Mouse-human Chimeric Antibody Research Articles

Epidermal Growth Factor Receptor Targeting in Colorectal Carcinoma: Antibodies and Patient-Derived Organoids as a Smart Model to Study Therapy Resistance.

Colorectal cancer (CRC) is the second leading cause of cancer-related death worldwide. Therefore, the need for new therapeutic strategies is still a challenge. Surgery and chemotherapy represent the first-line interventions; nevertheless, the prognosis for metastatic CRC (mCRC) patients remains unacceptable. An important step towards targeted therapy came from the inhibition of the epidermal growth factor receptor (EGFR) pathway, by the anti-EGFR antibody, Cetuximab, or by specific tyrosine kinase inhibitors (TKI). Cetuximab, a mouse-human chimeric monoclonal antibody (mAb), binds to the extracellular domain of EGFR thus impairing EGFR-mediated signaling and reducing cell proliferation. TKI can affect the EGFR biochemical pathway at different steps along the signaling cascade. Apart from Cetuximab, other anti-EGFR mAbs have been developed, such as Panitumumab. Both antibodies have been approved for the treatment of KRAS-NRAS wild type mCRC, alone or in combination with chemotherapy. These antibodies display strong differences in activating the host immune system against CRC, due to their different immunoglobulin isotypes. Although anti-EGFR antibodies are efficient, drug resistance occurs with high frequency. Resistant tumor cell populations can either already be present before therapy or develop later by biochemical adaptations or new genomic mutations in the EGFR pathway. Numerous efforts have been made to improve the efficacy of the anti-EGFR mAbs or to find new agents that are able to block downstream EGFR signaling cascade molecules. Indeed, we examined the importance of analyzing the anti-EGFR antibody-drug conjugates (ADC) developed to overcome resistance and/or stimulate the tumor host's immunity against CRC growth. Also, patient-derived CRC organoid cultures represent a useful and feasible in vitro model to study tumor behavior and therapy response. Organoids can reflect tumor genetic heterogeneity found in the tissue of origin, representing a unique tool for personalized medicine. Thus, CRC-derived organoid cultures are a smart model for studying the tumor microenvironment and for the preclinical assay of anti-EGFR drugs.

Advances of monoclonal antibodies and analysis of marketed antibody drugs

In recent years, there has been a frequent occurrence of various epidemics worldwide such as COVID-19, monkeypox, influenza, and others additionally, there has been an increase in the number of new patients diagnosed with various types of tumors. Traditional drugs have limited effectiveness against emerging infectious diseases, tumors, and autoimmune diseases. However, with the emergence of hybridoma technology, monoclonal antibodies have achieved extensive applications and antibody drugs are playing an important role in modern medicine. Monoclonal antibodies have undergone various development stages, starting from mouse-derived antibodies to human-mouse chimeric antibodies, humanized antibodies, and ultimately human antibodies. Throughout this process, their immunogenicity has gradually decreased, while their safety for human use steadily increased. Fully human antibodies are currently the safest form of antibody, because their sequences all come from human sources and they do not induce human anti-murine antibody reactions. With the advance of genetic engineering technology, flow cytometry coupled to single B cell gene amplification technology has made it easier to construct and screen for fully human monoclonal antibodies. The development of antibody drugs has provided new opportunities, and the market for monoclonal antibody drugs will further expand. This article reviews the research progress of monoclonal antibodies and presents information on the 163 monoclonal antibody drugs approved by the United States Food and Drug Administration (FDA) as of Oct 1st, 2023. The aim is to offer new insights for the development and production of monoclonal antibodies in China.

Abstract LB307: The role of tumor microenvironment derived growth factors in pediatric brain tumors

Abstract Introduction: Children with H3K27-altered diffuse midline gliomas (DMG-H3K27a), have a 5-year survival rate of only 2% following diagnosis. While in-depth genomic characterization has been performed in DMG-H3K27a, very few druggable targets have been identified. A common feature of DMG-H3K27a is infiltration of microglia, macrophages, other myeloid cells, collectively referred to as GAMs. Cellular crosstalk between non-tumor and tumor cells in the tumor microenvironment (TME) can both promote and or inhibit tumor growth, thus representing an opportunity in the pursuit of novel therapeutics. We have recently determined that DMG-H3K27a tumor cells stimulate microglial secretion of pro-tumor growth factors. Hypothesis: Microglial-derived growth factors activate receptor tyrosine kinases (RTKs) via paracrine signaling in DMG-H3K27a. Methods: We used scRNA-seq and RNA-seq datasets and, in parallel, in vitro studies, such as co-culture studies, ELISA assays, western blotting, and growth assays to test our hypothesis. In addition, we are developing a mouse-human chimeric neutralizing antibodies of our top targets for in vivo studies. Preliminary Results: Microglial cells secrete growth factors into the TME, which promotes DMG-H3K27a cell proliferation and growth. Targeting growth factors with neutralizing antibodies attenuates glioma cell proliferation in co-culture studies. Conclusion: This research uncovers an important signaling network between GAMs and DMG-H3K27a that can be therapeutically targeted. Citation Format: Andrea Cruz, Brenden Johnson, Taylor Gatesman, Mathew Halbert, Suchet Taori, Baoli Hu, Gary Kohanbash, Ian F. Pollack, Sameer Agnihotri. The role of tumor microenvironment derived growth factors in pediatric brain tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB307.

Preparation of HSV-IgM human-mouse chimeric antibody and development of stable recombinant cell line

In order to achieve large-scale production of HSV-IgM (HSV1, HSV2) human-mouse chimeric antibody in vitro, the gene sequence of the corresponding hybridoma cell was harvested by RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) technique to clone the chimeric antibody into eukaryotic expression vectors, and express the target proteins in CHO-S cells. At the same time, the screening process of stable cell lines was optimized, and the pressure conditions of pool construction stage and monoclonal screening stage were explored. Finally, the target protein was purified by protein L affinity purification method and the biological activity was detected. The recombinant IgM antibodies, HSV1 and HSV2, weighted at 899 kDa and 909 kDa respectively, were prepared. The optimal screening pressure was 20P200M (the first phase of pressure) and 50P1000M (the second phase of pressure). The final titer for the monoclonal expression of HSV1-IgM and HSV2-IgM was 1 620 mg/L and 623 mg/L, respectively. This study may facilitate the development of quality control products of HSV1 and HSV2 IgM series recombinant antibodies as well as efficient expression of IgM subtype antibodies in vitro.

Dosimetry of a Novel 111Indium-Labeled Anti-P-Cadherin Monoclonal Antibody (FF-21101) in Non-Human Primates.

P-cadherin is associated with a wide range of tumor types, making it an attractive therapeutic target. FF-21101 is a human-mouse chimeric monoclonal antibody (mAb) directed against human P-cadherin, which has been radioconjugated with indium-111 (111In) utilizing a DOTA chelator. We investigated the biodistribution of FF-21101(111In) in cynomolgus macaques and extrapolated the results to estimate internal radiation doses of 111In- and yttrium-90 (90Y)-FF-21101 for targeted radioimmunotherapy in humans. Whole-body planar and SPECT imaging were performed at 0, 2, 24, 48, 72, 96, and 120 h post-injection, using a dual-head gamma camera. Volumes of interest of identifiable source organs of radioactivity were defined on aligned reference CT and serial SPECT images. Organs with the highest estimated dose values (mSv/MBq) for FF-21101(111In) were the lungs (0.840), spleen (0.816), liver (0.751), kidneys (0.629), and heart wall (0.451); and for FF-21101(90Y) dose values were: lungs (10.49), spleen (8.21), kidneys (5.92), liver (5.46), and heart wall (2.61). FF-21101(111In) exhibits favorable biodistribution in cynomolgus macaques and estimated human dosimetric characteristics. Data obtained in this study were used to support the filing of an investigational new drug application with the FDA for a Phase I clinical trial.

Optimizing the use of vilobelimab for the treatment of COVID-19

ABSTRACT Introduction On 4 April 2023i4 April 2023, the United States Food and Drug Administration issued an emergency use authorization for the use of vilobelimab (GohibicTM) for the treatment of COVID-19 in hospitalized adults when initiated within 48 hours of receiving invasive mechanical ventilation or extracorporeal membrane oxygenation. Areas Covered Vilobelimab is a human-mouse chimeric IgG4 kappa antibody that targets human complement component 5a, a part of the immune system that is thought to play an important role in the systemic inflammation due to SARS-CoV-2 infection that leads to COVID-19 disease progression. Expert Opinion A pragmatic, adaptive, randomized, multicenter phase II/III study evaluating vilobelimab for the treatment of severe COVID-19 found that patients receiving invasive mechanical ventilation and usual care who were treated with vilobelimab had a lower risk of death by day 28 and day 60 compared to those receiving placebo. This manuscript explores what is known about vilobelimab and explores how this treatment may be used in the future to treat severe COVID-19.

Characterization of a human-mouse chimeric monoclonal antibody targeting rabies virus glycoprotein.

At present, the horse or human rabies immunoglobulin (RIG) used for postexposure prevention of human rabies (PEP) has high cost and limited availability. It is strongly encouraged to replace RIG with equivalent or more effective and safer products. Mouse and human monoclonal antibodies have been shown to protect rodents from lethal rabies virus (RABV) attacks. In this study, we reported a human-mouse chimeric monoclonal antibody, 12-2A12, which showed a strong neutralization potency and a wide breadth against multiple street viruses of RABV in vitro. The antibody binded the viral glycoprotein (G) with nanomolar affinity. The complex structure of 12-2A12 bound to RABV G revealed that the antibody recognizes an epitope that partially overlaps with the recognition region for the nicotinic acetylcholine receptor (nAChR). The antibody therefore would interfere with the nAChR/G interaction to block the viral receptor binding. In addition, comparison of our complex structure with the G structure in the acidic state reveals a clear steric clash, highlighting that the antibody would further prevent the conformational changes of the viral glycoprotein that are essential for membrane fusion. In light of these functional and structural data, we believe that 12-2A12 might be developed to be included in an antibody cocktail for potential use in human rabies PEP.

Novel neutralizing mouse-human chimeric monoclonal antibodies against the SARS-CoV-2 receptor binding domain.

Introduction. Neutralizing antibodies have been widely used for the prophylaxis and treatment of COVID-19.Hypothesis. The major target for these neutralizing antibodies is the receptor-binding domain (RBD) of the viral spike protein.Aim. In the present study, we developed and characterized three neutralizing chimeric mouse-human mAbs for potential therapeutic purposes.Methodology. Light and heavy chain variable region genes of three mouse mAbs (m4E8, m3B6, and m1D1) were amplified and ligated to human Cγ1 and Cκ constant region genes by PCR. After cloning into a dual promoter mammalian expression vector, the final constructs were transiently expressed in DG-44 cells and the purified chimeric antibodies were characterized by ELISA and Western blotting. The neutralizing potency of the chimeric mAbs was determined by three different virus neutralization tests including sVNT, pVNT, and cVNT.Results. All three recombinant chimeric mAbs display human constant regions and are able to specifically bind to the RBD of SARS-CoV-2 with affinities comparable to the parental mAbs. Western blot analysis showed similar epitope specificity profiles for both the chimeric and the parental mouse mAbs. The results of virus neutralization tests (sVNT, pVNT, and cVNT) indicate that c4E8 had the most potent neutralizing activity with IC50 values of 1.772, 0.009, and 0.01 µg ml-1, respectively. All chimeric and mouse mAbs displayed a similar pattern of reactivity with the spike protein of the SARS-CoV-2 variants of concern (VOC) tested, including alpha, delta, and wild-type.Conclusion. The chimeric mAbs displayed neutralizing potency similar to the parental mouse mAbs and are potentially valuable tools for disease control.

Standardization of HIT Diagnostic Assays, with K070, a chimeric human-mouse antibody, mimicking heparin dependent pathogenic antibodies

This study reports the K070 monoclonal antibody, a human-mouse recombinant chimeric monoclonal antibody, IgG1 isotype, targeted to Heparin and PF4 complexes, which mimics Heparin Induced Thrombocytopenia. This antibody has a strong reactivity to Heparin-PF4 complexes, and a much weaker one to PF4 alone. When plasmas spiked with this antibody at concentrations ≥ 0.5 µg/ml are tested at the standard immunoassay dilution, a dose dependent reactivity is obtained. This opens the perspective to use this material to calibrate immunoassays for a quantitative measurement of heparin dependent antibodies, when the disease is suspected. Furthermore, when plasmas are spiked at a higher concentration, from 20 to 200 µg/ml, they test positive in functional assays, like Platelet Aggregation/Activation Tests or Flow Cytometry methods, in presence of a low (0.1-1.0 IU/ml) but not a high (10-100 IU/ml) heparin concentration. In functional assays K070 reactivity level depends on the platelet rich plasmas used. In Platelet Aggregometry Tests some platelet rich plasmas are already positive when tested with plasmas supplemented with 20 µg/ml, whilst others require 100 to 200 µg/ml. At this latter concentration, all tested plasmas generate a strong positive responses with functional assays. K070 is then a useful antibody for calibration of immunoassays and standardization of functional methods used to diagnose Heparin Induced Thrombocytopenia.

Phase 1 trial of HS006, a recombinant human-mouse chimeric anti-CD20 monoclonal antibody to treat CD20 positive B-cell non-Hodgkin's lymphoma in China

Phase 1 trial of HS006, a recombinant human-mouse chimeric anti-CD20 monoclonal antibody to treat CD20 positive B-cell non-Hodgkin's lymphoma in China

Therapeutic Anti-Jagged1 Antibody Targeting Osteoblast-Related Myeloid Dysplasia to Overcome Standard of Care Resistance

Acute myeloid leukemia (AML) and myelodysplasia (MDS) are increasingly recognized as not only genetic but niche-dependent malignancies prompting the question of whether targeting signals from supporting stromal cells may provide a more stable target for therapy than emerging leukemic clones. Whereas standard of care and malignant cell targeted therapies fail to eliminate resistant clones or prevent the expansion of new ones, inhibition of oncogenic or tumor promoting signals from the stroma could overcome mutation dependance and clonal resistance and be an efficient approach to manage disease and prevent relapse. Here, we inhibited a potent, niche-driven tumorigenic pathway, constitutive activation of b-catenin/Jagged1 signaling in osteoblasts. This pathway is activated following hypermethylation of its regulators in MDS patients; is activated in approximately 40% of MDS and AML patients; its activation levels increase with disease severity and progression; correlate with MDS to AML transformation and del(5q)-associated myeloid malignancies and leads to MDS rapidly progressing to AML in mice. To examine the therapeutic potential of targeting this pathway we generated a human-mouse chimeric antibody that efficiently and specifically binds JAG1 (anti-JAG1) and inhibits Notch1 signaling. Administration of anti-JAG1 in leukemic mice with osteoblastic active b-catenin rescued anemia, thrombocytopenia, neutrophilia and lymphocytopenia, restored LT-HSCs (the leukemia initiating population), relieved myeloid differentiation block and eliminated blasts. Body weight increased and lethality was abrogated in treated mice. In contrast, chemotherapy at a dose simulating induction regimen used in humans, dramatically exacerbated anemia, thrombocytopenia and lymphocytopenia without decreasing blasts leading to increased lethality due to bone marrow failure. A lower chemotherapy dose induced a physiologically relevant response in wild type mice, but failed to improve hematopoiesis and myeloid differentiation block in leukemic mice and as a result AML progression and lethality was not affected. Emphasizing relevance to human disease, anti-JAG1 treatment of patient-derived samples with activated b-catenin in their osteoblasts, inhibited MDS and AML cell growth and survival and promoted myeloid and erythroid differentiation irrespective of disease category through its actions on osteoblasts. These findings suggest anti-JAG1 may provide a broad therapeutic approach that is effective in 1/3 of MDS and AML patients with cytogenetically distinct types of disease. The therapeutic potential of targeting the bone marrow stromal niche in hematological malignancies can be a treatment strategy that may evade relapse and overcome standard of care resistance and toxicity.

AML-327 Novel Anti–B7-H3 Blocking Antibody Enhances NK Cell–Mediated Cytotoxicity and Improves Outcomes in AML-Bearing Mice

B7-H3 is an immune checkpoint molecule that is overexpressed in various human malignancies. Several monoclonal antibodies (mAbs) targeting B7-H3 have shown promising results against solid tumors. However, B7-H3's role in AML remains unexplored. i) To analyze the prognostic significance of B7-H3 in AML. ii) To investigate the immunomodulatory role of B7-H3 in AML in vitro and in vivo. We analyzed B7-H3 expression in 100 AML patients and 20 healthy donors by flow cytometry and tested for associations with clinical features. To investigate the role of B7-H3 in immunomodulation, B7-H3 was knocked down in AML cell lines using shRNA or using anti-B7-H3 blocking mAbs (T1-A5, HEK1B3A3, and 58B1A2). A human-mouse chimeric (chT-1A5) antibody was generated, and its binding site on B7-H3 protein was characterized by epitope mapping. Finally, we treated mice bearing AML cells with anti-B7-H3 antibodies and measured human AML engraftment with flow cytometry and bioluminescence imaging. Expression of B7-H3 was significantly higher in AML patients than in healthy donors (p<0.01) and in CD34+ AML cells than in CD34- cells (p<0.01). Clinically, we observed that high B7-H3 expression was associated with a poor prognosis. Furthermore, we observed that inhibition of B7-H3 expression or blocking of its activity in AML cells using a novel monoclonal antibody (T-1A5) significantly enhanced natural killer (NK) cell-mediated cytotoxicity in vitro and in vivo. Moreover, we observed that the human-mouse chimera of this antibody (ChT-1A5) induced dose-dependent cell-mediated cytotoxicity (ADCC) in B7-H3+ primary AML cells but not in normal hematopoietic cells, suggesting the specify of this antibody for AML cells. Epitope mapping using peptides derived from the B7-H3 protein identified the FG loop region as the binding site for the T1-A5 antibody. Finally, treatment with T-1A5 or chT-1A5 in combination with human NK cells significantly reduced leukemia burden and extended survival in AML xenograft and patient-derived xenograft models. Our data indicate that B7-H3 is overexpressed in AML and that an anti-B7-H3 antibody (T1-A5) not only blocks B7-H3's immunomodulatory function but also induces ADCC in AML cells in vitro and in vivo.

CPGJ602 plus mFOLFOX6 as first-line treatment for patients with KRAS/NRAS/BRAF wild-type metastatic colorectal cancer: A randomized phase II study.

3574 Background: CPGJ602 is a recombinant anti-EGFR human-mouse chimeric monoclonal antibody. CPGJ602 plus modified fluorouracil, leucovorin, and oxaliplatin (mFOLFOX6) may have efficacy in KRAS/NRAS/BRAF wild-type metastatic colorectal cancer. Methods: In this open-label, randomized trial, patients who had received no previous treatment were randomly assigned (2:2:1) to receive CPGJ60（325mg/m2, q2w) plus mFOLFOX6 (biweekly group), CPGJ602 (400 mg/m 2 initial dose followed by 250 mg/m2 /week thereafter) plus mFOLFOX6 (weekly group) or cetuximab (400 mg/m2 initial dose followed by 250 mg/m2 /week thereafter) plus mFOLFOX6 (cetuximab group). All subjects received treatment up to 16 weeks. The primary endpoint was the best overall response (BOR) at 16 weeks. The second endpoints were DCR, DOR, PFS, safety. Results: As of Dec 30, 2021, 76 patients were enrolled (30 in biweekly group, 32 in weekly group and 14 in control group). The best overall response achieved at 16 weeks was 76.7% (23/30, 95% CI 60.3% - 92.0%),78.1% (25/32, 95% CI 62.5% - 92.5%) and 78.6% (11/14, 95% CI 49.2% - 95.3%) in the biweekly group, weekly group and cetuximab group, respectively. The confirmed overall response rate at 16 weeks was 60%(18/30), 71.9%(23/32) and 57.1%(8/14) in the biweekly group, weekly group and cetuximab group, respectively. The PFS rates at 16 weeks were 81.5% (95% CI 57.7% -92.6%), 96.8% (95% CI 79.2%-99.5%), 81.3% (95% CI 41.5%- 95.2%) in the biweekly group, weekly group and cetuximab group, respectively. The most common adverse events were a decreased neutrophil count (60%，78.1% and 71.4% in the biweekly group, weekly group and cetuximab group respectively), decreased white-cell count (53.3%, 78.1% and 71.4% respectively), decreased platelet count (40.0%, 40.6% and 42.9% respectively), and elevated serum aspartate aminotransferase (43.3%, 37.5% and 35.7% respectively). Conclusions: CPGJ602 plus mFOLFOX6 could be an option for KRAS/NRAS/BRAF wild-type metastatic colorectal cancer. Clinical trial information: NCT04466254.

Generation and characterization of humanized synergistic neutralizing antibodies against SARS-CoV-2.

The emerging coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), is the causative agent of coronavirus disease 2019 (COVID‐19), which has become a severe threat to global public health and local economies. In this study, several single‐chain antibody fragments that bind to the receptor‐binding domain (RBD) of the SARS‐CoV‐2 spike (S) protein were identified and used to construct human‐mouse chimeric antibodies and humanized antibodies. These antibodies exhibited strong binding to RBD and neutralization activity towards a SARS‐CoV‐2 pseudovirus. Moreover, these antibodies recognize different RBD epitopes and exhibit synergistic neutralizing activity. These provide candidate to combination use or bispecific antibody to potential clinical therapy for COVID‐19.

Potent anti-tumor activity of CD45RA-targeting Hm3A4-Ranpirnase against myeloid lineage leukemias

ABSTRACT CD45RA is a specific marker for leukemia stem cell (LSC) sub-populations in acute myeloid leukemia (AML). Ranpirnase (Rap), an amphibian RNase, has been extensively investigated in preclinical and clinical studies for its antitumor activity. Rap could be administered repeatedly to patients without inducing an immune response. Reversible renal toxicity has been reported to be dose-limiting. In this study, we generated a novel immunotoxin targeting LSCs: Hm3A4-Rap, which was composed of Rap and Hm3A4, a human-mouse chimeric antibody against CD45RA. This immunotoxin was generated recombinantly by fusing Rap to Hm3A4 at the Fc terminus and then produced by stably transfecting CHO cells. The immunotoxin was purified using Ni-NTA and then evaluated using RT-PCR, SDS-PAGE, antibody titer assays, competitive inhibition assays, and internalization assays. In addition, the purity, molecular integrity, and affinity to the CD45RA antigen were determined. In vitro studies demonstrated that Hm3A4-Rap could efficiently kill target cells. In vivo studies demonstrated that Hm3A4-Rap had potent anti-leukemia activity, with dosed mice showing a significant increase in survival time compared to control mice (P < 0.01). In summary, our immunotoxin had excellent biological activity suggesting its potential therapeutic value for treating AML patients. Additional preclinical and clinical studies are needed to develop this immunotoxin as a treatment option for patients with leukemia.

Efficacy and Safety of Recombinant Human-Mouse Chimeric Anti-CD20 Monoclonal Antibody (SCT400) Combined with CHOP in Patients with Treatment-Naïve Diffuse Large B-Cell Lymphoma: A Multicenter, Randomized, Single-Blind, Parallel Active-Controlled, Phase III Study

Background:SCT400 is a recombinant, human-mouse chimeric anti-CD20 IgG1 monoclonal antibody and has the same variable regions and antigen-binding site as rituximab. SCT400 has the heavy and κ light chain constant regions of human IgG1 allotype G1m (1,17), which is common in Asians. There is only one amino acid difference between SCT400 and rituximab (V219A in the CH1 domain of the heavy chain). The phase Ⅱ study showed that pharmacokinetics, pharmacodynamics and safety of SCT400 are equivalent to rituximab in patients with CD20-positive non-Hodgkin lymphoma.Objectives:This phase III study aimed to evaluate the safety and efficacy of SCT400 plus cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) compared with rituximab plus CHOP in patients with treatment-naïve diffuse large B-cell lymphoma (DLBCL) (ClinicalTrials.gov, identifier: NCT02772822).Methods:In this multicenter, randomized, single-blind, parallel active-controlled, non-inferiority, phase III study, patients were enrolled from 37 centers in China. Eligible patients were aged 18～75 years with histologically confirmed CD20-positive, treatment-naïve DLBCL; international prognostic index (IPI) 0～2; an Eastern Cooperative Oncology Group (ECOG) performance status (PS) of 0～2. Patients were randomly assigned in a 2:1 ratio to receive either SCT400 plus CHOP (S-CHOP) or rituximab plus CHOP (R-CHOP) every 21 days cycle treatment for six cycles.The primary endpoint was objective response rate (ORR) after six cycles of therapy, assessed by an independent review committee, with a non-inferiority margin of -12%. The secondary endpoints were complete response rate (CRR), progression-free survival (PFS), overall survival (OS), and safety.Results:Between October 19, 2016 and July 13, 2018, 534 patients were screened and 364 eligible patients were randomized to either the S-CHOP group (n=243) or R-CHOP group (n=121). Patient demographics and disease characteristics at baseline were well balanced between the two treatment groups.In the per protocol set (PPS), the best ORRs within six cycles of treatment were 94.5% (95% confidence interval [CI], 90.8% to 97.0%) and 94.1% (95%CI, 88.2% to 97.6%) in the S-CHOP and R-CHOP groups respectively, with an intergroup difference of 0.4% (95%CI, -4.7% to 5.6%, P=0.6569) (Figure 1A). The 95%CI lay on the positive side of the prespecified non-inferiority margin of -12%, meeting the criterion for non-inferiority. The results of using the full analysis set (FAS) were consistent with the primary efficacy analysis in the PPS. No significant differences were observed in the secondary efficacy endpoints, in either the PPS or the FAS (Figure 1B and Figure 2).There were no significant differences were observed in ≥1 adverse event (AE, 97.9% vs. 99.2%), grade≥3 AE (85.2% vs. 86.0%), serious AE (35.4% vs. 38.8%), treatment discontinuation due to AE (7.8% vs.12.4%) between S-CHOP and R-CHOP groups, respectively (Table 1).AEs of special interest occurred in 63.8% of patients in the S-CHOP group and in 59.5% of patients in R-CHOP group, including infection (46.5% vs. 46.3%), infusion-related reaction (20.2% vs. 17.4%), cardiotoxicity (17.7% vs. 11.6%), hepatitis B virus reactivation (1.2% vs. 3.3%), ileus and enterobrosis (1.2% vs. 0.8%), tumor lysis syndrome (0.0% vs. 0.8%) (Table 1).A total of 51 deaths were reported in the S-CHOP group (n=35, 14.4%) and R-CHOP group (n=16, 13.2%), and 10 deaths were due to AE (S-CHOP group: n=7, 2.9%; R-CHOP group: n=3, 2.5%) (Table 1).After treatment, study-drug related anti-drug antibodies (ADAs) were detected in 11(10.9%) patients in S-CHOP group and in 8(16.0%) patients in R-CHOP group (Table 1).Conclusion:SCT400 plus CHOP showed non-inferiority of efficacy to rituximab plus CHOP in patients with treatment-naïve DLBCL. The safety and immunogenicity profiles of SCT400 were comparable with rituximab, with no new treatment-related AE occurred. SCT400 could be a therapeutic option for DLBCL.Funding source:This study was supported by the sponsor, Sinocelltech Ltd., and by grants from the China National Major Project for New Drug Innovation (No. 2018ZX09736002 and No. 2017ZX09304015).Conflicts of interest:Liangzhi Xie is an employee of Sinocelltech Ltd. and has ownership interests in the company. All other authors declare no conflicts of interest. [Display omitted] DisclosuresHu: Astellas Pharma, Inc.: Research Funding. Xie: Sinocelltech Ltd.: Current Employment, Current equity holder in publicly-traded company.

Anti-B7-H3 Antibody (T-1A5) Blocks Immunomodulatory Function of B7-H3 and Enhances NK Cell-Mediated Cytotoxicity Against Acute Myeloid Leukemia Cells

Background: The immune checkpoint molecule B7-H3 (CD276) is overexpressed in various solid tumors and hematological malignancies; however, its expression is limited in normal tissue, which makes it an attractive therapeutic target in cancer. Several monoclonal antibodies (mAbs) targeting B7-H3 have shown promising results against solid tumors. However, B7-H3's role in acute myeloid leukemia (AML) remains unexplored. Here, we hypothesized that targeting B7-H3 using mAbs alters the immunomodulatory function of B7-H3 and enhances NK cell-mediated cytotoxicity against AML cells.Methods: B7-H3 protein expression was analyzed in the peripheral blood (PB) and bone marrow of 100 patients with AML and 20 healthy donors by flow cytometry and tested for associations with multiple clinical parameters and disease outcomes. To investigate B7-H3's role in immunomodulation, we stably knocked down B7-H3 in AML cell lines including OCI-AML3, MV4-11, and U937 and co-cultured them with activated human NK cells. NK cell-induced apoptosis was measured by annexin-v binding assay using an IncuCyte live-cell imaging system. B7-H3-blocking mAbs (clones T-1A5, HEK5-1B3, and 58B1) were tested for their effect on NK-cell-mediated cytotoxicity in AML cell lines using live-cell imaging. AML xenograft (OCI-AML3) or patient-derived xenograft (PDX) models were used to determine the effect of anti-B7-H3 antibodies on AML growth. In vivo AML growth was monitored by measuring human CD45 positive cells with flow cytometry. A human-mouse chimeric (ch) antibody was generated based on sequences from the T-1A5 antibody, and its binding site on the B7-H3 protein was characterized by epitope mapping. Further, we evaluated the effect of chT-1A5 on NK cell-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) in primary AML cells and healthy donor PB-derived mononuclear cells (PBMCs).Results: Expression of B7-H3 was significantly higher in AML patients than in healthy donors (p < 0.01) and was higher in CD34 positive than in CD34 negative AML cells (p < 0.01). High B7-H3 expression was associated with poor overall survival (p = 0.04) and prognostic risk scores (p = 0.05). NK cell-mediated apoptosis was 3-fold higher in all 3 B7-H3-knockdown AML cell lines than in scrambled control cells, suggesting that B7-H3 is an important immunomodulator of NK cells. Moreover, we observed a significant increase in NK cell-mediated killing of AML cells in the presence of anti-B7-H3 mAbs (p < 0.01). In vivo, anti-B7-H3 antibodies significantly inhibited AML growth and extended survival in PDX models compared to IgG control treatment. Among PDX-bearing mice treated with the three anti-B7-H3 antibodies, the T-1A5 antibody-treated group survived for longer than the other groups. In combination with NK cells, T-1A5 treatment also significantly increased the survival of AML xenograft-bearing mice compared to NK + IgG control treatment. These data suggest that the T-1A5 antibody blocks B7-H3 and enhances NK cell-mediated cytotoxicity in AML cells in vivo. Next, we found that a chT-1A5 antibody induced NK cell-mediated ADCC in primary AML cells and cell lines in a dose-dependent manner. In contrast, it did not induce any ADCC activity in healthy donor-derived PBMCs, suggesting that chT-1A5 is not toxic to healthy cells. Moreover, the chT-1A5 antibody combined with human NK cells dramatically inhibited leukemia growth and extended survival in B7-H3 positive AML PDX models (p < 0.001) compared to the control (rituximab-treated) group. Finally, epitope mapping using peptides derived from the B7-H3 protein identified the FG loop region of B7-H3 as the binding site for the T-1A5 antibody, which may be involved in the immunomodulatory function of B7-H3.Conclusion: B7-H3 is overexpressed in AML cells, and its expression is associated with poor overall survival. Anti-B7-H3 antibodies block B7-H3's immunomodulatory function and inhibit AML growth in vivo. A chT-1A5 antibody in combination with NK cells induced ADCC in primary AML cells in vitro and in vivo but had no effect on PBMCs from healthy donors. Therefore, targeting B7-H3 could benefit AML patients, specifically those with a poor clinical prognosis. DisclosuresBattula: Tolero Pharmaceuticals: Research Funding.

A FZD7-specific Antibody-Drug Conjugate Induces Ovarian Tumor Regression in Preclinical Models.

Although WNT signaling is frequently dysregulated in solid tumors, drugging this pathway has been challenging due to off-tumor effects. Current clinical pan-WNT inhibitors are nonspecific and lead to adverse effects, highlighting the urgent need for more specific WNT pathway-targeting strategies. We identified elevated expression of the WNT receptor Frizzled class receptor 7 (FZD7) in multiple solid cancers in The Cancer Genome Atlas, particularly in the mesenchymal and proliferative subtypes of ovarian serous cystadenocarcinoma, which correlate with poorer median patient survival. Moreover, we observed increased FZD7 protein expression in ovarian tumors compared with normal ovarian tissue, indicating that FZD7 may be a tumor-specific antigen. We therefore developed a novel antibody-drug conjugate, septuximab vedotin (F7-ADC), which is composed of a chimeric human-mouse antibody to human FZD7 conjugated to the microtubule-inhibiting drug monomethyl auristatin E (MMAE). F7-ADC selectively binds human FZD7, potently kills ovarian cancer cells in vitro, and induces regression of ovarian tumor xenografts in murine models. To evaluate F7-ADC toxicity in vivo, we generated mice harboring a modified Fzd7 gene where the resulting Fzd7 protein is reactive with the human-targeting F7-ADC. F7-ADC treatment of these mice did not induce acute toxicities, indicating a potentially favorable safety profile in patients. Overall, our data suggest that the antibody-drug conjugate approach may be a powerful strategy to combat FZD7-expressing ovarian cancers in the clinic.

Ralph A. Reisfeld, PhD: In Memoriam (1926–2020)

![][1] The sad news that Ralph A. Reisfeld, PhD, passed away on December 6, 2020 reached us early this year, and we would like to reflect on Ralph's long career and distinctive contribution to biomedical science and in particular to the field of cancer research. Ralph was well known

Inhibition of astrocyte hemichannel improves recovery from spinal cord injury.

Spinal cord injury (SCI) causes severe disability, and the current inability to restore function to the damaged spinal cord leads to lasting detrimental consequences to patients. One strategy to reduce SCI morbidity involves limiting the spread of secondary damage after injury. Previous studies have shown that connexin 43 (Cx43), a gap junction protein richly expressed in spinal cord astrocytes, is a potential mediator of secondary damage. Here, we developed a specific inhibitory antibody, mouse-human chimeric MHC1 antibody (MHC1), that inhibited Cx43 hemichannels, but not gap junctions, and reduced secondary damage in 2 incomplete SCI mouse models. MHC1 inhibited the activation of Cx43 hemichannels in both primary spinal astrocytes and astrocytes in situ. In both SCI mouse models, administration of MHC1 after SCI significantly improved hind limb locomotion function. Remarkably, a single administration of MHC1 30 minutes after injury improved the recovery up to 8 weeks post-SCI. Moreover, MHC1 treatment decreased gliosis and lesion sizes, increased white and gray matter sparing, and improved neuronal survival. Together, these results suggest that inhibition of Cx43 hemichannel function after traumatic SCI reduces secondary damage, limits perilesional gliosis, and improves functional recovery. By targeting hemichannels specifically with an antibody, this study provides a potentially new, innovative therapeutic approach in treating SCI.