Recombinant Monoclonal Antibody Research Articles

Isolation and characterization of rabies monoclonal antibody charge variants.

Current postexposure prophylaxis of rabies includes vaccines, human rabies immunoglobulin (RIG), equine RIG, and recombinant monoclonal antibodies (mAb). In the manufacturing of rabies recombinant mAb, charge variants are the most common source of heterogeneity. Charge variants of rabies mAb were isolated by salt gradient cation exchange chromatography (CEX) to separate acidic and basic and main charge variants. Separated variants were further extensively characterized using orthogonal analytical techniques, which include secondary and tertiary structure determination by far and near ultraviolet circular dichroism spectroscopy. Charge and size heterogeneity were evaluated using CEX, isoelectric focusing (IEF), capillary-IEF, size exclusion chromatography, sodium dodecyl sulfate polyacrylamide gel electrophoresis, and western blotting. Antigen binding affinity was assessed by enzyme linked immuno-sorbent assay and rapid florescence foci inhibition test. Results from structural and physicochemical characterizations concluded that charge variants are formed due to posttranslational modification demonstrating that the charge heterogeneity, these charge variants did neither show any considerable physicochemical change nor affect its biological function. This study shows that charge variants are effective components of mAb and there is no need of deliberate removal, until biological functions of rabies mAb will get affected.

Abstract PO1-27-01: Comparing the Efficacy and Safety of TQB2440 versus the Reference Pertuzumab for the Treatment of HER2-Positive Early or Locally Advanced Breast Cancer: A Multicenter, Randomized, Double-Blind, Parallel-Controlled Phase 3 Trial

Abstract Background: Pertuzumab is a recombinant humanized monoclonal antibody targeting the extracellular dimerization domain II of HER2. On September 30, 2013, the FDA have granted accelerated approval of a pertuzumab regimen for neoadjuvant treatment of patients (pts) with high-risk, HER2-positive early stage breast cancer. TQB2440 is a pertuzumab (Perjeta®, Roche) biosimilar. This study aimed to compare the efficacy and safety of TQB2440 and the reference pertuzumab combined with trastuzumab and docetaxel in pts with HER2-positive early or locally advanced breast cancer. Methods: In this multicenter, randomized, double-blind phase 3 study, eligible pts were aged 18-75 with operable HER2-positive (IHC 3+ or ISH+) clinical stage II-IIIC breast cancer negative for ER/PR and had an ECOG PS of 0-1. The pts were randomly assigned to receive either TQB2440 or the reference pertuzumab (Perjeta®) (840 mg on day 1, cycle 1, followed by 420 mg on cycle 2-4, q3w) added to trastuzumab (8 mg/kg on day 1, cycle 1, followed by 6 mg/kg for cycles 2-4, q3w) + docetaxel (75 mg/m2, cycle 1-4, q3w). The pts then underwent surgery followed by adjuvant treatment with FEC regimens (fluorouracil 600 mg/m², epirubicin 90 mg/m², cyclophosphamide 600 mg/m², cycle 5-7, q3w), then TQB2440 (840 mg on day 1, cycle 8, followed by 420 mg on cycle 9-20, q3w) + trastuzumab (8 mg/kg on day 1, cycle 8, followed by 6 mg/kg for cycles 9-20, q3w) or until disease progression or intolerable toxicity. The primary endpoint was total pathologic complete response (tpCR) by independent review committee (IRC). Equivalence was established if the 90% confdence intervals (CIs) of the relative ratio [RR] within the interval of 0.76 to 1.32. Secondary endpoints included breast pathologic complete response (bpCR) by IRC, tpCR & bpCR by investigator, breast conserving surgery (BCS) rates, objective response rate (ORR), event-free survival (EFS), disease-free survival (DFS), OS and safety. Results: Between October 21, 2020, and November 21, 2022, 412 pts were enrolled (TQB2440 group, n=207; the reference pertuzumab group, n=205). Data cutoff was November 30, 2022. In the intention-to-treat (ITT) population, the tpCR by IRC of the TQB2440 group and the reference drug group were 58.94% and 58.05%, respectively. The RR was 1.02 (90% CI, 0.89, 1.16), which was within the predefined equivalence interval of 0.76 to 1.32. There was no statistically significant difference in the bpCR by IRC between the TQB2440 group and the reference pertuzumab group (67.63% [95% CI, 60.80%, 73.95%] vs. 63.90% [95% CI, 56.92%, 70.48%], P=0.4249). The BCS rate also comparable between the two groups with 13.04% (95% CI, 8.77%, 18.41%) vs. 13.17% (95% CI, 8.86%, 18.58%) (P=0.9695). Additionally, the results of the PP (per-protocol) population were similar to those of the ITT population. The incidence of treatment-related adverse events (TRAEs) and grade ≥3 TRAEs were similar between the TQB2440 group and the reference pertuzumab group with 79.71% vs. 75.98% and 49.28% vs. 41.67%, respectively. Conclusion: In patients with HER2-positive early or locally advanced breast cancer, TQB2440 demonstrated equivalent efficacy and similar safety to the reference pertuzumab. Clinical trial information: NCT05985187. Research Sponsor: Chia Tai Tianqing Pharmaceutical Group Co., Ltd Citation Format: Qingyuan Zhang, Shusen Wang, Nanlin Li, Qiao Cheng, Yu ren, Xuchen Cao, Jianjun Huang, Caigang Liu, Hongwei Yang, Limin Wei, Zhangjun Song, Huadong Zhao, Fangling Ning, Xiaojia Wang, Dehong Zou, Xiaohua Zeng, Jie Hao, Yunjiang Liu, Huijuan Wang, Nie Jianyun, Liang Li, Lina Liu, Tao Sun, Xiaobo Hu, Zhenhua Zhai, Huihua Xiong, Yuanqi Zhang, Enxiang Zhou, Jing Sun, Zhenhai Cai, Antai Zhang, Shui Wang, Junyang Mo, Qun Su, Xiuheng Qi, Guoren Zhou, Shuqun Zhang, Guozhong Cui, Wei Wang, Mingjiang Fan, Xinyu Qian, Xinhong Wu, Zhihong Wang, Jiuda Zhao, Yonghui Luo, Yanming Zhang, Fuguo Tian, Jiye Zhang, Dongning Chai, Qingshan Li. Comparing the Efficacy and Safety of TQB2440 versus the Reference Pertuzumab for the Treatment of HER2-Positive Early or Locally Advanced Breast Cancer: A Multicenter, Randomized, Double-Blind, Parallel-Controlled Phase 3 Trial [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-27-01.

A Phase I Clinical Study Comparing the Pharmacokinetics, Safety, and Immunogenicity of GB221 Injection and Trastuzumab (Herceptin®) in Healthy Chinese Adults.

GB221 is a recombinant humanized anti-HER2 monoclonal antibody. The purpose of this study was to evaluate the pharmacokinetic, safety, and immunogenicity of GB221 in healthy Chinese adults in comparisontotrastuzumab (Herceptin®). In this randomized, double-blind, parallel-group phase I clinical trial, 88 subjects were randomized 1:1 to receive a single intravenous infusion (90-100 min) of GB221 or trastuzumab (6 mg/kg). The primary pharmacokinetic parameters-maximum observed serum concentration (Cmax), area under the serum concentration-time curve from zero to the last quantifiable concentration at time t (AUC0-t), and area under the serum concentration-time curve from time zero to infinity (AUC0-∞)-of GB221 and trastuzumab were compared to establish whether the 90% confidence interval (CI) attained the 80-125% bioequivalence standard. Safety and immunogenicity were also evaluated. The GB221 group (n=43) and the trastuzumab group (n=44) showed similar pharmacokinetic characteristics. The geometric mean ratios (90% CI) of Cmax, AUC0-t, and AUC0-∞ between the two groups were 107.53% (102.25-113.07%), 108.31% (103.57-113.26%), and 108.34% (103.57-113.33%), respectively. The incidence of treatment-emergent adverse events (TEAEs) was 83.7% (36/43) of the subjects in the GB221 group and 95.5% (42/44) of the subjects in the trastuzumab group. No subjects withdrew from the trial due to TEAEs, and there were no occurrences of serious adverse events. All subjects tested negative for antidrug antibodies (ADA). GB221 demonstrated similar pharmacokinetics to trastuzumab and comparable safety and immunogenicity in healthy Chinese adults.

Association between skin toxicity and efficacy of necitumumab in squamous non-small-cell lung cancer: a pooled analysis of two randomized clinical trials-SQUIRE and JFCM

Efficacy of necitumumab [recombinant human monoclonal antibody that blocks the ligand binding epidermal growth factor receptor (EGFR)] in patients with squamous (SQ) non-small-cell lung cancer (NSCLC) has been confirmed in two randomized clinical trials (SQUIRE and JFCM). This study evaluated the association between efficacy and initial skin toxicity with necitumumab treatment by analyzing pooled data from two clinical trials (SQUIRE and JFCM). Data of 635 patients with SQ-NSCLC (intent-to-treat population) treated with necitumumab plus gemcitabine and cisplatin (N+ GC) were pooled from two clinical trials (SQUIRE and JFCM). The relationship between skin toxicities developed by the end of the second cycle and efficacy was evaluated. Efficacy endpoints included overall survival (OS), progression-free survival (PFS), and objective response rate (ORR). Univariate and multivariate analyses were carried out for these endpoints. OS and ORR were associated with skin toxicity, whereas PFS was not. Patients with grade ≥2 or grade 1 skin toxicity had significantly longer OS compared to patients without skin toxicity (grade 0) in the N+ GC group [median= 15.0 (grade ≥2); 12.7 (grade 1); 9.4 (grade 0) months; hazard ratio (HR)= 0.51 (grade ≥2 to grade 0); 95% confidence interval (CI) 0.40-0.64, P < 0.001 and HR= 0.64 (grade 1 to grade 0); 95% CI 0.52-0.80, P < 0.001]. In multivariate analysis, OS was significantly associated with skin toxicity. A significant association was found between necitumumab-induced skin toxicity and efficacy. These results are consistent with the previously reported association between other EGFR inhibitors-induced skin toxicity and efficacy.

Efficacy and safety of ongericimab given by prefilled syringe or autoinjector in primary hypercholesterolemia and mixed hyperlipidemia

Background and AimsLimited evidence exist regarding the association between ongericimab, a novel recombinant humanized anti-PCSK9 monoclonal antibody, and primary hypercholesterolemia and mixed dyslipidemia. This study aimed to evaluate the efficacy and safety of ongericimab administered by prefilled syringe (PFS) or autoinjector (AI) in Chinese patients with primary hypercholesterolemia and mixed dyslipidemia on stable optimized lipid-lowering therapy. Methods and ResultsA total of 255 patients on stable optimized lipid-lowering therapy were randomized in a 2:1:2:1 ratio to receive PFS for the subcutaneous injection of ongericimab 150 mg every 2 weeks (Q2W) or a matching placebo, or AI for the subcutaneous injection of ongericimab 150 mg Q2W or a matching placebo. The primary efficacy endpoint was the percent change in low-density lipoprotein cholesterol (LDL-C) levels from baseline to week 12. Safety was also evaluated. At week 12, the least squares mean percent changes were -72.7% (3.9%) for PFS and -71.1% (3.8%) for AI (all P<0.001) compared to respective matching placebo groups. Beneficial effects were also seen for all secondary lipid parameters, notably with robust reduction in Lp (a). Treatment-emergent adverse events (TEAEs) and serious AEs with ongericimab were reported in 46.2% and 2.4% of patients, compared to 44.2% and 3.5% with placebo. ConclusionIn Chinese patients with primary hypercholesterolemia and mixed dyslipidemia, a 12-week treatment regimen with ongericimab administered by PFS or AI significantly reduced LDL-C and other lipid parameters, proving to be safe and well tolerated. Patients experienced consistent effects from PFS or AI devices. Clinical trial registrationCTR20220027; January 11, 2022; http://www.chinadrugtrials.org.cn/index.html.

Omalizumab (Xolair) for food allergy.

Omalizumab (Xolair – Genentech), a recombinant anti-IgE monoclonal antibody FDA-approved for treatment of allergic asthma, chronic rhinosinusitis with nasal polyps, and chronic urticaria,1,2 has now also been approved for use in conjunction with food allergen avoidance to reduce IgE-mediated food allergic reactions caused by accidental exposure in patients ≥1 year old. Omalizumab is the first drug to be approved in the US to reduce allergic reactions to more than one food. Palforzia, an oral peanut allergen powder, was approved in 2020 to mitigate allergic reactions caused by accidental peanut exposure in patients with a confirmed peanut allergy.

Real-Life Study of Patient Preference for Dupilumab or Revision Surgery for Recurrent Chronic Rhinosinusitis with Nasal Polyps.

(1) Background: Chronic rhinosinusitis with nasal polyps (CRSwNP) has a high rate of recurrence in patients, despite therapy with local corticosteroids and functional endoscopic sinus surgery. Dupilumab, a recombinant monoclonal human IgG4 antibody directed against the IL-4 receptor α that inhibits both IL-4 and IL-13 signal transduction, is available for symptomatic therapy. Patient preference between repeated surgery and injection therapy with Dupilumab is not known. (2) Methods: Patients who had experienced at least one surgical intervention for nasal polyps and were treated with Dupilumab for at least 3 months completed a retrospective patient questionnaire. (3) Results: In a cohort of 75 previously operated CRSwNP patients, 91.5% preferred therapy with Dupilumab to repeated surgery for nasal polyps. Preference for Dupilumab in the subgroups of patients with concomitant Non-steroidal Anti-inflammatory Drugs Exacerbated Respiratory Disease (N-ERD) (n = 32), patients with concomitant asthma (n = 25), and patients without concomitant disease (n = 18) was 100%, 96%, and 72%, respectively. (4) Conclusions: Patient preference for Dupilumab over repeat surgery is strongest in previously operated CRSwNP patients with concomitant asthma or N-ERD, but remains very high in patients without concomitant disease.

Abstract 6330: A novel monoclonal antibody for the detection, enrichment and activation of cells expressing scFv-based chimeric antigen receptors

Abstract Introduction: Chimeric Antigen Receptor (CAR)-T cell therapy is a highly innovative form of immunotherapy that has proven to be successful in the treatment of B cell malignancies and multiple myeloma. As this treatment modality continues to evolve toward the targeting of novel tumor antigens and engineering of CAR constructs that enhance cell persistence, there is a need for reagents that can be leveraged to selectively interrogate multiple aspects of CAR-T cell biology. Here, we report on a novel and highly versatile recombinant rabbit monoclonal antibody raised against the Gly4Ser peptide linker, which is commonly integrated into single-chain variable fragment (scFv)-based CARs. This antibody can be leveraged for flow cytometry-based CAR detection, bead-based CAR-T cell enrichment, and selective activation of CAR transduced cells. Methods: The recombinant monoclonal antibody, E7O2V, was generated by immunizing rabbits with a synthetic peptide containing multiple repeats of the Gly4Ser core pentapeptide. E7O2V was directly conjugated to a panel of fluorophores and validated for specificity in a live cell flow cytometry assay using non-transduced and CAR-transduced primary human T cells. To assess its utility in cell enrichment using bead-based sorting, a biotinylated conjugate of E7O2V was used in combination with streptavidin releasable magnetic meads to immunoaffinity purify CAR transduced cells from a mixed population. Finally, a plate-bound antibody stimulation assay was leveraged to assess the ability of E7O2V to selectively activate CAR transduced cells. Results: Live cell flow cytometric analysis of CAR-transduced primary human T cells revealed that E7O2V could detect surface expressed CARs, independent of scFv specificity. No specific staining was observed on non-transduced cells. Furthermore, flow cytometric analysis of bead-free, purified cells revealed that biotinylated E7O2V could enrich CAR transduced cells to a high degree of purity and viability. E7O2V was found to selectively activate CAR transduced cells, but not non-transduced cells, as demonstrated by upregulation of cell surface activation markers, including CD69 and CD25. Conclusions: Exploiting the commonly used Gly4Ser linker of scFv-based CARs for antibody discovery, we identified a novel and highly versatile monoclonal antibody, E7O2V. This antibody binds to CARs of varying antigen specificity and can be leveraged in multiple assay formats to interrogate various attributes of CAR transduced cells, including CAR expression, CAR signaling, and transcriptome profiling enabled by bead-based enrichment. Citation Format: Amrik Singh, Sarah L'Heureux, Jeremy Fisher. A novel monoclonal antibody for the detection, enrichment and activation of cells expressing scFv-based chimeric antigen receptors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6330.

Abstract 2760: Novel GPCR recombinant monoclonal antibodies for cancer biology research

Abstract Recombinant technology is becoming the preferred production mechanism for creation of antibody reagents used in academic and clinical cancer biology research. Recombinant antibodies (rAbs) are superior to standard polyclonal and hybridoma-generated monoclonal reagents as they offer reproducible performance and consistent, scalable supply. A major focus of antibody manufacturers is to leverage rAb production strategies to generate antibodies against challenging targets for which there are few, if any, reliable antibodies. G-protein coupled receptors (GPCRs) are encoded by more than 820 human genes and represent the largest membrane protein superfamily in the genome. These seven-pass transmembrane domain receptors are involved in a multitude of physiological and pathophysiological processes related to various chronic diseases, including an array of malignancies. Perhaps 30-40% of all therapeutics are directed against GPCR activity, and ~350 of the non-olfactory GPCRs are thought to be potential candidates for drug development. However, for a slew of technical reasons, the development and validation of high-quality antibodies against GPCRs has proven difficult. In an effort to facilitate further research into GPCRs, GeneTex is combining its recombinant antibody production platform with enhanced validation techniques to make specific monoclonal antibodies against GPCRs with direct relevance to cancer and other diseases. GeneTex’s rAb production protocol employs a multi-parameter fluorescence-activated single cell sorting (FACS)-based methodology to identify and select antigen-specific IgG+ memory B cells from an immunized rabbit (Starkie et al., 2016). The heavy and light chain variable region genes from single cells are amplified, cloned, and co-expressed in mammalian cells to generate a monoclonal IgG. Application-specific testing is performed in conjunction with knockout/knockdown (KO/KD), differential expression comparison in cells and tissues, cell fraction enrichment (i.e., membrane extracts versus whole cell extracts), and other methodologies. Antibodies directed against extracellular GPCR domains are validated for specificity using GPCR arrays (CDI Labs, Baltimore, MD). This workflow has resulted in the production of specific antibodies for a number of GPCRs studied by cancer biologists. These include novel recombinant monoclonal antibodies to the dopamine D2 receptor (DRD2), retinoic acid-induced protein 3 (RAI3), CXC motif chemokine receptor 2 (CXCR2), CXCR4, and CXCR7. All of these antibodies were validated for at least two applications, while the specificity of three (i.e., RAI3, CXCR4, and CXCR7) is supported by KO/KD data. GPCR array data argues for the specificity of the other two antibodies (i.e., DRD2 and CXCR2). This same production/validation approach will be applied as GeneTex targets the remaining GPCRs. Citation Format: Alexander Ball, Yung Lin Hsieh, Chun Kai Huang, Sega Huang, Shang Ru Wu, Yue Yun Chang, Jiming Liu, Po Shin Peng, Chia Yi Lin. Novel GPCR recombinant monoclonal antibodies for cancer biology research [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2760.

Abstract 1883: A MUC1 antibody-conjugated with a tubulysin B analog, DXC005, demonstrates excellent synergistic effect in combination with gemcitabine for treatment of pancreatic tumors

Abstract Pancreatic cancer is a malignant tumor with high incidence and mortality. It is difficult to diagnose and detect in the early stage, with low surgical resection rate and high recurrence and metastasis rate after surgery. At present, the clinical therapeutic strategy is extremely limited. The first-line Standard of Care (SOC) for unresectable pancreatic cancer is chemotherapy. The preferred regimen includes gemcitabine combined with albumin paclitaxel or FOLFIRINOX (5-FU+Oxaliplatin+Irinotecan). Due to the limited long-term benefits and toxic side effects of chemotherapy, targeted therapy combined with chemotherapy has become a new therapeutic strategy. MUC1 is a highly glycosylated transmembrane mucin located on the lumen surface of epithelial cells. It can protect cells from extreme factors and plays an important role in tumor cell metabolism, apoptosis, epithelial-mesenchymal transition (EMT) and metastasis. Previous studies have confirmed that MUC1 is highly expressed in a variety of tumors, including pancreatic cancer, and is closely related to poor prognosis. DXC005 is a novel MUC1-targeting antibody-drug conjugate (ADC), generated by conjugating a Tubulysin B analogue to a recombinant humanized anti-Muc1 monoclonal antibody. DXC005 is the first MUC1-ADC IND in China, and is currently in phase I clinical trials. Preclinical studies have confirmed the efficacy of DXC005 monotherapy (2.5 mg/kg, 5 mg/kg, 10 mg/kg in one administration) in the HuPrime ® pancreatic cancer PDX model (PA1194). The tumor growth inhibition (TGI) was 42.53 %, 70.77 %, and 95.58 %, respectively. We want to further clarify whether DXC005 combined with chemotherapeutic drug Gemcitabine can ensure or even improve the efficacy while reducing the dosage of Gemcitabine. In PA1194 xenograft model, DXC005 (3 mg/kg or 6 mg/kg) in combination with Gemcitabine (10 mg/kg) showed significant anti-tumor efficacy with 58.77% and 93.17% TGIs, respectively. In contrast, the treatment with 10 mg/kg of Gemcitabine alone exhibited much less TGI. Furthermore, complete response (CR) was observed in some animals after treatment with DXC005 (6 mg/kg) plus Gemcitabine (10 mg/kg). All groups of treatment are tolerated well, no abnormal animal behavior and body weight loss were observed in the study. The above results concluded that DXC005 combined with Gemcitabine can achieve synergistic effect even with reduced dose of Gemcitabine, which will serve as a support for synergistic application in clinical studies. Citation Format: Xiaobo Cai, Min Cao, Huihui Guo, Qingliang Yang, Yongxiang Chen, Xiangfei Kong, Yong Du, Zhicang Ye, Zhixiang Guo, Lingli Zhang, Lu Bai, Junxiang Jia, Yunxia Zheng, Wei Zheng, Jun Zheng, Wenjun Li, Yuanyuan Huang, Zhongliang Fan, Binbin Chen, Yanlei Yang, Meng Dai, Robert Y. Zhao. A MUC1 antibody-conjugated with a tubulysin B analog, DXC005, demonstrates excellent synergistic effect in combination with gemcitabine for treatment of pancreatic tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1883.

Tocilizumab in chronic active antibody-mediated rejection: rationale and protocol of an in-progress randomized controlled open-label multi-center trial (INTERCEPT study)

BackgroundChronic active antibody-mediated rejection (caAMR) in kidney transplants is associated with irreversible tissue damage and a leading cause of graft loss in the long-term. However, the treatment for caAMR remains a challenge to date. Recently, tocilizumab, a recombinant humanized monoclonal antibody directed against the human interleukin-6 (IL-6) receptor, has shown promise in the treatment of caAMR. However, it has not been systematically investigated so far underscoring the need for randomized controlled studies in this area.MethodsThe INTERCEPT study is an investigator-driven randomized controlled open-label multi-center trial in kidney transplant recipients to assess the efficacy of tocilizumab in the treatment of biopsy-proven caAMR. A total of 50 recipients with biopsy-proven caAMR at least 12 months after transplantation will be randomized to receive either tocilizumab (n = 25) added to our standard of care (SOC) maintenance treatment or SOC alone (n = 25) for a period of 24 months. Patients will be followed for an additional 12 months after cessation of study medication. After the inclusion biopsies at baseline, protocol kidney graft biopsies will be performed at 12 and 24 months. The sample size calculation assumed a difference of 5 ml/year in slope of estimated glomerular filtration rate (eGFR) between the two groups for 80% power at an alpha of 0.05.The primary endpoint is the slope of eGFR at 24 months after start of treatment. The secondary endpoints include assessment of the following at 12, 24, and 36 months: composite risk score iBox, safety, evolution and characteristics of donor-specific antibodies (DSA), graft histology, proteinuria, kidney function assessed by measured GFR (mGFR), patient- and death-censored graft survival, and patient-reported outcomes that include transplant-specific well-being, adherence to immunosuppressive medications and perceived threat of the risk of graft rejection.DiscussionNo effective treatment exists for caAMR at present. Based on the hypothesis that inhibition of IL-6 receptor by tocilizumab will reduce antibody production and reduce antibody-mediated damage, our randomized trial has a potential to provide evidence for a novel treatment strategy for caAMR, therewith slowing the decline in graft function in the long-term.Trial registrationClinicalTrials.gov NCT04561986. Registered on September 24, 2020

Abstract 6581: DX126-262 combined with chemotherapy (Cisplatin and 5-Fu) demonstrates promising antitumor efficacy in HER2-positive gastric cancer

Abstract Gastric cancer is a common malignant tumor of the digestive system, and its morbidity and mortality rank among the top five in the world. Although significant progress has been made in cytotoxic chemotherapy, immunotherapy and targeted therapy in recent years, the long-term prognosis of patients with advanced or metastatic gastric cancer is still poor. Trastuzumab combined with chemotherapy is the first-line standard of care (SOC) for HER2-positive advanced or metastatic gastric cancer. Two ADC drugs targeting HER2, DS-8201 and RC-48, have also been approved for the treatment of gastric cancer. However, their limited clinical benefits and occurrence of adverse events still present a significant challenge. Therefore, minimizing the off-target toxicity and collateral damage while achieving significant chemotherapy efficacy has become the focus of gastric cancer treatment. DX126-262 is a novel HER2-targeting antibody-drug conjugate (ADC), generated by conjugating a Tubulysin B analogue to a recombinant humanized anti-HER2 monoclonal antibody through a linker containing branched hydrophilic polyethylene glycol, using thiol-maleimide chemistry to achieve drug antibody ratio (DAR) of 3.8. Previous studies have confirmed that DX126-262 monotherapy showed excellent efficacy in HER2-positive gastric cancer NCI-N87 cells in vitro and in vivo. And the efficacy has also been demonstrated in clinical trials (now in phase II, CTR20211871). Based on above results, we intended to further explore whether combination therapy of DX126-262 with Cisplatin and 5-FU can improve the anti-tumor efficacy compared with SOC (Herceptin plus Cisplatin and 5-FU) or DS-8201 monotherapy. The triple-drug combination therapy demonstrated much better therapeutic efficacy than single drug (DX126-262, Cisplatin, 5-FU, Herceptin, or DS-8201) or double-drug combination (Cisplatin plus 5-FU) or SOC (Herceptin plus Cisplatin and 5-FU) on human gastric cancer cells in vitro and in vivo. Meanwhile, triple-drug combination therapy did not exhibit superimposed toxicity, judging by the body weight of mice and hemal biochemistry assays. These results suggested that DX126-262 plus Cisplatin and 5-FU might be a promising strategy for treatment of HER2-positive advanced or metastatic gastric cancer in clinic. Citation Format: Xiaobo Cai, Min Cao, Huihui Guo, Qingliang Yang, Xiangfei Kong, Yong Du, Zhicang Ye, Zhixiang Guo, Lingli Zhang, Lu Bai, Junxiang Jia, Yunxia Zheng, Yongxiang Chen, Miaomiao Chen, Wei Zheng, Jun Zheng, Wenjun Li, Yuanyuan Huang, Mengmeng Liu, Zhongliang Fan, Hangbo Ye, Yifang Xu, Binbin Chen, Meng Dai, Robert Y. Zhao. DX126-262 combined with chemotherapy (Cisplatin and 5-Fu) demonstrates promising antitumor efficacy in HER2-positive gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6581.

Reverse-engineering the anti-MUC1 antibody 139H2 by mass spectrometry-based de novo sequencing.

Mucin 1 (MUC1) is a transmembrane mucin expressed at the apical surface of epithelial cells at mucosal surfaces. MUC1 has a barrier function against bacterial invasion and is well known for its aberrant expression and glycosylation in adenocarcinomas. The MUC1 extracellular domain contains a variable number of tandem repeats (VNTR) of 20 amino acids, which are heavily O-linked glycosylated. Monoclonal antibodies against the MUC1 VNTR are powerful research tools with applications in the diagnosis and treatment of MUC1-expressing cancers. Here, we report direct mass spectrometry-based sequencing of anti-MUC1 hybridoma-derived 139H2 IgG, enabling reverse-engineering of the functional recombinant monoclonal antibody. The crystal structure of the 139H2 Fab fragment in complex with the MUC1 epitope was solved, revealing the molecular basis of 139H2 binding specificity to MUC1 and its tolerance to O-glycosylation of the VNTR. The available sequence of 139H2 will allow further development of MUC1-related diagnostic, targeting, and treatment strategies.

Adalimumab as a cause of kidney injury in patients with Crohn's disease.

Adalimumab (ADM) is a recombinant human monoclonal antibody (anti-TNFα) used to treat inflammatory bowel diseases. It can cause kidney injury (KI). We describe two pediatric patients with Crohn's disease (CD) in whom ADM therapy was associated with kidney injury (KI). The drug was discontinued in both cases. For the first patient, changes were irreversible despite intensive glucocorticosteroid (GCS) therapy. For the second patient, discontinuation of ADM led to an improvement in kidney function. Due to the risk of KI in patients undergoing ADM therapy, careful assessment of kidney function and early specialist referral are required. Timely withdrawal of ADM can significantly reduce kidney damage, but in some cases, the kidney damage can be irreversible.

Budget impact and cost-utility analysis of prophylactic emicizumab versus on-demand bypassing agents for adolescent severe haemophilia A patients with inhibitors in India

IntroductionSevere Haemophilia A patients with inhibitors are currently being treated with bypassing agents like activated prothrombin complex concentrates (aPCC) and recombinant factor VIIa. Emicizumab is a recombinant humanized monoclonal antibody, introduced to reduce the bleeding events, improve treatment adherence, and quality of life. However, cost-effectiveness and long-term sustainability of the intervention is not studied in a low middle income setting like India. AimThe primary objective of this study was to evaluate the cost-utility of Emicizumab compared to traditional bypassing agents in the treatment of severe haemophilia A patients with inhibitors in India. Secondary objective was to analyze the budgetary impact of introducing Emicizumab for this patient population from the perspective of public health system in India. MethodsMarkov model was created to compare the prophylactic emicizumab therapy against bypassing agents for a hypothetical cohort of 10-year-old adolescents in India. The time horizon was 10 years and model built based on health system perspective. Cost utility was expressed as costs per quality-adjusted life-years (QALYs) gained. All costs were expressed as 2021 US dollars. Probabilistic sensitivity analysis was performed to check the robustness of the estimates. ResultsProphylactic emicizumab was a cost saving intervention with negative Incremental Cost Utility Ratio (ICUR) against recombinant factor VIIa of −853,573 USD (INR -63,109,773), and negative ICUR of −211,675 USD (INR -15,650,403) against APCC. The estimated total budget for treating all the severe Haemophilia A patients with inhibitors in India was USD 59,042,000 (INR 4,365,329,312) for 10 years’ time horizon (per patient cost of USD 295,210 [INR 21,826,646.56]). ConclusionProphylactic emicizumab therapy is a cost saving intervention when compared to both the bypassing agents as it is less costly and more effective for severe Haemophilia A patients with inhibitors in India.

Impact of Excipient Extraction and Buffer Exchange on Recombinant Monoclonal Antibody Stability.

The foundation of a biosimilar manufacturer's regulatory filing is the demonstration of analytical and functional similarity between the biosimilar product and the pertinent originator product. The excipients in the formulation may interfere with characterization using typical analytical and functional techniques during this biosimilarity exercise. Consequently, the producers of biosimilar products resort to buffer exchange to isolate the biotherapeutic protein from the drug product formulation. However, the impact that this isolation has on the product stability is not completely known. This study aims to elucidate the extent to which mAb isolation via ultrafiltration-diafiltration-based buffer exchange impacts mAb stability. It has been demonstrated that repeated extraction cycles do result in significant changes in higher-order structure (red-shift of 5.0 nm in fluorescence maxima of buffer exchanged samples) of the mAb and also an increase in formation of basic variants from 19.1 to 26.7% and from 32.3 to 36.9% in extracted innovator and biosimilar Tmab samples, respectively. It was also observed that under certain conditions of tertiary structure disruptions, Tmab could be restabilized depending on formulation composition. Thus, mAb isolation through extraction with buffer exchange impacts the product stability. Based on the observations reported in this paper, we recommend that biosimilar manufacturers take into consideration these effects of excipients on protein stability when performing biosimilarity assessments.

Biological therapy of severe bronchial asthma in a child

Currently, bronchial asthma is a global health problem, due to its high prevalence, economic component, as well as a violation of the social adaptation of children suffering from this disease. This article is devoted to a detailed analysis of the clinical case of a patient born in 2009 with an established diagnosis of Bronchial asthma, atopic form, severe persistent partially controlled course, which initiated therapy with a genetically engineered biological preparation of a humanized monoclonal antibody against IgE – omalizumab. However, in subsequent years, the patient’s condition was unstable, severe seizures were repeated, she was repeatedly hospitalized in the department where infusion therapy was performed and basic therapy was reviewed, dose adjustments and administration regimens of monoclonal antibodies to IgE (omalizumab) were carried out. A retrospective analysis of the results of objective, instrumental and laboratory examinations of the patient was also carried out over the past three years, when the child received the genetically engineered drug omalizumab without interruption. Taking into account the severe course of AD, the lack of control over the disease against the background of basic therapy with combined drugs in combination with therapy with monoclonal antibodies to IgE (omalizumab), it was decided to correct treatment and initiate therapy with a genetically engineered drug, recombinant human monoclonal antibody IgG4 (dupilumab). This clinical example once again shows how personalized the approach should be when prescribing therapy to patients with severe asthma, and also dictates the need to develop new diagnostic methods and management tactics for patients with uncontrolled forms of this disease.

Dual CTLA-4 and PD-1 checkpoint blockade using CS1002 and CS1003 (nofazinlimab) in patients with advanced solid tumors: A first-in-human, dose-escalation, and dose-expansion study.

This study investigated the safety and efficacy of an anti-CTLA-4 monoclonal antibody (CS1002) as monotherapy and in combination with an anti-PD-1 monoclonal antibody (CS1003) in patients with advanced/metastatic solid tumors. The phase 1 study involved phase 1a monotherapy dose-escalation (part 1) and phase 1b combination therapy dose escalation (part 2) and expansion (part 3). Various dosing schedules of CS1002 (0.3, 1, or 3mg/kg every 3 weeks, or 3mg/kg every 9 weeks) were evaluated with 200mg CS1003 every 3 weeks in part 3. Parts 1, 2, and 3 included a total of 13, 18, and 61 patients, respectively. No dose-limiting toxicities or maximum tolerated doses were observed. Treatment-related adverse events (TRAEs) were reported in 30.8%, 83.3%, and 75.0% of patients in parts 1, 2, and 3, respectively. Grade ≥3 TRAEs were experienced by 15.4%, 50.0%, and 18.3% of patients in each part. Of 61 patients evaluable for efficacy, 23 (37.7%) achieved objective responses in multiple tumor types. Higher objective response rates were observed with conventional and high-dose CS1002 regimens (1mg/kg every 3 weeks or 3mg/kg every 9 weeks) compared to low-dose CS1002 (0.3mg/kg every 3 weeks) in microsatellite instability-high/mismatch repair-deficient tumors, melanoma, and hepatocellular carcinoma (50.0% vs. 58.8%, 14.3% vs. 42.9%, and 0% vs. 16.7%). CS1002, as monotherapy, and in combination with CS1003, had a manageable safety profile across a broad dosing range. Promising antitumor activities were observed in patients with immune oncology (IO)-naive and IO-refractory tumors across CS1002 dose levels when combined with CS1003, supporting further evaluation of this treatment combination for solid tumors. CS1002 is a human immunoglobulin (Ig) G1 monoclonal antibody that blocks the interaction of CTLA-4 with its ligands and increases T-cell activation/proliferation. CS1003, now named nofazinlimab, is a humanized, recombinant IgG4 monoclonal antibody that blocks the interaction between human PD-1 and its ligands. In this original article, we determined the safety profile of CS1002 as monotherapy and in combination with CS1003. Furthermore, we explored the antitumor activity of the combination in anti-programmed cell death protein (ligand)-1 (PD-[L]1)-naive microsatellite instability-high/mismatch repair-deficient (MSI-H/dMMR) pan tumors, and anti-PD-(L)1-refractory melanoma and hepatocellular carcinoma (HCC). CS1002 in combination with CS1003 had manageable safety profile across a broad dosing range and showed promising antitumor activities across CS1002 dose levels when combined with CS1003. This supports further assessment of CS1002 in combination with CS1003 for the treatment of solid tumors.

Development of Plant-Derived Bispecific Monoclonal Antibody Targeting PD-L1 and CTLA-4 against Mouse Colorectal Cancer.

Checkpoint blockade immunotherapy has revolutionized cancer treatment, with monoclonal antibodies targeting immune checkpoints, yielding promising clinical benefits. However, with the advent of resistance to immune checkpoint inhibitor treatment in clinical trials, developing next-generation antibodies with potentially increased efficacy is critical. Here, we aimed to generate a recombinant bispecific monoclonal antibody for dual inhibition of programmed cell death protein 1/programmed cell death ligand 1 and cytotoxic T-lymphocyte-associated protein 4 axes. The plant system was used as an alternative platform for bispecific monoclonal antibody production. Dual variable domain immunoglobulin atezolizumab × 2C8 is a plant-derived bispecific monoclonal antibody that combines both programmed cell death ligand 1 and cytotoxic T-lymphocyte-associated protein 4 blockade into a single molecule. Dual variable domain immunoglobulin atezolizumab × 2C8 was transiently expressed in Nicotiana benthamiana and the expression level was determined to be the highest after 4 days of infiltration. The size and assembly of the purified bispecific monoclonal antibody were determined, and its function was investigated in vitro and in vivo. The molecular structures of plant-produced dual variable domain immunoglobulin atezolizumab × 2C8 are as expected, and it was mostly present as a monomer. The plant-produced dual variable domain immunoglobulin atezolizumab × 2C8 showed in vitro binding to programmed cell death ligand 1 and cytotoxic T-lymphocyte-associated protein 4 proteins. The antitumor activity of plant-produced bispecific monoclonal antibody was tested in vivo by treating humanized Balb/c mice bearing a CT26 colorectal tumor. Plant-produced dual variable domain immunoglobulin atezolizumab × 2C8 significantly inhibited tumor growth by reducing tumor volume and weight. Body weight changes indicated that the plant-produced bispecific monoclonal antibody was safe and tolerable. Overall, this proof of concept study demonstrated the viability of plants to produce functional plant-based bispecific immunotherapy.

[Short-term substitution of calcineurin inhibitors (CNI) with recombinant humanized anti-CD25 monoclonal antibody (Basiliximab) as aGVHD prophylaxis in CNI intolerant patients after allogeneic hematopoietic stem cell transplantation].

Objectives: To investigate the efficacy of short-term substitution of recombinant humanized anti-CD25 monoclonal antibody (Basiliximab) as acute GVHD (aGVHD) prophylaxis in calcineurin inhibitors (CNI) intolerant patients following allogeneic hematopoietic stem cell transplantation (allo-HSCT) . Methods: This study included 17 patients with refractory malignant hematological disorders who underwent salvage allo-HSCT at the Bone Marrow Transplantation Department of Shanghai Zhaxin Traditional Chinese and Western Medicine Hospital from August 2021 to August 2022 and were treated with Baliximab to prevent aGVHD due to severe adverse reactions to CNI. There were seven men and ten women, with a median age of 43 years (18-67). Following the discontinuation of CNI, Basiliximab was administered at a dose of 1 mg/kg once weekly until CNI or mTOR inhibitors were resumed. Results: Basiliximab was started at an average of 5 (1-32) days after HSCT. The median duration of substitution was 20 (7-120) days. All had neutrophil engraftment within a median of 12 (10-17) days. Thirteen patients had platelet engraftment after a median of 13 (11-20) days. Four patients did not develop stable platelet engraftment. Eight patients (47.1% ) developed Grade Ⅱ-Ⅳ aGVHD, while four (23.6% ) developed Grade Ⅲ/Ⅳ aGVHD. Only one patient died from aGVHD. Before the end of the followup period, seven of 17 patients died. The longest followup period of the survivors was 347 days, and the median survival rate was not met. The overall survival (OS) rate at six months was 62.6%. Among the 17 patients, 13 (76.4% ) experienced cytomegalovirus reactivation, 7 (41.2% ) experienced EB virus activation, and no cytomegalovirus disease was observed. Conclusions: When CNI intolerance occurs during allo-HSCT, short-term replacement with Baliximab can be used as an alternative to prevent aGVHD.