Complement-dependent Cytotoxicity Research Articles

Abstract 1237: IGM-2644, a CD38xCD3 bispecific IgM T cell engager, shows enhanced anti-tumor activity compared to daratumumab in preclinical models of multiple myeloma

Abstract Multiple myeloma (MM) is a hematologic malignancy characterized by the accumulation of monoclonal plasma cells in the bone marrow. CD38 is an attractive therapeutic target for MM due to its high expression on plasma cells, but it is also expressed on multiple subsets of normal immune cells. Approved therapies including anti-CD38 monoclonal antibodies such as daratumumab are available, however resistance inevitably develops, and toxicities such as reduced immune cell counts put patients at increased risk for infections. To address this and other issues associated with current therapies, we are developing IGM-2644, an engineered high affinity, high avidity bispecific anti-CD38xCD3 IgM T cell engager (TCE) antibody for the treatment of plasma cell disorders. IGM-2644 kills CD38-expressing tumor cells through complement-dependent cytotoxicity (CDC) and T cell-dependent cellular cytotoxicity (TDCC). Live cell imaging of tumor cells in the presence of normal human serum as a source of complement has shown that IGM-2644, compared to daratumumab, has exhibited superior CDC with faster kinetics and higher maximal killing, as well as increased activity on tumor cell lines that express low levels of CD38. IGM-2644 also has displayed enhanced cellular dependent cytotoxicity over daratumumab across tumor cell lines with a range of CD38 expression levels in vitro. In ex vivo assays with MM patient bone marrow mononuclear cells (BMMC), IGM-2644 has shown superior tumor cell depletion compared to daratumumab even with low T cell to tumor cell ratios. Furthermore, IGM-2644 can deplete tumor cells in BMMC from daratumumab-refractory MM patients. Similarly, using in vivo humanized xenograft tumor mouse models with various levels of CD38 expression, greater anti-tumor activity with IGM-2644 over daratumumab was observed. When compared to daratumumab, IGM-2644 demonstrated lower immune cell fratricide in an in vivo xenograft tumor mouse model, suggesting a potentially differentiated safety profile. In summary, IGM-2644 is a bispecific anti-CD38xCD3 IgM TCE antibody that demonstrates superior tumor cell killing when compared to daratumumab in vitro and especially in xenograft tumor models in vivo with low CD38 expression while having low immune cell depletion, potentially reducing the risk of infections. The safety and efficacy of IGM-2644 has been initially studied in a phase 1 clinical trial for relapsed/refractory (r/r) MM. Applicability of IGM-2644 for the treatment of other plasma cell disorders is currently being explored, including autoimmune diseases. Citation Format: Kevin C. Hart, Daniel Santos, Keyu Li, Rui Yun, Min Chai, Andras Kabai, Gene Li, Poonam Yakkundi, Roel P. Funke, Yinghui Guan, Thomas Manley, Liqin Liu, Angus M. Sinclair, Albert F. Candia, Bruce Keyt, Maya F. Kotturi. IGM-2644, a CD38xCD3 bispecific IgM T cell engager, shows enhanced anti-tumor activity compared to daratumumab in preclinical models of multiple myeloma [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 1237.

Abstract 2356: NAV-006: A next-generation rituximab targeting CD20 for the treatment of B-cell lymphomas immunosuppressed by MUC16/CA125

Abstract Rituximab is a CD20-targeting antibody that is the standard-of-care for patients with Non-Hodgkin Lymphoma (NHL) cases. Rituximab’s mechanism of action includes complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC), herein referred to as Humoral Immuno-Oncology (HIO) mechanisms. Recent clinical evidence suggest that high serum levels of the MUC16 (CA125) protein inversely correlate with the effectiveness of rituximab clinical activity in 40-50% of follicular lymphoma (FL) as well as diffuse large B-cell lymphoma patients with newly diagnosed or relapsed/refractory (R/R) disease. In this study we demonstrated that CA125 binds to and suppresses the activity of rituximab, tafasitamab as well as the bispecific mosunetuzumab and glofitamab antibodies, which are approved for treating cases with indolent disease. These therapies have an overall response rate around 50-60%, suggesting the presence of a mechanism that allows lymphoma cells to escape these agents’ immune-mediated killing effects. CA125 may facilitate this immunosuppressive mechanism, and in fact, upon binding rituximab, CA125 reduces its affinity for the Fc receptor, CD16a, thus suppressing ADCC. Using a proprietary technology called Block-Removed Immunoglobulin Technology (BRITE) that employs randomized amino acid substitution and HIO effector function screening, we have identified a rituximab variant named NAV-006. NAV-006 is refractory to the immunosuppressive effects mediated by CA125 via its reduced CA125 interaction and increased HIO activity as compared to parent rituximab as well as the other antibodies used to treat R/R FL. In an in vivo model of human B-cell lymphoma, NAV-006 showed superior efficacy compared to parent rituximab. These data warrant further investigation of NAV-006 as a next generation anti-CD20 antibody that could improve upon the efficacy of antibody-mediated therapies used to treat NHL patients with high levels of CA125. Citation Format: Luigi Grasso, J. Bradford Kline, Nicholas C. Nicolaides. NAV-006: A next-generation rituximab targeting CD20 for the treatment of B-cell lymphomas immunosuppressed by MUC16/CA125 [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 2356.

Abstract 6344: Engineering of ICAM-1 refractory antibodies for the development of therapeutic antibodies and antibody drug conjugates (ADCs) in ICAM-1 over-expressing cancers

Abstract Human cancers employ a number of mechanisms to evade host immune responses against novel antigens generated from aberrant over-expression, mutations and/or epigenetic alterations. Humoral immunity utilizes antibodies and immune-effector cells as well as molecular immune complexes involving the complement system to mediate the killing of dysregulated cancer cells. We refer to these anti-cancer mechanisms as Humoral Immuno-Oncology (HIO). HIO suppression is mediated by tumor-produced proteins called HIO factors. One such factor is CA125, which was previously shown to bind IgG-type antibodies and inhibit their immune-effector activities, including antibody dependent (ADCC) and complement dependent (CDC) cellular cytotoxicity. Using a combination of experimental screening and literature searches, a second protein produced by tumors and associated with a variety of cancer indications was discovered and found to be soluble ICAM-1 (sICAM-1). Through various functional studies we reported that sICAM-1 and membrane ICAM-1 (mICAM-1) are capable of binding IgG1-type antibodies that in turn inhibits their immune-effector activity. Through a combination of truncation and substitution mutagenesis, we identified a four amino acid motif within the CH3 domain of IgG1 essential for ICAM-1 binding, resulting in the inhibition of ADCC activity. While traditional glycine and alanine substitutions in this region abrogated ICAM-1 binding, these modifications caused tertiary structural changes in the Fc domain that resulted in loss of ADCC activity. Through a combinatorial amino acid substitution approach, we identified a four amino acid combination within the 407YSKL410 to 407FARV410 motif that resulted in antibodies refractory to sICAM-1 binding with robust ADCC activity. Additionally, isogenic wildtype and ICAM-1 knockdown target cell lines showed mICAM-1 inhibited target cell killing of a saporin antibody drug conjugate (ADC) and this was the direct result of mICAM-1 reducing antibody internalization, a requisite for maximal ADC target cell killing. The inhibitory activity of mICAM-1 could be overcome employing antibodies containing the 407FARV410 substitution. These findings highlight yet another mechanism by which tumors can suppress the host’s immune system for survival and offer new concepts for developing antibody-based therapies that can aid in the treatment of various cancer indications, especially those over-expressing ICAM-1. Moreover, the findings here offer diagnostic and therapeutic clinical design opportunities to improve upon existing approved immune-mediated therapies for which this factor is present. Citation Format: Brad Kline, Luigi Grasso, Nicolas Nicolaides. Engineering of ICAM-1 refractory antibodies for the development of therapeutic antibodies and antibody drug conjugates (ADCs) in ICAM-1 over-expressing cancers [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 6344.

Abstract 2915: Targeting complement factor H renders STK11-mutant lung adenocarcinoma sensitive to anti-PD-1 therapy in mice

Abstract STK11 mutations occurs in 15-20% of lung adenocarcinoma (LUAD) and is associated with KRAS mutations, poor survival and resistance to immune checkpoint inhibitors (ICI). We observed tumor cell lines and primary human STK11-mutant LUAD upregulates of expression of C3 and complement factor H (FH). Deletion of C3 in STK11 knock-out tumor cells resulted in reduced FH expression. FH is a co-factor of Factor I, which converts C3b to proteolytically inactive iC3b thereby limiting activation of the alternative pathway (AP) C3 convertase. FH binds to proteoaminoglycans on the cell membrane and protects cells from AP complement-dependent cytotoxicity. With increased C3 generation by STK11-mutant tumor cells, we reasoned that systemic or tumor-derived FH protects tumor cells from complement attack. We hypothesized that tumor-derived C3 would promote STK11-mutant LUAD growth in immune intact mice by promoting immune evasion and would drive ICI resistance. Based on the expected tumor-protective effects of FH, we also hypothesized that anti-FH antibodies would control tumor growth and render tumor more sensitive to ICI. We evaluated growth of subcutaneously administered CMT167-STK11KO with and without C3 deletion (CMT167-STK11KO is murine KRASG12V LUAD with CRISPR/Cas9 deletion of STK11). Deletion of C3 in CMT167-STK11KO (CMT167-STK11KO/C3KO) resulted in dramatic inhibition of tumor growth and in sensitivity to anti-PD-1 therapy in vivo. In contrast, deletion of C3 in STK11-KO tumors had no effect on tumor growth in nude mice or following CD8 depletion in WT mice. These findings support a role of tumor-derived C3 in evasion of T cell anti-tumor immunity and as an obstacle to anti-PD-1 therapy. GT103 is a therapeutic antibody that binds tumor cell-attached CFH, causes complement activation, kills tumor cells, initiates a shift from a pro- to an anti-tumor immune microenvironment, and is currently being evaluated in a Phase 1b-2 clinical trial in combination with pembrolizumab (NCT05617313). We observed that single agent GT103 or anti-PD-1 has limited efficacy in CMT167-STK11KO tumor in WT mice. However, the combination of GT103 and anti-PD-1 was very effective in limiting tumor growth. Our results support a role of tumor-derived C3 in suppressing CD8 T-cell immunity and in driving ICI resistance and that the addition of GT103 renders STK11-KO tumors sensitive to anti-PD-1 in mice. These studies establish rationale for evaluating this combination in patients with STK11-mutant LUAD. Citation Format: Sora Suzuki, Anm Nazmul Khan, Thejaswini Giridharan, Catrina Ting, Ryan Bushey, Elizabeth Gottlin, Michael Campa, Edward F. Patz, Edwin H. Yau, Brahm Segal. Targeting complement factor H renders STK11-mutant lung adenocarcinoma sensitive to anti-PD-1 therapy in mice [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 2915.

Pretransplant Screening for Prevention of Hyperacute Graft Loss in Pig-to-primate Kidney Xenotransplantation.

Xenotransplantation using pig organs is now a clinical reality. However, the process for xenograft recipient screening lacks clarity and scientific rigor: no established thresholds exist to determine which levels of preformed antipig natural antibodies (Nabs) will be safe for clinical xenograft transplantation, and hyperacute rejection (HAR) or acute humoral xenograft rejection (AHXR), which still impacts pig-to-primate kidney xenograft survivals, may impede broader application of pig-to-human clinical xenograft transplantation. We retrospectively examined 28 cases of pig-to-baboon kidney xenotransplantation using GalTKO±human complement regulatory protein (hCRP)-transgenic (Tg) pig donors, as well as 6 cases of triple-KO multi-Tg (10GE) pig donors, and developed screening algorithms to predict risk of HAR/AHXR based on recipient antipig Nab levels. Preformed Nabs were evaluated using both complement-dependent cytotoxicity and antibody (IgM and IgG) binding flow-cytometry assays. High complement-dependent cytotoxicity was associated with HAR/AHXR as expected. However, we also found that high levels of IgG were independently associated with HAR/AHXR, and we developed 2 indices to interpret and predict the risk of IgG-mediated HAR/AHXR. Based on the data in this study, we have established a new 2-step screening, which will be used for future clinical kidney xenotransplantation trials.

Development of ISB 1442, a CD38 and CD47 bispecific biparatopic antibody innate cell modulator for the treatment of multiple myeloma

Antibody engineering can tailor the design and activities of therapeutic antibodies for better efficiency or other advantageous clinical properties. Here we report the development of ISB 1442, a fully human bispecific antibody designed to re-establish synthetic immunity in CD38+ hematological malignancies. ISB 1442 consists of two anti-CD38 arms targeting two distinct epitopes that preferentially drive binding to tumor cells and enable avidity-induced blocking of proximal CD47 receptors on the same cell while preventing on-target off-tumor binding on healthy cells. The Fc portion of ISB 1442 is engineered to enhance complement dependent cytotoxicity, antibody dependent cell cytotoxicity and antibody dependent cell phagocytosis. ISB 1442 thus represents a CD47-BsAb combining biparatopic targeting of a tumor associated antigen with engineered enhancement of antibody effector function to overcome potential resistance mechanisms that hamper treatment of myeloma with monospecific anti-CD38 antibodies. ISB 1442 is currently in a Phase I clinical trial in relapsed refractory multiple myeloma.

Hypoxia-activated ADCC-enhanced humanized anti-CD147 antibody for liver cancer imaging and targeted therapy with improved selectivity.

Therapeutic antibodies (Abs) improve the clinical outcome of cancer patients. However, on-target off-tumor toxicity limits Ab-based therapeutics. Cluster of differentiation 147 (CD147) is a tumor-associated membrane antigen overexpressed in cancer cells. Ab-based drugs targeting CD147 have achieved inadequate clinical benefits for liver cancer due to side effects. Here, by using glycoengineering and hypoxia-activation strategies, we developed a conditional Ab-dependent cellular cytotoxicity (ADCC)-enhanced humanized anti-CD147 Ab, HcHAb18-azo-PEG5000 (HAP18). Afucosylated ADCC-enhanced HcHAb18 Ab was produced by a fed-batch cell culture system. Azobenzene (Azo)-linked PEG5000 conjugation endowed HAP18 Ab with features of hypoxia-responsive delivery and selective targeting. HAP18 Ab potently inhibits the migration, invasion, and matrix metalloproteinase secretion, triggers the cytotoxicity and apoptosis of cancer cells, and induces ADCC, complement-dependent cytotoxicity, and Ab-dependent cellular phagocytosis under hypoxia. In xenograft mouse models, HAP18 Ab selectively targets hypoxic liver cancer tissues but not normal organs or tissues, and has potent tumor-inhibiting effects. HAP18 Ab caused negligible side effects and exhibited superior pharmacokinetics compared to those of parent HcHAb18 Ab. The hypoxia-activated ADCC-enhanced humanized HAP18 Ab safely confers therapeutic efficacy against liver cancer with improved selectivity. This study highlights that hypoxia activation is a promising strategy for improving the tumor targeting potential of anti-CD147 Ab drugs.

Structural Basis for Multivalent MUC16 Recognition and Robust Anti-Pancreatic Cancer Activity of Humanized Antibody AR9.6.

Mucin-16 (MUC16) is a target for antibody-mediated immunotherapy in pancreatic ductal adenocarcinoma (PDAC) among other malignancies. The MUC16-specific monoclonal antibody AR9.6 has shown promise for PDAC immunotherapy and imaging. Here, we report the structural and biological characterization of the humanized AR9.6 antibody (huAR9.6). The structure of huAR9.6 was determined in complex with a MUC16 SEA (Sea urchin sperm, Enterokinase, Agrin) domain. Binding of huAR9.6 to recombinant, shed, and cell-surface MUC16 was characterized, and anti-PDAC activity was evaluated in vitro and in vivo. HuAR9.6 bound a discontinuous, SEA domain epitope with an overall affinity of 88 nmol/L. Binding affinity depended on the specific SEA domain(s) present, and glycosylation modestly enhanced affinity driven by favorable entropy and enthalpy and via distinct transition state thermodynamic pathways. Treatment with huAR9.6 reduced the in vitro growth, migration, invasion, and clonogenicity of MUC16-positive PDAC cells and patient-derived organoids (PDO). HuAR9.6 blocked MUC16-mediated ErbB and AKT activation in PDAC cells, PDOs, and patient-derived xenografts and induced antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. More importantly, huAR9.6 treatment caused substantial PDAC regression in subcutaneous and orthotopic tumor models. The mechanism of action of huAR9.6 may depend on dense avid binding to homologous SEA domains on MUC16. The results of this study validate the translational therapeutic potential of huAR9.6 against MUC16-positive PDACs.

GlYcoLISA: antigen-specific and subclass-specific IgG Fc glycosylation analysis based on an immunosorbent assay with an LC-MS readout.

Immunoglobulin G (IgG) fragment crystallizable (Fc) glycosylation modulates effector functions such as antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. Consequently, assessing IgG Fc glycosylation is important for understanding the role of antibodies in infectious, alloimmune and autoimmune diseases. GlYcoLISA determines the Fc glycosylation of antigen-specific IgG by an immunosorbent assay with a liquid chromatography-mass spectrometry (LC-MS) readout. Detection of antigen-specific IgG glycosylation in a subclass- and site-specific manner is realized by LC-MS-based glycopeptide analysis after proteolytic cleavage. GlYcoLISA addresses challenges related to the low abundance of specific IgG and the high background of total IgG by using well-established immunosorbent assays for purifying antibodies of the desired specificity using immobilized antigen. Alternative methods with sufficient glycan resolution lack these important specificities. GlYcoLISA is performed in a 96-well plate format, and the analysis of 160 samples takes ~5 d, with 1 d for sample preparation, 2 d of LC-MS measurement and 2 d for partially automated data processing. GlYcoLISA requires expertise in LC-MS operation and data processing.

Impact of antibody architecture and paratope valency on effector functions of bispecific NKp30 x EGFR natural killer cell engagers.

Natural killer (NK) cells emerged as a promising effector population that can be harnessed for anti-tumor therapy. In this work, we constructed NK cell engagers (NKCEs) based on NKp30-targeting single domain antibodies (sdAbs) that redirect the cytotoxic potential of NK cells toward epidermal growth factor receptor (EGFR)-expressing tumor cells. We investigated the impact of crucial parameters such as sdAb location, binding valencies, the targeted epitope on NKp30, and the overall antibody architecture on the redirection capacity. Our study exploited two NKp30-specific sdAbs, one of which binds a similar epitope on NKp30 as its natural ligand B7-H6, while the other sdAb addresses a non-competing epitope. For EGFR-positive tumor targeting, humanized antigen-binding domains of therapeutic antibody cetuximab were used. We demonstrate that NKCEs bivalently targeting EGFR and bivalently engaging NKp30 are superior to monovalent NKCEs in promoting NK cell-mediated tumor cell lysis and that the architecture of the NKCE can substantially influence killing capacities depending on the NKp30-targeting sdAb utilized. While having a pronounced impact on NK cell killing efficacy, the capabilities of triggering antibody-dependent cellular phagocytosis or complement-dependent cytotoxicity were not significantly affected comparing the bivalent IgG-like NKCEs with cetuximab. However, the fusion of sdAbs can have a slight impact on the NK cell release of immunomodulatory cytokines, as well as on the pharmacokinetic profile of the NKCE due to unfavorable spatial orientation within the molecule architecture. Ultimately, our findings reveal novel insights for the engineering of potent NKCEs triggering the NKp30 axis.

Assessment of IgM DSAs in Transplant Recipients: Relationship to De Novo IgG DSAs and Risk for Antibody Rejection.

The presence of anti-HLA donor-specific antibodies (DSAs) is associated with antibody-mediated rejection (AMR) and inferior graft survival. However, recent data suggest that ~50% of AMR episodes are IgG DSA negative and possibly related to non-HLA DSAs. After the initial activation of B cells to alloantigen, IgM is the first immunoglobulin produced. In addition, both IgM and IgG isotopes can activate the classic complement pathway and induce complement-dependent cytotoxicity to allograft targets. Current practices focus on the assessment of IgG DSAs with little concern for the assessment of IgM DSAs. Here, we examined anti-HLA IgM in a cohort of 22 patients who developed de novo IgG DSAs by a modified single-antigen bead-based test. We found IgM HLA DSAs developed before IgG DSAs. The median time from the detection of IgM DSAs to the appearance of de novo IgG DSAs was 461 d. Most patients had IgM DSAs against the same HLA-DQ antigens, for which IgG de novo DSAs were also later detected. IgM DSAs were detected in patients with biopsies suspected of AMR. The detection of IgM DSAs could be an early indicator of alloimmune responses to allografts before IgG de novo DSAs appear.

CD46 and CD59 inhibitors enhance complement-dependent cytotoxicity of anti-CD38 monoclonal antibodies daratumumab and isatuximab in multiple myeloma and other B-cell malignancy cells

ABSTRACT Multiple myeloma (MM) is an incurable malignancy of the B-cell lineage. Remarkable progress has been made in the treatment of MM with anti-CD38 monoclonal antibodies such as daratumumab and isatuximab, which can kill MM cells by inducing complement-dependent cytotoxicity (CDC). We showed that the CDC efficacy of daratumumab and isatuximab is limited by membrane complement inhibitors, including CD46 and CD59, which are upregulated in MM cells. We recently developed a small recombinant protein, Ad35K++, which is capable of transiently removing CD46 from the cell surface. We also produced a peptide inhibitor of CD59 (rILYd4). In this study, we tested Ad35K++ and rILYd4 in combination with daratumumab and isatuximab in MM cells as well as in cells from two other B-cell malignancies. We showed that Ad35K++ and rILYd4 increased CDC triggered by daratumumab and isatuximab. The combination of both inhibitors had an additive effect in vitro in primary MM cells as well as in vivo in a mouse xenograft model of MM. Daratumumab and isatuximab treatment of MM lines (without Ad35K++ or rILYd4) resulted in the upregulation of CD46/CD59 and/or survival of CD46high/CD59high MM cells that escaped the second round of daratumumab and isatuximab treatment. The escape in the second treatment cycle was prevented by the pretreatment of cells with Ad35K++. Overall, our data demonstrate that Ad35K++ and rILYd4 are efficient co-therapeutics of daratumumab and isatuximab, specifically in multi-cycle treatment regimens, and could be used to improve treatment of multiple myeloma.

Using capillary electrophoresis sodium dodecyl sulfate (CE-SDS) and liquid chromatograph mass spectrometry (LC-MS) to identify glycosylated heavy chain heterogeneity in the anti-VEGFR-2 monoclonal antibody.

The size variant, which can be measured by capillary electrophoresis sodium dodecyl sulfate (CE-SDS), is a critical quality attribute of monoclonal antibodies (mAbs). The CE-SDS size heterogeneity can hardly be identified by tandem mass spectrometry, which is an intractable obstacle of mAb development and quality control across the industry. We analyzed the purity of an anti-vascular endothelial growth factor receptor 2 (VEGFR-2) mAb, an antagonist of the human VEGFR-2, through reduced CE-SDS and observed glycosylated heavy chain heterogeneity. The heterogeneity has potential impact on safety, efficacy, and stability of drugs for clinical use. Therefore, it should be characterized so as to evaluate its potential risk. In order to identify the heterogeneity, we used mass spectrometry to confirm that the molecular size heterogeneity was not due to peptide bond cleavage in the heavy chain. Subsequently, we employed mass-spectrometry-glycosylation profiling and CE-SDS analysis of various glycosidase-treated samples, in addition to the preparation of mAb references with different glycoforms. Ultimately, we demonstrated that the heavy chain heterogeneity was induced by different levels of galactosylation modifications which will potentially impact the efficacy of antibody drugs (i.e., complement-dependent cytotoxicity). In this study, potential risk caused by heavy chain size heterogeneity was evaluated, which addressed the obstacle of mAb development and quality control. Therefore, this study offers a feasible approach for the investigation and identification of heavy chain heterogeneity in reduced CE-SDS, providing a novel strategy for mAb quality control and evaluation.

Combinatory glycoengineering of monoclonal antibodies and its application in cancer therapy: a narrative review.

The application of immunotherapy, especially in terms of recombinant monoclonal antibodies (mAbs), is a revolution in the pharmaceutical industry field. Glycosylation plays an essential role in the structures and functions of mAbs, which must be carefully monitored and designed throughout their entire lifespan to ensure safety and efficacy. This review aimed to summarize the current status of the glycoengineering of mAbs for providing reference for the pharmaceutical industry. The application of glycoengineering of mAbs in cancer therapy will also be discussed in this article. We searched the PubMed/MEDLINE database to identify studies published between 1970 and 2023 that investigated glycoengineering of recombinant mAbs and its applications. The major findings of these studies were summarized. This article reviews the relationship between glycosylation profiles and antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), shelf-life, and other properties of mAbs. Furthermore, the rational design of glycosylation profiles provides solutions to the unmet needs of new drug development and biosimilar manufacturers. This review also emphatically describes various feasible strategies to optimize the glycosylation pattern in biomanufacturing reported in recent years, especially the approaches of combinatory glycoengineering explored in this field. Finally, we share examples of glycoengineering of mAbs applied in cancer therapy. Glycan modifications have been achieved through genetic and metabolic glycoengineering. A better understanding of the interplay between cells and the exogenous environment help the combinatorial strategy for glycoengineering in precise control mode. Furthermore, new high-tech approaches for glycoengineering are leading biological engineering and biotherapeutics to a new stage.

Role of Complement-dependent Cytotoxicity Crossmatch and HLA Typing in Solid Organ Transplant.

Solid organ transplantation is a life-saving medical operation that has progressed greatly because of developments in diagnostic tools and histocompatibility tests. Crossmatching for complement-dependent cytotoxicity (CDC) and human leukocyte antigen (HLA) typing are two important methods for checking graft compatibility and reducing the risk of graft rejection. HLA typing and CDC crossmatching are critical in kidney, heart, lung, liver, pancreas, intestine, and multi-organ transplantation. A systematic literature search was conducted on the internet, using PubMed, Scopus, and Google Scholar databases, to identify peer-reviewed publications about solid organ transplants, HLA typing, and CDC crossmatching. Recent advances in HLA typing have allowed for high-resolution evaluation, epitope matching, and personalized therapy methods. Genomic profiling, next-generation sequencing, and artificial intelligence have improved HLA typing precision, resulting in better patient outcomes. Artificial intelligence (AI) driven virtual crossmatching and predictive algorithms have eliminated the requirement for physical crossmatching in the context of CDC crossmatching, boosting organ allocation and transplant efficiency. This review elaborates on the importance of HLA typing and CDC crossmatching in solid organ transplantation.

Recombinant therapeutic proteins degradation and overcoming strategies in CHO cells.

Mammalian cell lines are frequently used as the preferred host cells for producing recombinant therapeutic proteins (RTPs) having post-translational modified modification similar to those observed in proteins produced by human cells. Nowadays, most RTPs approved for marketing are produced in Chinese hamster ovary (CHO) cells. Recombinant therapeutic antibodies are among the most important and promising RTPs for biomedical applications. One of the issues that occurs during development of RTPs is their degradation, which caused by a variety of factors and reducing quality of RTPs. RTP degradation is especially concerning as they could result in reduced biological functions (antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity) and generate potentially immunogenic species. Therefore, the mechanisms underlying RTP degradation and strategies for avoiding degradation have regained an interest from academia and industry. In this review, we outline recent progress in this field, with a focus on factors that cause degradation during RTP production and the development of strategies for overcoming RTP degradation. KEY POINTS: • The recombinant therapeutic protein degradation in CHO cell systems is reviewed. • Enzymatic factors and non-enzymatic methods influence recombinant therapeutic protein degradation. • Reducing the degradation can improve the quality of recombinant therapeutic proteins.

A phase I study of EO-3021 in adult patients with solid tumors likely to express CLDN18.2.

TPS429 Background: Claudin 18 isoform 2 (CLDN18.2) belongs to a family of tight junction proteins with broad expression in gastric and gastroesophageal junction (GEJ), pancreatic, esophageal, and other solid tumors. Expression of CLDN18.2 in normal tissue is limited to the gastric mucosa, making it a promising antibody-drug conjugate (ADC) therapeutic target. Currently, there are no approved therapies targeting CLDN18.2. EO-3021 (also known as SYSA1801) is an ADC comprised of a fully human CLDN18.2 monoclonal antibody (mAb) specifically conjugated at glutamine 295 with a cleavable linker and MMAE with a homogenous drug-to-antibody ratio (DAR) of 2. In preclinical models, EO-3021 selectively delivers a potent cytotoxic MMAE payload directly to cancer cells expressing CLDN18.2, retains antibody-dependent cellular toxicity (ADCC) and complement-dependent cytotoxicity (CDC) activity, and exhibits a bystander effect.1 In an ongoing Phase 1 study in China (NCT05009966), EO-3021 exhibited signs of anti-tumor activity in patients with gastric cancer and had an acceptable safety profile.2 Outside of Greater China, EO-3021 is being evaluated by Elevation Oncology for the treatment of patients with advanced or metastatic solid tumors likely to express CLDN18.2, particularly gastric and GEJ adenocarcinomas. Methods: This is a Phase I, open-label, multi-center, dose escalation and expansion study to investigate the safety, tolerability, pharmacokinetics (PK) and preliminary anti-tumor activity of EO-3021 in patients with solid tumors likely to express CLDN18.2 (NCT05980416). Patients whose tumors have progressed on or after standard therapy, or who are intolerable for available standard therapy are eligible for participation. Approximately 120 patients will be enrolled in this study. There are 4 planned dose levels in the dose finding portion of the study. Patients will receive EO-3021 intravenously (IV) once every three weeks until disease progression or unacceptable toxicity. The expansion cohort will evaluate EO-3021 in patients with gastric/GEJ adenocarcinoma. Provision of tumor samples (archived and fresh biopsy) is required for study enrollment. CLDN18.2 expression in tumor tissue via will be assessed by retrospective evaluation via central immunohistochemistry assay; results will be explored for possible correlation with tumor response. Enrollment in the dose escalation portion of the study began in August 2023. 1. Dan M, et al. Cancer Res. 83, 2023. 2. Wang Y, et al. J Clin Oncol. 41, 2023. Clinical trial information: NCT05980416 .

Dynamics of HLA and angiotensin II type 1 receptor antibodies during pregnancy

BackgroundAlloantibodies, especially anti-human leukocyte antigen antibodies (HLA antibodies), and autoantibodies, as angiotensin II type 1 receptor antibodies (AT1R antibodies), may complicate the access and the course of transplantation. Pregnancy is a known source of HLA antibodies, with most studies evaluating pregnancy-induced sensitization by complement-dependent cytotoxicity assays, mainly after childbirth. AT1R antibodies have been evaluated in the context of preeclampsia. We aimed to evaluate pregnancy as a natural source of HLA antibodies and AT1R antibodies, their dynamics along gestation and the potential factors involved in antibody appearance. MethodsSerum samples from pregnant women were collected during the three trimesters of pregnancy (1T, 2T, 3T). Presence of HLA antibodies was assessed by screening beads on Luminex and AT1R antibodies by ELISA. ResultsA cohort of 138 pregnant women were included. Samples from all were tested in 1T, 127 in 2T and 102 in 3T. HLA antibodies increased from 29.7 % (1T) to 38.2 % (3T). AT1R antibodies were stable around 30 % along pregnancy. Up to 43.2 % multiparous women had HLA antibodies, with a similar proportion of class I and class II antibodies. In primiparous women HLA antibodies increased along pregnancy (from 17.6 % to 34.1 %), with predominance of class II HLA antibodies. AT1R antibodies were not different in primiparous and multiparous women. ConclusionsPregnancy is a relevant source of HLA antibodies sensitization, but not of AT1R antibodies. HLA antibodies increased clearly in primiparous women with predominance of class II. The use of newer solid-phase techniques on Luminex evidence a higher degree of HLA sensitization during pregnancy.

Rhamnose modified antibodies show improved immune killing towards EGFR-positive solid tumor cells

Therapeutic monoclonal antibodies (mAbs) against the epidermal growth factor receptor (EGFR) have shown clinical efficacy in colorectal cancer and other solid cancers. Enhancing the effector functions of these anti-EGFR mAbs is believed to be a valuable approach to achieve improved efficacy in clinical setting. Here, we report the development of an effector function-enhanced antibody by rhamnose (Rha) functionalization. Cetuximab, a human/mouse chimeric anti-EGFR mAb, was selected and site-specifically conjugated with Rha haptens. The obtained cetuximab-Rha conjugate was shown to be able to selectively redirect amounts of endogenous anti-Rha antibodies onto EGFR-positive solid tumor cells and thereby provide more Fc domains to achieve enhancement of effector functions including complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated phagocytosis (ADCP). Particularly, CDC, one powerful cell killing mechanism which is inactive in cetuximab, was dramatically improved. This study demonstrates the potential of rhamnose-modified antibody for EGFR-positive solid tumor immunotherapy.

A Review of Anti-CD20 Antibodies in the Management of B-Cell Lymphomas

Anti-CD20 monoclonal antibodies (mAbs) have revolutionized the treatment of lymphomas by improving the survival of patients, particularly in conjunction with chemotherapy. Until recently, the gold standard was based on the utilization of Rituximab (RTX) combined with chemotherapy. With our better understanding of monoclonal antibody (mAb) engineering, anti-CD20 mAb therapy has evolved to enhance clinical outcomes by improving pharmacokinetics, safety, activity and immunogenicity. Efforts to improve the on-targeting CD20 expressed on lymphomas through novel bioengineering techniques have led to the development of newer anti-CD20 mAbs that have accentuated complement-dependent cytotoxicity (CDC), antibody-dependent cell medicated cytotoxicity (ADCC), and/or a direct killing effect. There are several anti-CD20 monoclonal antibodies that have been evaluated for the treatment of lymphomas, some of which are now approved in addition to RTX.