High-affinity Monoclonal Antibody Research Articles

A Sensitive Monoclonal-Antibody-Based ELISA for Forchlorfenuron Residue Analysis in Food Samples.

In this study, forchlorfenuron (CPPU) was coupled with succinic anhydride to yield a CPPU hapten (CPPU-COOH), and a high-affinity monoclonal antibody (mAb) that can specifically recognize CPPU was produced. Using this mAb as a recognition reagent, a sensitive indirect competitive enzyme-linked immunosorbent assay (icELISA) for CPPU was optimized, which exhibits an IC50 of 1.04 ng/mL, a limit of detection of 0.16 ng/mL, and a linear range of 0.31–3.43 ng/mL for CPPU. Cross-reactivity percentages with six analogues were all below 6%. The average recovery rates for cucumber and orange samples were from 85.23% to 119.14%. The analysis results of this icELISA showed good consistency with those from liquid chromatography mass spectrometry. These results suggest that the proposed icELISA provides a sensitive, specific, and reliable strategy for CPPU detection in food samples.

Competitively disrupting the neutrophil-specific receptor–autoantigen CD177:proteinase 3 membrane complex reduces anti-PR3 antibody-induced neutrophil activation

CD177 is a neutrophil-specific receptor presenting the proteinase 3 (PR3) autoantigen on the neutrophil surface. CD177 expression is restricted to a neutrophil subset, resulting in CD177pos/mPR3high and CD177neg/mPR3low populations. The CD177pos/mPR3high subset has implications for antineutrophil cytoplasmic autoantibody (ANCA)–associated autoimmune vasculitis, wherein patients harbor PR3-specific ANCAs that activate neutrophils for degranulation. Here, we generated high-affinity anti-CD177 monoclonal antibodies, some of which interfered with PR3 binding to CD177 (PR3 “blockers”) as determined by surface plasmon resonance spectroscopy and used them to test the effect of competing PR3 from the surface of CD177pos neutrophils. Because intact anti-CD177 antibodies also caused neutrophil activation, we prepared nonactivating Fab fragments of a PR3 blocker and nonblocker that bound specifically to CD177pos neutrophils. We observed that Fab blocker clone 40, but not nonblocker clone 80, dose-dependently reduced anti-PR3 antibody binding to CD177pos neutrophils. Importantly, preincubation with clone 40 significantly reduced respiratory burst in primed neutrophils challenged with either monoclonal antibodies to PR3 or PR3–ANCA immunoglobulin G from ANCA-associated autoimmune vasculitis patients. After separating the two CD177/mPR3 neutrophil subsets from individual donors by magnetic sorting, we found that PR3–ANCAs provoked significantly more superoxide production in CD177pos/mPR3high than in CD177neg/mPR3low neutrophils, and that anti-CD177 Fab clone 40 reduced the superoxide production of CD177pos cells to the level of the CD177neg cells. Our data demonstrate the importance of the CD177:PR3 membrane complex in maintaining a high ANCA epitope density and thereby underscore the contribution of CD177 to the severity of PR3–ANCA diseases.

Higher rates of skin clearance and efficacy in challenging body areas are associated with better health‐related quality of life following ixekizumab maintenance treatment in pediatric patients with plaque psoriasis

Background/ObjectivesInformation is limited on the relationship between skin clearance, resolution of challenging body areas, and improvement of patient‐reported outcomes (PROs) in pediatric psoriasis. Ixekizumab, a high‐affinity monoclonal antibody that selectively targets interleukin‐17A, is approved for the treatment of moderate‐to‐severe psoriasis in patients aged 6 to <18 years. This study examines improvement in psoriasis clearance in challenging body areas in pediatric patients relative to health‐related quality of life.MethodsData from the IXORA‐PEDS trial (NCT03073200) were analyzed, and changes from baseline were measured for overall Psoriasis Area and Severity Index (PASI), static Physicians' Global Assessment of psoriasis (sPGA), Psoriasis Scalp Severity Index (PSSI), Palmoplantar Psoriasis Area and Severity Index (PPASI), and Nail Psoriasis Severity Index. Rates of Dermatology Life Quality Index (DLQI), or Children's DLQI (CDLQI), scores of 0 or 1 were evaluated using the Cochran‐Armitage trend test.ResultsHigher rates of DLQI/CDLQI (0,1) scores were significantly associated with greater PASI and PSSI responses at both Week 12 and Week 48 (p < .0001). A significant association was also observed between DLQI/CDLQI (0,1) and sPGA scores (p < .0001). Significantly higher rates of DLQI/CDLQI (0,1) scores were achieved in patients with greater levels of palmoplantar clearance as measured by PPASI at Week 12 (p = .0139), but significance was not sustained at Week 48 (p = .0896).ConclusionsGreater skin clearance and scalp resolution are associated with better PROs over a short‐term (12‐week) and long‐term (48‐week) period. This demonstrates that greater improvement of skin clearance and scalp resolution may benefit quality of life in pediatric patients with psoriasis.

Diagnosis and Stratification of Pseudomonas aeruginosa Infected Patients by Immunochemical Quantitative Determination of Pyocyanin From Clinical Bacterial Isolates.

The development of a highly sensitive, specific, and reliable immunochemical assay to detect pyocyanin (PYO), one of the most important virulence factors (VFs) of Pseudomonas aeruginosa, is here reported. The assay uses a high-affinity monoclonal antibody (mAb; C.9.1.9.1.1.2.2.) raised against 1-hydroxyphenazine (1-OHphz) hapten derivatives (PC1; a 1:1 mixture of 9-hydroxy- and 6-hydroxy-phenazine-2-carobxylic acids). Selective screening using PYO and 1-OHphz on several cloning cycles allowed the selection of a clone able to detect PYO at low concentration levels. The microplate-based ELISA developed is able to achieve a limit of detection (LoD) of 0.07 nM, which is much lower than the concentrations reported to be found in clinical samples (130 μM in sputa and 2.8 μM in ear secretions). The ELISA has allowed the investigation of the release kinetics of PYO and 1-OHphz (the main metabolite of PYO) of clinical isolates obtained from P. aeruginosa-infected patients and cultured in Mueller–Hinton medium. Significant differences have been found between clinical isolates obtained from patients with an acute or a chronic infection (~6,000 nM vs. ~8 nM of PYO content, respectively) corroborated by the analysis of PYO/1-OHphz levels released by 37 clinical isolates obtained from infected patients at different stages. In all cases, the levels of 1-OHphz were much lower than those of PYO (at the highest levels 6,000 nM vs. 300 nM for PYO vs. 1-OHphz, respectively). The results found point to a real potential of PYO as a biomarker of P. aeruginosa infection and the possibility to use such VF also as a biomarker for patient stratification[2] and for an effective management of these kinds of infections.

Measurement of total and free prostate specific antigen (PSA) in human serum samples using an ultra-microanalytical system

Prostate specific antigen (PSA) is a serine protease used for the screening of prostate cancer. The total portion of PSA (tPSA) can be found in its free form (fPSA), or bound to other proteins forming a stable complex. A heterogeneous sandwich-type UltraMicro Enzyme-Linked ImmunoSorbent Assay (UMELISA) has been developed for the measurement of tPSA and fPSA in human serum samples. Strips coated with a high affinity monoclonal antibody (MAb) directed against PSA are used as solid phase, to ensure the specificity of the assay. Biotinylated MAbs specific for tPSA and fPSA ensured sensitivity, given the high affinity binding to streptavidin. The assay was completed in 1.5 h, with a measuring range 0.019–20 µg/L (tPSA), and 0.009–20 µg/L (fPSA). The intra- and inter-assay CV were lower than 9%. Recovery percentages were 96–105%. High correlations were found between the values of the UMELISA PSA standards and the International Reference Standards 96/670 (R2 = 0.9996) and 96/688 (R2 = 0.9989). The assay did not recognize any of the interfering molecules tested. Regression analysis of serum samples showed a good correlation with Roche Elecsys total PSA (n = 631, R2 = 0.986, ρc = 0.992), BioMérieux VIDAS TPSA (n = 631, R2 = 0.989, ρc = 0.993) and Roche Elecsys free PSA (n = 164, R2 = 0.973, ρc = 0.979), all with a relative difference below 15%, and a p < 0.001. A retrospective study of the use of UMELISA PSA in Cuba was carried out. The analytical performance characteristics of UMELISA PSA support its use for the quantification of tPSA and fPSA in human serum samples in a single kit, making it an affordable diagnostic assay available to Cuban Public Health System and developing countries. Between the years 2014–2020, more than 3 million Cuban patients have benefited from the test for free.

Safety and Efficacy of Cusatuzumab in Combination with Venetoclax and Azacitidine (CVA) in Patients with Previously Untreated Acute Myeloid Leukemia (AML) Who Are Not Eligible for Intensive Chemotherapy; An Open-Label, Multicenter, Phase 1b Study

Safety and Efficacy of Cusatuzumab in Combination with Venetoclax and Azacitidine (CVA) in Patients with Previously Untreated Acute Myeloid Leukemia (AML) Who Are Not Eligible for Intensive Chemotherapy; An Open-Label, Multicenter, Phase 1b Study

Preparation and characterization of a high-affinity monoclonal antibody against nerve growth factor

Nerve growth factor (NGF) is produced and released in injured tissues or chronic pain tissues caused by other diseases. Studies have shown that monoclonal antibodies targeting NGF have a good efficacy in the treatment of osteoarthritis (OA), low back pain and chronic pain, which may be a promising therapy. In this study, DNA sequences of NGF-his and NGF-hFc were synthesized using eukaryotic expression system and subcloned into pTT5 expression vector. After that, NGF proteins were expressed by transient expression in HEK293E cells. We immunized mice with NGF-hFc protein and fused mouse spleen cells to prepare hybridomas. NGF-His protein was used to screen out the hybridoma supernatant that could directly bind to NGF. Antibodies were purified from hybridioma supernatant. Futhermore, via surface plasmon resonance (SPR) screening, six anti-NGF mAbs were screened to block the binding of NGF and TrkA receptor in the treatment of chronic pain. Among them, 58F10G10H showed high affinity (KD = 1.03 × 10−9 M) and even better than that of positive control antibody Tanezumab (KD = 1.53 × 10−9 M). Moreover, the specific reactivity of 58F10G10H was demonstrated by TF-1 cell proliferation activity experiments, competitive binding Enzyme-linked immunosorbent assay (ELISA) and the arthritis animal models in mice, respectively. In conclusion, in this study, a method for the preparation of high-yield NGF-HFC and NGF-His proteins was designed, and a high-affinity monoclonal antibody against NGF with potential for basic research and clinical application was prepared.

Tissue biodistribution and tumor targeting of near-infrared labelled anti-CD38 antibody-drug conjugate in preclinical multiple myeloma.

Daratumumab (DARA) is an FDA-approved high-affinity monoclonal antibody targeting CD38 that has shown promising therapeutic efficacy in double refractory multiple myeloma (MM) patients. Despite the well-established clinical efficacy of DARA, not all heavily pretreated patients respond to single-agent DARA, and the majority of patients who initially respond eventually progress. Antibody-drug conjugates (ADCs) combine the highly targeted tumor antigen recognition of antibodies with the cell killing properties of chemotherapy for effective internalization and processing of the drug. In this study, we evaluated the anti-tumor efficacy of DARA conjugated to the maytansine derivative, mertansine (DM1), linked via a non-cleavable bifunctional linker. The ADC was labelled with the near-infrared (NIR) fluorophore IRDye800 (DARA-DM1-IR) to evaluate its stability, biodistribution and pharmacokinetics in vitro and in vivo. We demonstrated the conjugation of: 1) DM1 enhanced tumor-killing efficacy of the native DARA and 2) IRDye800 allowed for visualization of uptake and tumor targeting ability of the ADC. With the advent of other classes of immunoconjugates for use in MM, we reasoned that such imaging techniques can be utilized to evaluate other promising conjugates in preclinical MM models on a whole-body and cellular level.

Preclinical Molecular PET-CT Imaging Targeting CDCP1 in Colorectal Cancer.

Colorectal cancer (CRC) is the third most common malignancy in the world, with 22% of patients presenting with metastatic disease and a further 50% destined to develop metastasis. Molecular imaging uses antigen-specific ligands conjugated to radionuclides to detect and characterise primary cancer and metastases. Expression of the cell surface protein CDCP1 is increased in CRC, and here we sought to assess whether it is a suitable molecular imaging target for the detection of this cancer. CDCP1 expression was assessed in CRC cell lines and a patient-derived xenograft to identify models suitable for evaluation of radio-labelled 10D7, a CDCP1-targeted, high-affinity monoclonal antibody, for preclinical molecular imaging. Positron emission tomography-computed tomography was used to compare zirconium-89 (89Zr)-10D7 avidity to a nonspecific, isotype control 89Zr-labelled IgGκ1 antibody. The specificity of CDCP1-avidity was further confirmed using CDCP1 silencing and blocking models. Our data indicate high avidity and specificity for of 89Zr-10D7 in CDCP1 expressing tumors at. Significantly higher levels than normal organs and blood, with greatest tumor avidity observed at late imaging time points. Furthermore, relatively high avidity is detected in high CDCP1 expressing tumors, with reduced avidity where CDCP1 expression was knocked down or blocked. The study supports CDCP1 as a molecular imaging target for CRC in preclinical PET-CT models using the radioligand 89Zr-10D7.

Population pharmacokinetic and exposure-efficacy analysis of ixekizumab in paediatric patients with moderate-to-severe plaque psoriasis (IXORA-PEDS).

AimsIxekizumab is a high‐affinity monoclonal antibody that selectively targets interleukin‐17A used in the treatment of adult and paediatric patients with moderate‐to‐severe psoriasis. This analysis evaluated the pharmacokinetics (PK) of ixekizumab and the exposure–efficacy relationship in paediatric patients aged 6 to <18 years with psoriasis.MethodsPopulation PK and exposure–efficacy models were developed. The models used data from paediatric patients with psoriasis participating in the Phase 3 IXORA‐PEDS trial in which patients were dosed according to weight categories. The exposure–efficacy model is a Psoriasis Area and Severity Index (PASI) time course model using data up to Week 12, a co‐primary efficacy endpoint.ResultsA 2‐compartment population PK model describes the PK of ixekizumab in paediatric patients with the effect of body weight incorporated on clearance and volume terms using an allometric relationship. The weight category‐based dosing ensured that ixekizumab mean trough serum concentrations in paediatric patients with psoriasis (3.20–3.33 μg/mL) were within the range of concentrations observed in adult patients with psoriasis (mean [standard deviation]: 3.48 [2.16] μg/mL) administered an efficacious dosing regimen. The observed PASI response rates at Week 12 in paediatric patients (91.9/81.8/52.5% for PASI75/90/100) are well predicted by the final exposure–efficacy model and response rates are similar or higher than those achieved in adults (86.2/66.6/35.0% for PASI75/90/100).ConclusionThis analysis is the first to describe the PK and exposure–efficacy relationship of ixekizumab in paediatric patients with psoriasis. The analyses support the selection of the weight category‐based ixekizumab dosing regimens approved for use in paediatric patients with psoriasis.

Detection of Borrelia burgdorferi antigens in tissues and plasma during early infection in a mouse model

Borrelia burgdorferi is the causative agent of Lyme borreliosis, which is the most common tick-borne human disease in Europe and North America. Currently, the diagnosis of Lyme borreliosis is based on serological tests allowing indirect detection of anti-Borrelia antibodies produced by patients. Their main drawback is a lack of sensitivity in the early phase of disease and an incapacity to prove an active infection. Direct diagnostic tests are clearly needed. The objectives of this study were to produce tools allowing sensitive detection of potential circulating Borrelia antigens and to evaluate them in a mouse model. We focused on two potential early bacterial makers, the highly variable OspC protein and the conserved protein FlaB. High-affinity monoclonal antibodies were produced and used to establish various immunoassays and western blot detection. A very good limit of detection for OspC as low as 17 pg/mL of sample was achieved with SPIE-IA. In infected mice, we were able to measure OspC in plasma with a mean value of 10 ng/mL at 7 days post-inoculation. This result suggests that OspC could be a good blood marker for diagnosis of Lyme borreliosis and that the tools developed during this study could be very useful.

Tralokinumab in atopic dermatitis.

Atopic dermatitis (AD) is a common chronic inflammatory disease characterized by recurrent eczematous lesions and intense pruritus, and it can have marked negative impact on those affected. Pathophysiologically, AD is complex with genetic predisposition and environmental provocation being important contributors. Mechanistically these can promote epidermal barrier dysfunction, skin microbiome abnormalities and a skewed immune response which is predominantly type-2 immunity-based. Our increased understanding of the immunological processes involved highlight a key role for interleukin-13 (IL-13). This mini-review evaluates tralokinumab, a high-affinity monoclonal antibody that specifically binds to and inhibits IL-13. Based on dose-finding study results, tralokinumab 300mg every two weeks (Q2W) subcutaneously (SC) was investigated in three pivotal phaseIII clinical trials in adults with moderate-to-severe AD not adequately controlled on topical corticosteroids alone. Tralokinumab was significantly superior to placebo regarding the proportion of patients achieving IGA 0/1 and EASI-75 at week 16 (primary endpoints), as well as improving scores for worst daily pruritus, Dermatology Life Quality Index (DLQI), and Scoring Atopic Dermatitis (SCORAD) (secondary endpoints). The week 16 response was sustained during follow-up, and treatment with tralokinumab was found to be well-tolerated with an overall frequency and severity of adverse events comparable to placebo.

28171 Assessing long-term maintenance of efficacy with tralokinumab monotherapy in patients with moderate-to-severe atopic dermatitis: Combined results from two phase 3, randomized, double-blind, placebo-controlled trials (ECZTRA 1 and 2)

Tralokinumab is a fully human, high-affinity monoclonal antibody that specifically neutralizes interleukin-13, a key driver of skin inflammation and barrier dysfunction in atopic dermatitis (AD). In two pivotal trials, ECZTRA 1 and 2, tralokinumab monotherapy was superior to placebo for all primary and secondary endpoints at week 16 in adults with moderate-to-severe AD. High-responding patients achieving IGA 0/1 or EASI-75 at week 16 on tralokinumab q2w were rerandomized in the maintenance phase 2:2:1 to tralokinumab q2w, q4w, or placebo for 36 weeks.

Abstract 1841: IMGC936, an investigational ADAM9-targeting antibody drug conjugate, is active against patient-derived ADAM9-expressing xenograft models

Abstract Background: A disintegrin and metalloprotease (ADAM) 9 is a member of the ADAM family of transmembrane proteins. ADAM9 overexpression correlates with tumor progression, metastasis and poor prognosis in multiple cancers. IMGC936, an ADAM9-targeting antibody drug conjugate (ADC), is comprised of a high-affinity humanized monoclonal antibody site-specifically coupled to DM21 at a drug-antibody ratio of 2.0. DM21 is a next-generation linker-payload that combines a maytansinoid microtubule-disrupting payload with a stable tripeptide linker. IMGC936 is in a phase 1 dose escalation study evaluating safety and pharmacokinetics in cancer patients. The goals of this study were: 1) to explore ADAM9 expression in various solid tumors, and 2) to evaluate the activity of IMGC936 in clinically relevant patient-derived xenograft (PDX) models with ADAM9 expression similar to that observed in human tumors. Methods: ADAM9 expression was evaluated on primary patient and PDX formalin-fixed, paraffin-embedded samples with the anti-ADAM9 antibody D64B5 (Cell Signaling Technology). Expression of ADAM9 was quantitated by H-score to capture the frequency and intensity of staining. Mice bearing PDX tumors were dosed once at 8.6 mg/kg of IMGC936, which delivered 100 µg/kg of DM payload. Anti-tumor activity was defined by NCI standards: median tumor volume of treated mice over control mice (%) &amp;gt; 42% (inactive), ≤ 42% (active), and &amp;lt;10% (highly active). Results and Conclusions: ADAM9 was highly expressed in multiple tumor types. A majority of the tumor samples had medium to high levels of ADAM9 with 62% of non-small cell lung carcinoma (NSCLC), 65% of triple negative breast cancer (TNBC), 73% of gastric cancer, and 85% of pancreatic cancer samples having H- scores of 101 to 300. The remaining tumor samples had lower levels of ADAM9 expression (H-score 1 to 100) with only 1.2% of NSCLC samples being ADAM9-negative. Activity of IMGC936 was analyzed in PDX models derived from NSCLC, TNBC, pancreatic and gastric cancers. The range of ADAM9 expression was 27 to 226 by H-score, with 80% of the samples having H-scores above 101. A single dose of IMGC936 at 8.6 mg/kg was well tolerated. Across the tumor types tested, IMGC936 was active or highly active in 24 out of 35 models (69%) with complete regressions in 6 models (4 NSCLC, 2 TNBC). The 24 IMGC936 sensitive models had H-scores between 65 and 224. The 11 non-sensitive models had H-scores between 27 and 226. The data suggest that factors independent of ADAM9 expression contribute to model sensitivity toward IMGC936 and warrant further biomarker exploration. These studies demonstrate that ADAM9 is highly expressed in a large number of solid tumor indications and show that IMGC936 has activity against multiple solid tumor types with a wide range of clinically relevant ADAM9 levels. These data support the clinical evaluation of IMGC936 (NCT04622774). Citation Format: Olga Ab, Juniper A. Scribner, Kerstin Sinkevicius, Deryk Loo, Stuart W. Hicks, Krystal Watkins, Francine Z. Chen, Christopher Espelin, Marian Themeles, Ying Li, Chet Bohac, Patrick Zweidler McKay, Paul A. Moore, Callum M. Sloss, Ezio Bonvini, Eric H. Westin. IMGC936, an investigational ADAM9-targeting antibody drug conjugate, is active against patient-derived ADAM9-expressing xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1841.

Wnt5a Induces ROR1 to Bind and Activate Cortactin, Which Functions in Planar Cell Polarity and Migration of Chronic Lymphocytic Leukemia Cells

Studies have revealed a dynamic trafficking of chronic lymphocytic leukemia cells (CLL) between the blood and lymphoid compartments. Targeted therapies that impair chemokine-receptor signaling, such as ibrutinib, disrupt the capacity of leukemia cells to re-enter lymphoid compartments, precluding CLL cells from receiving growth/survival signals from the accessory cells within the lymphoid-tissue microenvironment, thereby causing attrition in the leukemia-cell population over time in a patient undergoing such targeted therapy. Studying the signaling pathways and intracellular proteins that influence/govern leukemia-cell migration may identify additional agents that can further disrupt CLL-cell trafficking and potentially enhance the therapeutic activity of drugs such as ibrutinib. Factoring in leukemia-cell migration may be the cytoskeletal protein cortactin (also called EMS1), which possesses a SH3 domain and a F-actin-binding domain, allowing it link cell-surface receptor-complexes to the actin cytoskeleton. Upon tyrosine phosphorylation, cortactin is activated, thereby facilitating RhoGTPase activation and polymerization and rearrangement of the actin cytoskeleton, especially the actin cortex around the cellular periphery, which is required for planar cell polarity, lamellipodia formation, invadopodia formation, cell migration, and endocytosis. We find cortactin abundantly is expressed in CLL B cells. Moreover, in freshly isolated CLL cells, we find that cortactin associates with ROR1, an evolutionarily conserved, oncoembryonic surface-protein expressed on many cancers, including CLL. Culture of CLL cells in serum-free medium results in the dissociation/inactivation of the ROR1-cortactin complex unless the leukemia cells are stimulated with the ligand for ROR1, namely Wnt5a, which, relative to the plasma of age-matched healthy adults, is present at high levels in the plasma of patients with CLL, including patients undergoing therapy with ibrutinib. Moreover, we find that Wnt5a induces ROR1 to complex with cortactin, which then undergoes tyrosine phosphorylation at Y421, recruit ARHGEF1, and activate RhoA, thereby enhancing/enabling leukemia-cell migration. These effects of Wnt5a on CLL cells could not be inhibited by ibrutinib, but could be blocked by cirmtuzumab, a humanized, high-affinity monoclonal antibody specific for ROR1 that is undergoing clinical evaluation in patients with CLL. We corroborated these findings using the CLL cell line MEC1, which also expresses cortactin, but does not express ROR1. MEC1 cells can be transduced to express ROR1 or various mutant forms of ROR1, allowing us to examine the structure-function relationships required for ROR1-cortactin complex formation. We confirmed that cortactin complexes with ROR1 and undergoes activation in response to Wnt5a in MEC1-ROR1 cells. We generated truncated forms of ROR1 and found the cytoplasmic proline-rich domain (PRD) of ROR1 was required for ROR1 to complex with cortactin upon stimulation with Wnt5a. We next introduced single amino-acid substitutions of proline (P) to alanine (A) in the ROR1-PRD at positions 784, 808, 826, 841, or 850 in potential SH3-binding sites. In contrast to wild-type ROR1, or other ROR1P==>A mutants, ROR1P(841)A had impaired capacity to recruit cortactin to ROR1 in response to Wnt5a. Moreover, Wnt5a could not induce cells expressing ROR1P(841)A to phosphorylate cortactin, and was not able to enhance MEC1-ROR1-cell F-actin polymerization. Collectively, these studies reveal that cortactin plays an important role in Wnt5a-enhanced CLL cell migration/F-actin polymerization via a ROR1-dependent signaling pathway that cannot be inhibited by ibrutinib or other inhibitors of BTK. DisclosuresKipps:AbbVie: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria; Oncternal: Research Funding; Gilead: Consultancy, Honoraria, Speakers Bureau; Pharmacyclics, LLC, an AbbVie Company: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding.

Dynamic Visual Representation of Clinical Efficacy of Ixekizumab in Psoriasis.

IntroductionIxekizumab, a high-affinity monoclonal antibody that selectively targets interleukin-17A, is an approved treatment for plaque psoriasis. This study aimed to use animated visualizations as a tool to simplify complex data from ixekizumab clinical trials.MethodsAnimated visualizations were developed to show outcomes from ixekizumab clinical trials and a Bayesian network meta-analysis of 11 approved biologics. The visualizations simultaneously highlighted both aggregate scores and the individual progression of patients over the course of treatment.ResultsThe animations provided key messages and information from the complex data in efficient and scientific ways that were also visually pleasing and simple to understand. The animations highlighted (1) rapid reduction in disease severity from baseline; (2) sustained efficacy of ixekizumab in the treatment of skin and nail psoriasis; (3) side-by-side comparisons of treatment efficacy and clinical improvement across trials; (4) simultaneous visual presentation of individual results with summary response over time; and (5) indirect comparison of relative treatment effects with other biologics based on Bayesian network meta-analysis.ConclusionThe rapid and sustained efficacy of ixekizumab in the treatment of psoriasis was demonstrated using multiple dynamic visualizations with different clinical endpoints. Animated visualizations provided a simpler and more comprehensive understanding of complex data than conventional static figures.Supplementary InformationThe online version contains supplementary material available at 10.1007/s13555-021-00548-2.

Folate Receptor Beta as a Direct and Indirect Target for Antibody-Based Cancer Immunotherapy.

Folate receptor beta (FRβ) is a folate binding receptor expressed on myeloid lineage hematopoietic cells. FRβ is commonly expressed at high levels on malignant blasts in patients with acute myeloid leukemia (AML), as well as on M2 polarized tumor-associated macrophages (TAMs) in the tumor microenvironment of many solid tumors. Therefore, FRβ is a potential target for both direct and indirect cancer therapy. We demonstrate that FRβ is expressed in both AML cell lines and patient-derived AML samples and that a high-affinity monoclonal antibody against FRβ (m909) has the ability to cause dose- and expression-dependent ADCC against these cells in vitro. Importantly, we find that administration of m909 has a significant impact on tumor growth in a humanized mouse model of AML. Surprisingly, m909 functions in vivo with and without the infusion of human NK cells as mediators of ADCC, suggesting potential involvement of mouse macrophages as effector cells. We also found that TAMs from primary ovarian ascites samples expressed appreciable levels of FRβ and that m909 has the ability to cause ADCC in these samples. These results indicate that the targeting of FRβ using m909 has the potential to limit the outgrowth of AML in vitro and in vivo. Additionally, m909 causes cytotoxicity to TAMs in the tumor microenvironment of ovarian cancer warranting further investigation of m909 and its derivatives as therapeutic agents in patients with FRβ-expressing cancers.

Anlotinib plus sintilimab in patients with recurrent advanced cervical cancer: A prospective, multicenter, single-arm, phase II clinical trial.

5524 Background: It is difficult for patients with recurrent advanced cervical cancer to obtain clinical benefits after the failure of standard chemotherapy. However, antiangiogenic therapy combined with immune checkpoint inhibitors have become a promising strategy for advanced cervical cancer. Anlotinib is a novel multi-target tyrosine kinase inhibitor, inhibiting tumour angiogenesis and proliferative signalling. Sintilimab is a fully humanized, high-affinity monoclonal antibody against programmed cell death-1 (PD-1). This phase II, single-arm study (ChiCTR1900023015) aims to evaluate the efficacy and safety of anlotinib plus sintilimab in patients with recurrent advanced cervical cancer. Methods: Patients who have received at least once platinum-based chemotherapy, histopathologically confirmed recurrent advanced cervical cancer (including squamous cell carcinoma, adenocarcinoma or adenosquamous carcinoma), more than 1% PD-L1 expression, ECOG 0-1 were considered eligible for enrollment. Anlotinib was taken orally (10mg mg qd, d1-14, 21 days per cycle), and sintilimab was administered intravenously (200mg once every 3 weeks). The treatment was continued until disease progression, death or intolerant toxicity. The primary endpoint was objective response rate (ORR) and the secondary endpoints included disease control rate (DCR), progression free survival (PFS), overall survival (OS) and safety. Results: Between September 2019 and February 2021, 42 patients with a median age of 52 years (range:47-58), FIGO histopathological stage I (11.9%), II (31.0%), III (33.3%), IV (9.5%) and undiagnosed (14.3%) were enrolled. 39 of these patients were evaluable. In the efficacy-evaluable population (n = 39), the therapeutic evaluation showed that 2 and 20 patients achieved complete response and partial response respectively, yielding the ORR of 56.4% (22/39, 95% CI:40.2 to 71.5). The DCR was 94.9% (37/39, 95% CI:80.7 to 98.8). The median response time was 1.6 months. The median PFS was not reached. The most common adverse events (AEs) were grade 1 or 2, which included hypothyroidism (33.3%), hypertension (23.8%), AST (21.4%), diarrhea (19.0%), ALT (16.7%), hand-foot syndrome(14.3%), hypertriglyceridemia (14.3%) and anemia (11.9%). The grade 3 AEs were hypertension (4.8%), hyponatremia (4.8%), immune pneumonia (2.4%) and immune myocarditis (2.4%). No higher AEs and treatment-related death were observed. Conclusions: Anlotinib plus sintilimab showed a promising efficacy with a favorable toxicity profile for patients with recurrent advanced cervical cancer. We will report more data in the future. Clinical trial information: ChiCTR1900023015.

P178 The impact of peripheral articular manifestations on the efficacy of ixekizumab in patients with radiographic axial spondyloarthritis

Abstract Background/Aims Axial spondyloarthritis (axSpA) is characterised by chronic inflammation of the axial skeleton and is commonly associated with peripheral articular manifestations (PA), which further increases disease burden. Ixekizumab (IXE), a high-affinity monoclonal antibody that selectively targets IL-17A, is approved for treating both nonradiographic and radiographic (r-) forms of axSpA. We explored the efficacy of IXE in patients with raxSpA with (PA+) and without (PA−) PA at baseline. Methods Week 16 data from two Phase 3, randomized, double-blind, placebo (PBO)-controlled trials, with patients who fulfilled the Assessment of Spondylo-Arthritis International Society (ASAS) criteria for r-axSpA and were either biologic-naïve (COAST-V, NCT02696785) or TNFi-experienced (COAST-W, NCT02696798), were analysed. Patients were randomly assigned 1:1:1:1 to subcutaneous PBO, 80 mg IXE every 4 (Q4W) or 2 (Q2W) weeks (80 mg or 160 mg starting dose, assigned 1:1) or 40 mg adalimumab (ADA) Q2W in COAST-V or 1:1:1 in COAST-W. We present a post-hoc analysis of data from COAST-V and -W and an integrated dataset, with patients categorized by baseline PA status. Presence of PA was defined as a 44-joint swollen joint count (SJC) ≥1 or a 46-joint tender joint count (TJC) ≥1. Efficacy and health-related quality of life (HRQoL) outcomes were analysed at week 16. Results Data from 656 patients (COAST-V: N = 146 PA−/194 PA+; COAST-W: N = 93 PA−/223 PA+) were analyzed. At baseline, PA+ patients had a higher mean age, more enthesitis (higher Spondyloarthritis Research Consortium of Canada [SPARCC] scores), and higher disease activity (higher CRP levels, Ankylosing Spondylitis Disease Activity Score [ASDAS] and BASDAI). ASAS40, ASDAS &amp;lt;2.1, and BASDAI50 responder rates (Table, section a) were all significantly higher for both IXE dose regimens versus PBO in both PA− and PA+ patients in the integrated dataset and numerically higher in the separate datasets. Furthermore, short-form-36 physical component summary (Table, section b) showed significantly greater improvements from baseline in both IXE groups vs PBO in the separate and integrated datasets, regardless of baseline PA status. Conclusion IXE led to significant improvements versus PBO in all efficacy and HRQoL endpoints at Week 16 in the integrated dataset, irrespective of PA manifestations at baseline. P178 Table 1:a) Responder rates at Week 16 for the intent-to-treat population, number (%)No peripheral articular manifestations at baselinePeripheral articular manifestations at baselinePBOADAQ2WIEQ4WIXEQ2WPBOADAQ2WIXEQ4WIXEQ2WIntegrated datasetaN=71N/AN=63N=66N=119N/AN=132N=115ASAS4011.5N/A29 (46.0)‡28 (42.4)‡18 (15.1)N/A39 (29.5)†45 (39.1)‡ASDAS &amp;lt;2.18 (11.3)N/A24 (38.1)‡23 (34.8)†8 (6.7)N/A31 (23.5)‡28 (24.3)‡BASDAI5010 (14.1)N/A21 (33.3)*26 (39.4)†15 (12.6)N/A38 (28.8)‡33 (28.7)†COAST-VbN=34N=39N=35N=38N=52N=51N=46N=45ASAS408 (23.5)17 (43.6)18 (51.4)*18 (47.4)8 (15.4)15 (29.4)21 (45.7)†25 (55.6)‡ASDAS &amp;lt;2.17 (20.6)18 (46.2)*14 (40.0)17 (44.7)*4 (7.7)16 (31.4)†21 (45.7)†18 (40.0)‡BASDAI507 (20.6)16 (41.0)13 (37.1)16 (42.1)8 (15.4)13 (25.5)21 (45.7)†20 (44.4)†COAST-WbN=37N/AN=28N=28N=67N/AN=86N=70ASAS403 (8.1)N/A11 (39.3)†10 (35.7)*10 (35.7)*N/A18 (20.9)20 (28.6)ASDAS &amp;lt;2.11 (2.7)N/A10 (35.7)‡6 (21.4)*4 (6.0)N/A10 (11.6)10 (14.3)BASDAI503 (8.1)N/A8 (28.6)*10 (35.7)*7 (10.4)N/A17 (19.8)13 (18.6)Non-responder imputation was used for missing responses.P-values vs. PBO (aCochran-Mantel-Haenszel test, adjusted by study;bFisher’s exact test):*P&amp;lt;0.05,†P&amp;lt;0.01,‡P&amp;lt;0.001.b) Change from baseline in SF-36 PCS scores at Week 16 for the intent-to-treat population, LSM (SE)No peripheral articular manifestations at baselinePeripheral articular manifestations at baselinePBOADAQ2WIXEQ4WIXEQ2WPBOADAQ2WIXEQ4WIXEQ2WIntegrated dataseta1.6 (0.90)N/A7.2 (0.95)‡6.1 (0.92)‡3.8 (0.69)N/A7.7 (0.67)‡8.2 (0.71)‡COAST-Vb2.3 (1.15)7.0 (1.10)†6.9 (1.13)†6.4 (1.07)*4.6 (1.00)6.9 (1.01)8.3 (1.07)*9.3 (1.10)†COAST-Wc0.64 (1.57)N/A7.1 (1.75)†5.4 (1.72)*1.9 (0.96)N/A6.4 (0.85)‡6.3 (0.97)‡P-values vs. PBO (MMRM analysis with treatment,a,b,c study,a baseline value,a,b,c visit,a,b,c baseline value-by-visit,a,b,c treatment-by-visit interaction,a,b,c geographic region,b,c baseline CRP status,b,c and number of prior TNF inhibitorsc included as fixed factors):*P &amp;lt; 0.05,†P &amp;lt; 0.01,‡P &amp;lt; 0.001.ADAQ2W = adalimumab every 2 weeks; ASAS = Assessment of Spondyloarthritis International Society criteria; ASDAS = ankylosing spondylitis disease activity score; BASDAI = Bath Ankylosing Spondylitis Disease Activity Index; CRP = c reactive protein; IXEQ2W = ixekizumab every 2 weeks; IXEQ4W = ixekizumab every 4 weeks; MMRM = mixed-effects model of repeated measures; N/A = not applicable; PBO = placebo; PCS = physical component summary; SE = standard error; SF-36 = short-form-36; TNF = tumour necrosis factor. Disclosure R. Sengupta: None. P. Bird: Grants/research support; Pfizer, Eli Lilly, Gilead, Abbvie, Novartis. D. Aletaha: Consultancies; Abbvie, Amgen, Celgene, Eli Lilly, Medac, Merck, Novartis, Pfizer, Roche, Sandoz, Sanofi, Genzyme. Grants/research support; Abbvie, Novartis, Roche. M. Magrey: Consultancies; Novartis, Pfizer, Eli Lilly, Abbvie, UCB. Other; Clinical trials with: Abbvie and UCB. Y. Kadono: Member of speakers’ bureau; Abbie, Lilly, Novartis. E. Soriano: Consultancies; Abbvie, Eli Lilly, GlaxoSmithKline, Novartis, Pfizer, Sandoz. Member of speakers’ bureau; Abbvie, Amber, Bristol-Myers Squibb, Eli Lilly, Novartis, Pfizer, Roche, UCB. Grants/research support; Abbvie, Eli Lilly, GlaxoSmithKline, Novartis, Pfizer, Sandoz, UCB. A.J. Bradley: Shareholder/stock ownership; Eli Lilly. Other; Employee of Eli Lilly and Company. S. Liu-Leage: Shareholder/stock ownership; Eli Lilly. Other; Employee of Eli Lilly and Company. Y. Schymura: Other; Employee of Eli Lilly and Company. M.J. Nissen: Corporate appointments; Abbvie, Celgene, Lilly, Novartis, Pfizer. Member of speakers’ bureau; Abbvie, Celgene, Novartis, Pfizer. Other; Member of EuroSpA (supported by Novartis).

P194 Response to ixekizumab by C-reactive protein level in patients with aadiographic axial spondyloarthritis: results from the COAST-V (biological-naïve) and COAST-W (TNF-α inhibitor-experienced) trials at 52 weeks

Abstract Background/Aims Tumor necrosis factor inhibitors (TNFis) are effective treatments for radiographic axial spondyloarthritis (r-axSpA), but may be less effective in patients (pts) without elevated C-reactive protein (CRP). This study evaluated the efficacy of ixekizumab (IXE), a high-affinity monoclonal antibody selectively targeting interleukin-17A, in pts with r-axSpA at 52 weeks (wks) with nonelevated (≤5mg/L) and elevated (&amp;gt;5mg/L) baseline (BL) CRP. Methods COAST-V (NCT02696785) and COAST-W (NCT02696798) were phase 3, multicenter, randomised, double-blind, PBO-controlled trials investigating efficacy of 80-mg IXE every 4 wks and every 2 wks in pts who met ASAS criteria for raxSpA, had radiographic sacroiliitis according to mNY criteria, and were biological diseasemodifying antirheumatic drug (bDMARDs)-naïve (COAST-V) or TNFiexperienced (COAST-W). Data from 157 COAST-V pts and 188 COAST-W pts treated with IXE from Wk0 to 52 were analyzed. Patients were stratified based on nonelevated (≤5mg/L) vs elevated (&amp;gt;5 mg/L) BL CRP. Additional analysis was done with BL CRP ≤10.0mg/L vs &amp;gt; 10.0mg/L. Efficacy was assessed by ASAS40, ≥50% improvement in Bath Ankylosing Spondylitis Disease Activity Index (BASDAI50), and change in Short Form 36 physical component summary (SF36 PCS) score. Missing data were imputed by nonresponder imputation for binary measures and modified BL observation carried forward for continuous measure. Week 16 data are presented for comparison. Results Of pts treated with IXE through Wk52, 34.4% had CRP ≤5.0mg/L, 65.6% had CRP &amp;gt;5.0mg/L, 61.8% had CRP ≤10.0mg/L, and 38.2% had CRP &amp;gt;10.0mg/L at BL in COASTV and 33.0% had CRP ≤5.0mg/L, 67.0% had CRP &amp;gt;5.0mg/L, 55.9% had CRP ≤10.0mg/L, and 44.1% had CRP &amp;gt;10.0mg/L at BL in COAST-W.At Wk16, the proportion of pts achieving ASAS40 in COAST-V was numerically higher with IXE in the ≤5mg/L group and significantly higher with IXE in the &amp;gt;5mg/L group vs PBO, as previously shown, and was significantly higher with IXE in the ≤10mg/L and &amp;gt;10mg/L groups vs PBO. Results were similar in COAST-W and significant in the &amp;gt;5mg/L and ≤10mg/L groups vs PBO. At Wk52, greater than 45% of COAST-V pts and greater than 35% of COAST-W pts treated with IXE achieved an ASAS40 response, greater than 40% of COAST-V pts and greater than 25% of COAST-W patients treated with IXE achieved a BASDAI50 response, and change from baseline in SF-36 PCS score in patients treated with IXE was greater than 5 points in both studies regardless of the BL CRP cutoffs evaluated. Conclusion A higher proportion of ASAS40 responders was observed in IXE treated arms versus PBO among bDMARD-naïve and TNFi-experienced pts with r-axSpA when the CRP cutoff of 10mg/L was evaluated, and the responses were consistent through Wk52. Furthermore, similar proportions of pts achieved BASDAI50 and SF-36 responses within each patient population regardless of the BL CRP cutoff evaluated. Disclosure J.D. Reveille: None. P. Rahman: None. D.M. Sandoval Calderon: Shareholder/stock ownership; D. Sandoval Calderon is a shareholder and employee of Eli Lilly and Company. T. Muram: Shareholder/stock ownership; T. Muram is a shareholder and employee of Eli Lilly and Company. A. Kronbergs: Shareholder/stock ownership; A. Kronbergs is a shareholder and employee of Eli Lilly and Company. R. Bolce: Shareholder/stock ownership; R.Bolce is a shareholder and employee of Eli Lilly and Company. V. Geneus: Shareholder/stock ownership; V. Geneus is a shareholder and employee of Eli Lilly and Company. T. Hunter: Shareholder/stock ownership; T. Hunter is a shareholder and employee of Eli Lilly and Company. S. Liu-Leage: Shareholder/stock ownership; S.L. League is a shareholder and employee of Eli Lilly and Company. M. Rudwaleit: None. J.A. Maldonado-Cocco: None. F. van den Bosch: None.