (mAb) inhibits their growth. A limited number of clinical responses in patients treated with the anti-EGFR mAb, (cetuximab), may reflect variability in EGFR type or signaling in neoplastic cells. This study combines EGFR-targeting with the non-MHC–restricted cytotoxicity of anti-CD3 activated T cells (ATC) to enhance receptor-directed cytotoxicity. Experimental Design: ATC from normal and patient donors were expanded ex vivo. Specific cytolytic activity of ATC armed with anti-CD3 × anti-EGFR (EGFRBi) against EGFR-expressing cancer cells derived from lung, pancreas, colon, prostate, brain, skin, or EGFR-negative breast cancer cells was evaluated in 51Cr release assays. In vivo studies comparing tumor growth delay induced by EGFRBi-armed ATCs or cetuximab were done in severe combined immunodeficient/Beige mice (SCID-Beige) bearing COLO 356/FG pancreatic and LS174T colorectal tumors. Results: At effector/target ratios from 3.125 to 50, both EGFRBi-armed normal and patient ATC were significantly more cytotoxic, by 23% to 79%, against EGFR-positive cells over ATC, cetuximab, anti-CD3 alone, or ATC armed with irrelevant BiAb directed at CD20. EGFRBi-armed ATC also secreted significantly higher levels of some TH1/TH2 cytokines compared with ATC alone. In mice, i.v. infusions of EGFRBi-armed ATC (0.001 mg equivalent/infusion) were equally effective as cetuximab (1 mg/infusion) alone for significantly delaying growth of established COLO 356/FG but not LS174T tumors compared with mice that received ATC alone or vehicle (P < 0.001). Conclusions: Combining EGFR antibody targeting with T cell–mediated cytotoxicity may overcome some limitations associated with EGFR-targeting when using cetuximab alone.

Liposome‐mediated transfer of vascular endothelial growth factor cDNA augments survival of random‐pattern skin flaps in the rat

Tissue engineering is an application for gene therapy that is in its infancy. We show that simple liposomal‐mediated gene transfer could result in a potentially useful biological effect in the field of wound healing. cDNA encoding the 165 amino acid form of vascular endothelial growth factor complexed to commercially available liposomes was injected into rat skin 1 week before raising a random pattern 3u2003×u200310u2003cm flap. The flap survival was enhanced by 14 percent, and was accomplished without accessing the arterial inflow of the territory. These results were statistically significant (pu2003<u20030.002) and reproducible. No adverse effects were seen. Histological analysis of the angiogenesis localized much of the new vessel formation to the area around the hair follicles. Polymerase chain reaction amplification of extracted flap tissue confirmed the presence of the transgene.

Basic FGF or VEGF gene therapy corrects insufficiency in the intrinsic healing capacity of tendons

Tendon injury during limb motion is common. Damaged tendons heal poorly and frequently undergo unpredictable ruptures or impaired motion due to insufficient innate healing capacity. By basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF) gene therapy via adeno-associated viral type-2 (AAV2) vector to produce supernormal amount of bFGF or VEGF intrinsically in the tendon, we effectively corrected the insufficiency of the tendon healing capacity. This therapeutic approach (1) resulted in substantial amelioration of the low growth factor activity with significant increases in bFGF or VEGF from weeks 4 to 6 in the treated tendons (pu2009<u20090.05 or pu2009<u20090.01), (2) significantly promoted production of type I collagen and other extracellular molecules (pu2009<u20090.01) and accelerated cellular proliferation, and (3) significantly increased tendon strength by 68–91% from week 2 after AAV2-bFGF treatment and by 82–210% from week 3 after AAV2-VEGF compared with that of the controls (pu2009<u20090.05 or pu2009<u20090.01). Moreover, the transgene expression dissipated after healing was complete. These findings show that the gene transfers provide an optimistic solution to the insufficiencies of the intrinsic healing capacity of the tendon and offers an effective therapeutic possibility for patients with tendon disunion.

Cellular Apoptosis and Proliferation in the Middle and Late Intrasynovial Tendon Healing Periods

PURPOSEnCellular apoptosis might be an important molecular event in the middle or late healing periods of intrasynovial tendons, but this has not been studied. We aimed to investigate cellular apoptosis and corresponding cellular proliferation in the middle and late healing stages of intrasynovial tendons.nnnMETHODSnThe flexor digitorum profundus tendons of 48 long toes (24 chickens) were completely transected within the sheath region and were repaired surgically. At days 28, 42, 56, and 84 after surgery, tendons were harvested and sectioned. In situ terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed to detect apoptotic cells. The sections were stained immunofluorescently with antibodies to proliferating cell nuclear antigen to assess proliferation and to Bcl-2 (an anti-apoptotic protein). Positively stained tenocytes were counted, and their distributional differences were verified in 3-dimensional images.nnnRESULTSnThe repaired intrasynovial tendons exhibited generally greater apoptosis in the surface region than in the core. The differences were more remarkable in the extended region than in the junction region of the cut tendon. At the core of the junction site, apoptosis of tenocytes was pronounced at all time points, but it was less severe at the core of the extended region. The proliferating cell nuclear antigen-positive and Bcl-2-positive tenocytes decreased significantly and continually at days 28, 42, and 56, respectively; these tenocytes were at a minimum at days 56 and 84.nnnCONCLUSIONSnApoptotic changes of tenocytes are most marked in the surface region and in the junction region of the healing tendon in the middle and late healing stages. Apoptosis in the core is less dramatic compared to that in the surface in the extended tendon regions. Cellular proliferation declines drastically and is minimal at days 56 and 84.nnnCLINICAL RELEVANCEnTenocyte apoptosis in the middle and late stages might be an important event contributing to intrasynovial tendon remodeling, which affects the healing strength and formation of adhesions.

Enhancement of flap survival and changes in angiogenic gene expression after AAV2‐mediated VEGF gene transfer to rat ischemic flaps

Necrosis of surgically transferred flaps due to ischemia is a serious wound problem. We evaluated the improvement of flap survival and changes in angiogenic gene expression profiles after transfer of the VEGF gene by means of adeno‐associated virus type 2 (AAV2) vector to rat ischemic flaps. Thirty rats were divided into one experimental group, one AAV2‐GFP group, and one saline group. AAV2‐VEGF or AAV2‐GFP were injected intradermally into the rat dorsum in the AAV2‐VEGF or AAV2‐GFP group. The saline group received saline injection. A 3 × 10u2003cm flap was raised in each rat two weeks post‐injection. One week after surgery, flap viability was evaluated. Angiogenesis real‐time PCR array was performed to analyze the expression of angiogenesis‐associated genes. The AAV2‐VEGF treatment significantly improved flap survival (p<0.05). Immunohistochemical staining showed increased VEGF expression in AAV2‐VEGF treated flaps. The PCR array identified remarkable changes in 6 out of the 84 angiogenesis‐associated genes in AAV2‐VEGF treated flaps. Particularly, EGF, PDGF‐A and VEGF‐B genes were up‐regulated in these flaps. In contrast, FGF2 gene expression was down‐regulated. In conclusion, AAV2‐VEGF improves flap survival and affects the expression of a series of endogenous growth factor genes, which likely play critical roles in the enhancement of ischemic flap survival.