Generation of dual specific bivalent BiTEs (dbBIspecific T-cell engaging antibodies) for cellular immunotherapy.

BACKGROUND: Bispecific T-cell engaging antibodies (BiTES), comprising dual anti-CD3 and anti-tumor antigen scFv fragments, are important therapeutic agents for the treatment of cancer. The dual scFv construct for BiTES requires proper protein folding while their small molecular size leads to rapid kidney clearance. METHODS: An intact (150 kDa) anti-tumor antigen antibody to CEA was joined in high yield (ca. 30%) to intact (150 kDa) anti-murine and anti-human CD3 antibodies using hinge region specific Click chemistry to form dual-specific, bivalent BiTES (dbBiTES, 300 kDa). dbBiTEs were tested in vitro by EM, flow cytometry and cell cytoxicity and in vivo by PET tumor imaging and redirected T-cell therapy. RESULTS: The interlocked hinge regions are compatible with a structural model that fits the electron micrographs of 300 kDa particles. Compared to intact anti-CEA antibody, dbBiTES exhibit high in vitro cytotoxicity, high in vivo tumor targeting as demonstrated by PET imaging, and redirected dbBiTE coated T-cells (1 microgram/10 million cells) that kill CEA+ target cells in vivo in CEA transgenic mice. CONCLUSION: dbBiTE redirected T-cell therapy is a promising, efficient approach for targeting and killing cancer cells.

Low Vision Practice and Service Provision Among Optometrists in Ghana: A Nationwide Survey.

PURPOSE: To characterize practice patterns of low vision services among Optometrists in Ghana. METHODS: The nationwide cross-sectional survey identified entities through the Ghana Optometrists Association (GOA) registry and utilized a semi-structured questionnaire to consolidate survey information that comprises practitioners' demographics, available services, diagnostic equipment, barriers to service provision and utilization, and interventions. RESULTS: 300 Optometrists were identified, with 213 surveyed (71% response rate). About fifty percent (52.6%) were in private practice, and more than two-thirds (77%) did not provide low vision services. Most (≥70%) reported lack of assistive devices, and basic eye care examination kits as the main barriers to low vision service provision. Similarly, practitioners reported unawareness of the presence of low vision centres (76.1%), and high cost of low vision aids (75.1%) as the prime perceived barriers for patients to utilize low vision services. Continuous professional development and public education (89-90%) were suggested as interventions to improve the uptake of low vision services. After statistical adjustment, private facility type (Adjusted odds ratio [AOR] = 0.35, p = 0.010) and lack of basic eye examination kits (AOR = 0.32, p = 0.002) were significantly associated with reduced odds of low vision service provision. Conversely, ≥15 years of work experience (AOR = 6.37, p = 0.011) was significantly associated with increased odds of low vision service provision. CONCLUSIONS: Overall, the results indicate inadequate low vision coverage and service delivery. Government policies must be directed towards equipping practitioners with equipment and subsidize patient cost of treatment to optimize low vision care.

Comparison of the performance of the dry eye questionnaire (DEQ-5) to the ocular surface disease index in a non-clinical population.

OBJECTIVE: To compare the performance of the dry eye questionnaire (DEQ-5) with the Ocular Surface Disease Index (OSDI) and further validate the DEQ-5 questionnaire. METHODS: A population-based cross-sectional study conducted in Ghana. OSDI and DEQ-5 questionnaires were administered to participants. Cronbach's alpha was used to evaluate the reliability of the OSDI and DEQ-5 questionnaires. Analysis of variance was used to evaluate the discriminant validity of DEQ-5. Concurrent validity was evaluated using the Spearman correlation analysis. A receiver operating characteristic (ROC) curve was generated to describe the sensitivity and specificity of the DEQ-5 questionnaire for diagnosis of dry eye symptoms. Cohen Kappa was used to evaluate agreement between the two questionnaires. RESULTS: The reliability of the overall OSDI and DEQ-5 scores were 0.919 and 0.819 respectively. The mean (SD) DEQ-5 scores for asymptomatic, mild, moderate and severe dry eye symptoms as defined by the OSDI grading were 3.05 (2.73), 5.13 (3.69), 7.65 (3.30) and 9.77 (4.16) respectively. There was a statistically significant correlation between total OSDI and total DEQ-5 scores (rs = 0.649, p < 0.0001). The area under the curve (AUC) of the ROC curve for DEQ-5 was 0.835 (95 % CI: 0.796 - 0.875). A DEQ-5 threshold of 5.5 yielded maximum sensitivity (0.712) and specificity (0.827). The Cohen kappa using a the DEQ-5 total score threshold of 5.5 was K = 0.539 (p < 0.0001). CONCLUSION: In conclusion, performance of the DEQ-5 questionnaire in discriminating symptoms of dry eye is comparable to the OSDI questionnaire. The DEQ-5 questionnaire is a valid measure of dry eye symptoms and can be used as a dry eye symptoms assessment tool in both clinical and epidemiological studies.

Association between diabetes and keratoconus-a systematic review and meta-analysis.

PURPOSE: To assess the association between diabetes mellitus and keratoconus. METHODS: PubMed, Google Scholar, Web of Science, and Scopus databases were searched for literature on the association between diabetes and keratoconus. The last literature search was conducted on April 4, 2021. A secondary form of the literature search was conducted by manually scanning the reference list of retrieved eligible articles. Included studies were cohort, case-control, or cross-sectional study design that used odds ratio or risk ratio to evaluate the relationship between keratoconus and diabetes. Egger's test was used to assess the presence of publication bias. The quality of eligible studies was assessed using the Newcastle-Ottawa Scale. RESULTS: Nine studies (six case-control and three cohort studies) published between 2000 and 2021 were included. The total number of keratoconus patients and controls were 27,311 and 53,732. respectively. Meta-analysis revealed no significant association between diabetes mellitus and keratoconus; the pooled odds ratio was 0.87 (95% confidence interval: 0.66-1.14; p = 0.314). There was significant heterogeneity (Q (df = 7) = 33.36, p < 0.001;I2 = 79.01, p < 0.001). Age of participants (p < 0.0001), study design (p < 0.001), and sample size (p = 0.024) were significant sources of heterogeneity. There was no evidence of publication bias. CONCLUSION: The current meta-analysis revealed no significant association between diabetes mellitus and keratoconus. Well-designed longitudinal prospective studies are, however, needed to investigate any association between diabetes mellitus and keratoconus.

Phase I Study of Yttrium-90 Radiolabeled M5A Anti-Carcinoembryonic Antigen Humanized Antibody in Patients with Advanced Carcinoembryonic Antigen Producing Malignancies.

Background: M5A is a humanized monoclonal antibody (mAb) directed against carcinoembryonic antigen (CEA) The purpose of this first in human phase I dose-escalation trial was to characterize the toxicities and determine the maximum tolerated dose (MTD) of yttrium-90 (90Y)-DOTA-M5A as a single agent and in combination with gemcitabine (gem). Methods: Patients with advanced metastatic CEA-producing malignancies who had progressed on standard therapies were first administered indium-111 (111In)-DOTA-M5A. If tumor targeting was observed, the patient then received the therapy dose of 90Y-DOTA-M5A. Serial scans, blood sampling, and 24 h urine collections were then performed to estimate radiation doses to organs and total body. Assays for human antihuman antibody (HAHA) responses were performed out to 6 months. Results: Of the 18 patients who received 111In-DOTA-M5A, 16 received 90Y-DOTA-M5A therapy; 1 patient at 14 mCi/m2 with gem (150 mg/m2 days 1and 3), 3 patients at 12 mCi/m2 with gem, 6 patients at 12 mCi/m2 without gem, and 6 at 10 mCi/m2 without gem. Prolonged cytopenias resulted in discontinuation of dose escalation with gemcitabine. A single agent MTD of 10 mCi/m2 was established based on dose-limiting hematopoietic toxicities. HAHA immune response was identified in 2 of 16 patients (12.5%). Stable disease at 3 months was seen in 10 patients and 2 patients demonstrated an 88% and 64% decrease in CEA back to normal levels. In 2 patients 111In-DOTA-M5A imaging revealed previously unknown brain metastases. Conclusion: This study demonstrates the potential utility of the 90Y-DOTA-M5A anti-CEA mAb as a therapeutic antibody. There is decreased immunogenicity compared with murine and chimeric mAbs, allowing for the potential of multiple administrations. Combined modality therapy approaches incorporating this agent should continue to be evaluated.

Potent immunomodulatory effects of an anti-CEA-IL-2 immunocytokine on tumor therapy and effects of stereotactic radiation.

While anti-CEA antibodies have no direct effect on CEA-positive tumors, they can be used to direct potent anti-tumor effects as an antibody-IL-2 fusion protein (immunocytokine, ICK), and at the same time reduce the toxicity of IL-2 as a single agent. Using a fusion protein of humanized anti-CEA with human IL-2 (M5A-IL-2) in a transgenic murine model expressing human CEA, we show high tumor uptake of the ICK to CEA-positive tumors with additional lymph node targeting. ICK treated CEA-positive tumors exhibit significant tumor eradication. Analysis of tumor-infiltrating lymphocytes shows a high frequency of both CD8+ and CD4+ T cells along with CD11b positive myeloid cells in ICK treated mice. The frequency of tumor-infiltrating FoxP3+ CD4+ T cells (Tregs) is significantly reduced vs anti-CEA antibody-treated controls, indicating that ICK did not preferentially stimulate migration or proliferation of Tregs to the tumor. Combination therapy with anti-PD-1 antibody did not improve tumor reduction over ICK therapy alone. Since stereotactic tumor irradiation (SRT), commonly used in cancer therapy has immunomodulatory effects, we tested combination SRT+ICK therapy in two tumor model systems. Use of fractionated vs single high dose SRT in combination with ICK resulted in greater tumor inhibition and immunity to tumor rechallenge. In particular, tumor microenvironment and myeloid cell composition appear to play a significant role in the response rate to ICK+SRT combination therapy.

ImmunoPET, [64Cu]Cu-DOTA-Anti-CD33 PET-CT, Imaging of an AML Xenograft Model.

PURPOSE: Acute myeloid leukemia (AML) is a highly aggressive form of leukemia, which results in poor survival outcomes. Currently, diagnosis and prognosis are based on invasive single-point bone marrow biopsies (iliac crest). There is currently no AML-specific noninvasive imaging method to detect disease, including in extramedullary organs, representing an unmet clinical need. About 85% to 90% of human myeloid leukemia cells express CD33 cell surface receptors, highlighting CD33 as an ideal candidate for AML immunoPET. EXPERIMENTAL DESIGN: We evaluated whether [64Cu]Cu-DOTA-anti-CD33 murine mAb can be used for immunoPET imaging of AML in a preclinical model. MicroCT was adjusted to detect spatial/anatomical details of PET activity. For translational purposes, a humanized anti-CD33 antibody was produced; we confirmed its ability to detect disease and its distribution. We reconfirmed/validated CD33 antibody-specific targeting with an antibody-drug conjugate (ADC) and radioimmunotherapy (RIT). RESULTS: [64Cu]Cu-DOTA-anti-CD33-based PET-CT imaging detected CD33+ AML in mice with high sensitivity (95.65%) and specificity (100%). The CD33+ PET activity was significantly higher in specific skeletal niches [femur (P < 0.00001), tibia (P = 0.0001), humerus (P = 0.0014), and lumber spine (P < 0.00001)] in AML-bearing mice (over nonleukemic control mice). Interestingly, the hybrid PET-CT imaging showed high disease activity in the epiphysis/metaphysis of the femur, indicating regional spatial heterogeneity. Anti-CD33 therapy using newly developed humanized anti-CD33 mAb as an ADC (P = 0.02) and [225Ac]Ac-anti-CD33-RIT (P < 0.00001) significantly reduced disease burden over that of respective controls. CONCLUSIONS: We have successfully developed a novel anti-CD33 immunoPET-CT-based noninvasive modality for AML and its spatial distribution, indicating a preferential skeletal niche.