Therapeutic Antibody Drug Research Articles

Considerations for Using Phage Display Technology in Therapeutic Antibody Drug Discovery.

Phage display is a versatile and effective platform for the identification and engineering of biologic-based therapeutics. Using standard molecular biology laboratory techniques, one can create a highly diverse and functional antibody phage-displayed library, and rapidly identify antibody fragments that bind to a target of interest with exquisite specificity and high affinity. Here, we discuss key aspects for the development of an antibody discovery strategy to harness the power of phage display technology to obtain molecules that can successfully be developed into therapeutics, including target validation, antibody design goals, and considerations for preparing and executing phage panning campaigns. Careful design and implementation of discovery campaigns-regardless of the target-provides the best chance of identifying desirable antibody fragments for further therapeutic development, so these principles can be applied to any new discovery project.

Discovery of a common light chain bispecific antibody targeting PD-1 and PD-L1 by Hybridoma-to-phage-to-yeast (H2PtY) platform

Abstract Background Therapeutic antibody drugs targeting the PD-1 pathway are generally characterized by relatively low response rates and susceptibility to drug resistance during clinical application. Therefore, there is an urgent need for alternative therapeutic strategies to increase the immune response rate. Bispecific antibodies co-targeting PD-1 and PD-L1 may have greater potential to improve the efficacy of the immune checkpoint pathway. Method In this study, we developed a potent humanized common light chain IgG shape bispecific antibody, named JMB2005, based on Hybridoma-to-Phage-to-Yeast platform. The platform allowed us to discover common light chain bispecific antibody from traditional mice for any pair of given targets. Results JMB2005 exhibited favorable developability, good manufacturing property, and satisfactory efficacy, which could be given via subcutaneous injection at the concentration of 120 mg/mL. Mechanistically, JMB2005 could bridge tumor cells and T cells with both Fab arms and promote T-cells to function as direct tumor cell killers. It could also promote T cell activation by blocking the binding of PD-L1 to CD80. Furthermore, JMB2005 has exhibited a favorable half-life and has demonstrated promising anti-tumor therapeutic efficacy in vivo. Conclusion Consequently, the present study showed that the novel humanized common light chain bispecific antibody JMB2005 may represent a novel therapeutic agent of great clinical potential.

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.

Potent and broadly neutralizing antibodies against sarbecoviruses induced by sequential COVID-19 vaccination

The current SARS-CoV-2 variants strikingly evade all authorized monoclonal antibodies and threaten the efficacy of serum-neutralizing activity elicited by vaccination or prior infection, urging the need to develop antivirals against SARS-CoV-2 and related sarbecoviruses. Here, we identified both potent and broadly neutralizing antibodies from a five-dose vaccinated donor who exhibited cross-reactive serum-neutralizing activity against diverse coronaviruses. Through single B-cell sorting and sequencing followed by a tailor-made computational pipeline, we successfully selected 86 antibodies with potential cross-neutralizing ability from 684 antibody sequences. Among them, PW5-570 potently neutralized all SARS-CoV-2 variants that arose prior to Omicron BA.5, and the other three could broadly neutralize all current SARS-CoV-2 variants of concern, SARS-CoV and their related sarbecoviruses (Pangolin-GD, RaTG13, WIV-1, and SHC014). Cryo-EM analysis demonstrates that these antibodies have diverse neutralization mechanisms, such as disassembling spike trimers, or binding to RBM or SD1 to affect ACE2 binding. In addition, prophylactic administration of these antibodies significantly protects nasal turbinate and lung infections against BA.1, XBB.1, and SARS-CoV viral challenge in golden Syrian hamsters, respectively. Importantly, post-exposure treatment with PW5-5 and PW5-535 also markedly protects against XBB.1 challenge in these models. This study reveals the potential utility of computational process to assist screening cross-reactive antibodies, as well as the potency of vaccine-induced broadly neutralizing antibodies against current SARS-CoV-2 variants and related sarbecoviruses, offering promising avenues for the development of broad therapeutic antibody drugs.

Monoclonal antibody precision therapy targeting inflammation for bipolar disorder: a narrative review.

Bipolar disorder (BD) is a severe mental disorder with various hypotheses regarding its pathogenesis. This article provides a summary of numerous studies on the variations in inflammatory cytokine levels in patients with BD and the effects of treatment with antipsychotics, mood stabilizers, and antidepressants on these levels. In addition, patients with autoimmune diseases who use anti-inflammatory monoclonal antibodies experience symptoms, such as depression, anxiety, and insomnia. These pieces of evidence suggest a potential association between immune inflammation and BD and offer new possibilities for therapy. Building upon this relationship, the authors propose an innovative approach for treating BD through individualized and precise therapy using anti-inflammatory monoclonal antibody drugs. To support this proposal, the authors compile information on pharmacological effects and relevant studies, including trials of various anti-inflammatory therapeutic monoclonal antibody drugs (e.g. infliximab, tocilizumab, and canakinumab) for the potential treatment of BD and its associated side effects in psychiatry. The authors categorize these anti-inflammatory monoclonal antibody drugs into levels I-IV through a comprehensive analysis of their advantages and disadvantages. Their potential is examined, and the need for further exploration of their pharmaceutical effects is established.

Evaluation of the In-Use Stability of Monoclonal Antibody IV Admixtures Prepared from Drug Products Containing Polysorbate 20 Degraded by Host-Cell Lipases

Host-cell lipases can be present in monoclonal antibody drug products and can degrade polysorbates present in the formulations as stabilizers. We hypothesized that the in-use stability of the IV admixture prepared from such a drug product might be impacted by decreasing levels of polysorbate 20. Host-cell lipase activity has, in fact, been observed during development of one of our therapeutic monoclonal antibody drug products. Throughout the course of the product shelf life, polysorbate 20 levels decreased but no other quality attributes of the drug product were impacted. An experimental approach was developed to simulate how the prepared IV admixture in-use stability is affected as polysorbate 20 concentration in the drug product decreased over the shelf life, and from that a minimum level of polysorbate 20 required in the drug product was determined to estimate the in-use stability of the IV admixture as the polysorbate 20 in the drug product degrades. The results indicate that although the observed degradation of polysorbate 20 does not affect quality attributes of this drug product, in-use stability of the IV admixture as a function of polysorbate degradation can be impacted and should be assessed to ensure sufficient quality.

Receptor-mediated drug delivery of bispecific therapeutic antibodies through the blood-brain barrier.

Therapeutic antibody drug development is a rapidly growing sector of the pharmaceutical industry. However, antibody drug development for the brain is a technical challenge, and therapeutic antibodies for the central nervous system account for ~3% of all such agents. The principal obstacle to antibody drug development for brain or spinal cord is the lack of transport of large molecule biologics across the blood-brain barrier (BBB). Therapeutic antibodies can be made transportable through the blood-brain barrier by the re-engineering of the therapeutic antibody as a BBB-penetrating bispecific antibody (BSA). One arm of the BSA is the therapeutic antibody and the other arm of the BSA is a transporting antibody. The transporting antibody targets an exofacial epitope on a BBB receptor, and this enables receptor-mediated transcytosis (RMT) of the BSA across the BBB. Following BBB transport, the therapeutic antibody then engages the target receptor in brain. RMT systems at the BBB that are potential conduits to the brain include the insulin receptor (IR), the transferrin receptor (TfR), the insulin-like growth factor receptor (IGFR) and the leptin receptor. Therapeutic antibodies have been re-engineered as BSAs that target the insulin receptor, TfR, or IGFR RMT systems at the BBB for the treatment of Alzheimer's disease and Parkinson's disease.

A Novel Conserved Linear Neutralizing Epitope on the Receptor-Binding Domain of the SARS-CoV-2 Spike Protein.

The continuous emergence of new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has made it challenging to develop broad-spectrum prophylactic vaccines and therapeutic antibodies. Here, we have identified a broad-spectrum neutralizing antibody and its highly conserved epitope in the receptor-binding domain (RBD) of the spike protein (S) S1 subunit of SARS-CoV-2. First, nine monoclonal antibodies (MAbs) against the RBD or S1 were generated; of these, one RBD-specific MAb, 22.9-1, was selected for its broad RBD-binding abilities and neutralizing activities against SARS-CoV-2 variants. An epitope of 22.9-1 was fine-mapped with overlapping and truncated peptide fusion proteins. The core sequence of the epitope, 405D(N)EVR(S)QIAPGQ414, was identified on the internal surface of the up-state RBD. The epitope was conserved in nearly all variants of concern of SARS-CoV-2. MAb 22.9-1 and its novel epitope could be beneficial for research on broad-spectrum prophylactic vaccines and therapeutic antibody drugs. IMPORTANCE The continuous emergence of new variants of SARS-CoV-2 has caused great challenge in vaccine design and therapeutic antibody development. In this study, we selected a broad-spectrum neutralizing mouse monoclonal antibody which recognized a conserved linear B-cell epitope located on the internal surface of RBD. This MAb could neutralize all variants until now. The epitope was conserved in all variants. This work provides new insights in developing broad-spectrum prophylactic vaccines and therapeutic antibodies.

Evaluation of novel anti-CEACAM6 antibody-based conjugates for radioimmunotheranostics of pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a highly malignant solid tumor that lacks early diagnostic methods. Recently, targeted immunotherapy and radiotherapy have been integrated with radionuclide-antibody conjugate drugs, which can be used for targeted diagnosis and dynamic imaging of tumors. CEACAM6 is overexpressed in pancreatic tumors and is a potential theranostic target for PDAC. We aimed to develop a novel targeted carrier for theranostics of PDAC and other solid tumors. Based on camelid heavy-chain-only antibodies, we developed a CEACAM6-targeting recombinant antibody NY004, and evaluated it as a novel antibody-carrier for imaging and therapy of cancer in tumor models. We labeled NY004 with theranostic nuclides and applied this self-developed antibody platform in diagnostic imaging and antitumor assessment in PDAC models. Through microPET, IHC, and biodistribution assays, targeting and biodistribution of [89Zr]-NY004 in solid tumors including PDAC was examined, and the investigated tumors were all CEACAM6-positive malignancies. We found that NY004 was suitable for use as a drug carrier for radioimmunotheranostics. Our study showed that NY004 was characterized by high targeted uptake and a long retention time in PANC-1 tumors (up to 6days post-injection), with good specificity and high imaging efficiency. Therapeutic evaluation of the radionuclide-labeled antibody drug [177Lu]-NY004 in PDAC tumor-bearing model revealed that NY004 had high and prolonged uptake in tumors, relatively low non-target organ uptake, and good anti-tumor efficacy. As a drug platform for radiotheranostics, CEACAM6-specific antibody NY004 met the requirements of easy-labeling, targeting specificity, and effective persistence in pancreatic adenocarcinoma tissues. • [89Zr]-NY004 has good specificity and high imaging efficiency, and is characterized by high tumor-targeting uptake and a long tumor retention time as a PET molecular imaging tracer. • Therapeutic radionuclide-conjugated antibody drug [177Lu]-NY004 has high uptake and prolonged uptake duration in tumors, low non-target organ uptake, and significant tumor-inhibiting efficacy in PDAC model. • The self-developed antibody structure NY004 is a promising drug platform for radioimmunotheranostics of CEACAM6-positive tumors including pancreatic ductal adenocarcinoma.

Latest Findings on the Role of CD47 in Tumor Immune Evasion and Related Targeted Therapies

CD47 is an immunoglobulin that is overexpressed on the surface of a variety of cancer cells. CD47 forms a signaling complex with signal regulatory protein alpha (SIRPα), prompting the escape of cancer cells from macrophage-mediated phagocytosis. In recent years, CD47 has been shown to be highly expressed in many types of solid tumors and is associated with poor prognosis in patients. More and more studies have shown that inhibition of the CD47-SIRPα signaling pathway can promote adaptive immune responses and enhance the phagocytosis of tumor cells by macrophages. Humanized anti-CD47 IgG4 monoclonal antibody has been studied in clinical trials for the treatment of a variety of advanced solid tumors and lymphomas, demonstrating a sound safety profile and achieving partial remission in some patients. In this review we discuss the structure and function of CD47 and the mechanism of CD47 regulation in tumors, summarize the research progress in therapeutic antibody drugs targeting CD47 and a bottleneck in research that targeted drugs are more prone to result in serious adverse effects, and evaluated the potential of the applying CD47-SIRPα signaling pathway in anti-cancer therapy.

Exploring the sheep (Ovis aries) immunoglobulin repertoire by next generation sequencing.

Next-generation sequencing (NGS) has revolutionized the way we determine the antibody repertoires encoded by B cells in the blood or lymphoid organs and transformed our understanding of adaptive immune responses in many species. Sheep (Ovis aries) have been widely used as a host for therapeutic antibody production since the early 1980s, however, little is known about their immune repertoires or immunological processes affecting the antibody generation. The objective of this study was to employ NGS for a comprehensive analysis of immunoglobulin heavy and light chain repertoires in four healthy sheep. We obtained >90% complete antibody sequences and nearly 130,000, 48,000 and 218,000 unique CDR3 reads for the heavy chain (IGH), kappa chain (IGK), and lambda chain (IGL) loci, respectively. Consistent with other species, we observed biased usage of germline variable (V), diversity (D) and joining (J) genes in the heavy and kappa loci, but not in the lambda loci. Moreover, the enormous diversity of CDR3 sequences was observed through sequence clustering and convergent recombination. These data will build a foundation for future studies investigating immune repertoires in health and disease as well as contribute to further refinement of ovine-derived therapeutic antibody drugs.

Optimized ACE2 decoys neutralize antibody-resistant SARS-CoV-2 variants through functional receptor mimicry and treat infection in vivo.

As severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) evolves to escape natural antibodies, it also loses sensitivity to therapeutic antibody drugs. By contrast, evolution selects for binding to ACE2, the cell-surface receptor required for SARS-CoV-2 infection. Consistent with this, we find that an ACE2 decoy neutralizes antibody-resistant variants, including Omicron, with no loss in potency. To identify design features necessary for in vivo activity, we compare several enzymatically inactive, Fc effector-silenced ACE2-Fc decoys. Inclusion of the ACE2 collectrin-like domain not only improves affinity for the S protein but also unexpectedly extends serum half-life and is necessary to reduce disease severity and viral titer in Syrian hamsters. Fc effector function is not required. The activity of ACE2 decoy receptors is due, in part, to their ability to trigger an irreversible structural change in the viral S protein. Our studies provide a new understanding of how ACE2 decoys function and support their development as therapeutics to treat ACE2-dependent coronaviruses.

Challenges and Strategies for a Thorough Characterization of Antibody Acidic Charge Variants.

Heterogeneity of therapeutic Monoclonal antibody (mAb) drugs are due to protein variants generated during the manufacturing process. These protein variants can be critical quality attributes (CQAs) depending on their potential impact on drug safety and/or efficacy. To identify CQAs and ensure the drug product qualities, a thorough characterization is required but challenging due to the complex structure of biotherapeutics. Past characterization studies for basic and acidic variants revealed that full characterizations were limited to the basic charge variants, while the quantitative measurements of acidic variants left gaps. Consequently, the characterization and quantitation of acidic variants are more challenging. A case study of a therapeutic mAb1 accounted for two-thirds of the enriched acidic variants in the initial characterization study. This led to additional investigations, closing the quantification gaps of mAb1 acidic variants. This work demonstrates that a well-designed study with the right choices of analytical methods can play a key role in characterization studies. Thus, the updated strategies for more complete antibody charge variant characterization are recommended.

Host cell protein identification in monoclonal antibody high molecular weight species

High molecular weight (HMW) species are product-related variants that may impact therapeutic product safety and efficacy. Therefore, HMW species and aggregates are considered critical quality attributes and their levels should be closely monitored and controlled during drug development, commercial manufacturing, and shelf-life storage period for therapeutic monoclonal antibody drug products. Various biophysical and analytical methods have been developed to characterize the HMW species to understand their mechanisms of formation and assess potential product risk. However, host cell protein (HCP) analysis has seldom been conducted to characterize the impurities in aggregates. In this work, HCP analysis of enriched HMW species and drug substance (DS) from five different monoclonal antibodies (mAbs) was performed. More HCPs are identified in the enriched HMW than in the DS, thus demonstrating a potential interaction between HCPs and HMW. Certain HCPs, including commonly detected HCPs and problematic HCPs, were enriched in HMW fractions. Especially, the most abundant HCP from mAb1, CC motif chemokine, was 46 times more abundant in enriched HMW than DS. The enriched HMW was further fractionated into enriched dimers and enriched very HMW (vHMW) fractions. The CC motif chemokine was found to interact mainly with mAb1 dimer species rather than vHMW fraction. Removing the HMW species from mAb1 significantly decreased the CC motif chemokine level in the final mAb1 DS. Our findings demonstrate that some HCPs are more preferentially bound to HMW species and this finding may provide a new opportunity for removing HCPs in downstream purification steps.

Structure-guided and phage-assisted evolution of a therapeutic anti-EGFR antibody to reverse acquired resistance

Acquired resistance to cetuximab in colorectal cancers is partially mediated by the acquisition of mutations located in the cetuximab epitope in the epidermal growth factor receptor (EGFR) ectodomain and hinders the clinical application of cetuximab. We develop a structure-guided and phage-assisted evolution approach for cetuximab evolution to reverse EGFRS492R- or EGFRG465R-driven resistance without altering the binding epitope or undermining antibody efficacy. Two evolved cetuximab variants, Ctx-VY and Ctx-Y104D, exhibit a restored binding ability with EGFRS492R, which harbors the most common resistance substitution, S492R. Ctx-W52D exhibits restored binding with EGFR harboring another common cetuximab resistance substitution, G465R (EGFRG465R). All the evolved cetuximab variants effectively inhibit EGFR activation and downstream signaling and induce the internalization and degradation of EGFRS492R and EGFRG465R as well as EGFRWT. The evolved cetuximab variants (Ctx-VY, Ctx-Y104D and Ctx-W52D) with one or two amino acid substitutions in the complementarity-determining region inherit the optimized physical and chemical properties of cetuximab to a great extent, thus ensuring their druggability. Our data collectively show that structure-guided and phage-assisted evolution is an efficient and general approach for reversing receptor mutation-mediated resistance to therapeutic antibody drugs.

Abstract 332: Development of a novel therapeutic CLDN18.2 monoclonal antibody and antibody drug conjugate for the treatment of CLDN18.2 positive cancers

Abstract Background: Claudin 18 is a member of the claudin family of tight junction tetraspan cell surface proteins. The 18.2 isoform of claudin 18 (CLDN18.2) is expressed in healthy gastric mucosa cells and largely absent from other normal adult cell types. In contrast to the low expression in healthy tissues, CLDN18.2 is highly overexpressed in many cancer types including gastric and pancreatic malignancies at high frequencies (50-80%). The high tumor-normal expression differential of CLDN18.2 make it an attractive target for both therapeutic monoclonal antibodies (mAbs) and antibody drug conjugate (ADCs). Here, we describe the preclinical development of a novel therapeutic CLDN18.2 mAb and ADC. Materials and Methods: CLDN18.2 specific mAbs were generated through a multiplexed immunization strategy by which mice were immunized with either peptides spanning loop 2 of the CLDN18.2 extracellular domain or mouse NIH3T3 cells overexpressing the full length CLDN18.2 protein. Antibody clones were screened by flow cytometry for selective binding to cell surface CLDN18.2. A CLDN18.2-directed ADC was generated from the fully humanized clinical candidate CLDN18.2 mAb by MMAE conjugation with a cleavable linker. CLDN18.2 positivity in cell line (CDX) and patient derived xenograft (PDX) models was determined by IHC assay. Results: Selective binding of the CLDN18.2-mAb to CLDN18.2, without cross reactivity to the more widely expressed CLDN18.1 isoform, was confirmed in human cancer cell lines and cells engineered to overexpress each isoform. The CLDN18.2-mAb demonstrated strong induction of both antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP) in in vitro assays; suggesting that Fc effector function is a key component of the mechanism of action of the mAb. Treatment of CLDN18.2 positive gastric and pancreatic CDXs with CLDN18.2-mAb monotherapy induced significant inhibition of tumor growth in CD-1 nude mice. Binding of CLDN18.2-mAb or CLDN18.2-ADC to the cell surface CLDN18.2 induced internalization of the protein-antibody complex. Complete regression of CDX tumors was observed in response to treatment with the CLDN18.2-ADC in three separate CLDN18.2 positive models (two pancreas, one gastric). Moreover, complete loss of xenograft tumor burden was also observed in 4/5 CLDN18.2 positive pancreas PDX models. No impact on xenograft tumor growth as observed in response to treatment with either the CLDN18.2-mAb or CLDN18.2-ADC in CLDN18.2 negative CDX/PDX models. Discussion: These data support the clinical development of this CLDN18.2-mAb and CLDN18.2-ADC for the treatment of CLDN18.2 positive cancers. Each of these molecules will begin phase 1 clinical testing in early 2022. Citation Format: Neil A. O'brien, Martina S. McDermott, Jun Zhang, Ke Wei Gong, Ming Lu, Benjamin G. Hoffstrom, Dylan Conklin, Tong Luo, Kevin Chau, Min Liang, Timothy R. Donahue, John A. Glaspy, Leonard Presta, Dennis J. Slamon. Development of a novel therapeutic CLDN18.2 monoclonal antibody and antibody drug conjugate for the treatment of CLDN18.2 positive cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 332.

Abstract 6020: Preclinical activity of LM-305 targeting G-protein-coupled receptor class 5 member D (GPRC5D) antibody drug conjugate for the treatment of multiple myeloma

Abstract Emerging therapies including chimeric antigen receptor (CAR) T therapy targeting B cell maturation antigen (BCMA) have shown promising clinical outcomes in relapsed and refractory multiple myeloma (RRMM). However, majority of patients eventually develop progressive disease due to heterogeneity in BCMA expression or loss of antigen during treatment. Therefore, new targets are required to overcome the problem of resistance encountered with these agents. The expression of GPRC5D (G-protein-coupled receptor class 5 member D) is high on MM cells and low in normal tissues. Although high levels of GPRC5D and BCMA were found in similar proportions of MM patients, they are independently expressed by MM cells. GPRC5DxCD3 bispecific T-cell-redirecting antibody is currently under clinical investigation. Preliminary results have shown early evidence in efficacy with tolerable safety profile. These findings identified GPRC5D as an ideal therapeutic antibody drug conjugate (ADC) target for the treatment of patients with RRMM. Therefore, the present study was aimed at developing a novel anti-GPRC5D ADC drug LM-305 and evaluating its preliminary efficacy by performing preclinical activity using in vitro and in vivo mouse models. The binding affinity of LM-305 was evaluated by flow cytometry in engineered GPRC5D over-expressing MM tumor cells and endogenous GPRC5D-expressing MM tumor cells. The internalization of LM-305 was assessed with pH-dependent dye in NCI-H929 and MM.1R cell lines. The cytotoxicity of LM-305 was assessed by cell viability assay in MM tumor cell lines. In vivo anti-tumor activity of multiple doses of LM-305 (1mg/kg, 3mg/kg, 10mg/kg) was evaluated in NCI-H929 and MM.1R tumor cell line derived xenograft (CDX) models. In vitro studies revealed that LM-305 binds with high affinity to GPRC5D over-expressing cell lines and GPRC5D endogenously expressing MM cells in a dose-dependent manner. It can be efficiently internalized by GPRC5D expressing cells and further lysosomal lysis was detected by pHrodo dye. LM-305 displayed potent cytotoxicity when co-cultured with MM tumor cells (NCI-H929 and MM.1R) with IC50 values ranging from 0.1 to 0.3 nM. In vivo tumor xenograft models suggested that treatment with LM-305 resulted in dose-dependent inhibition of tumor growth in tumor bearing mice. Additionally, LM-305 exhibited complete response (CR) in the GPRC5D high-expressing MM CDX models at a dose of 3mg/kg. Moreover, LM-305 showed good safety profile in the animal studies. In conclusion, this preclinical data suggested that LM-305 is a novel GPRC5D targeting ADC with best-in-class potential, and therefore it can be a promising therapeutic candidate for the treatment of RRMM patients expressing GPRC5D. Citation Format: Wentao Huang, Jie Luo, Yifan Li, Da Fei, Xia Qin, Runsheng Li. Preclinical activity of LM-305 targeting G-protein-coupled receptor class 5 member D (GPRC5D) antibody drug conjugate for the treatment of multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6020.

A Novel and Secure Pseudovirus Reporter System Based Assay for Neutralizing and Enhancing Antibody Assay Against Marburg Virus.

Marburg virus (MARV) is one of the principal members of the filovirus family, which can cause fatal hemorrhagic fever in humans. There are currently no prophylactic and therapeutic drugs on the market, and the high pathogenicity and infectivity of MARV make its research highly dependent on biosafety level 4 conditions, severely hindering the development of vaccines and therapies. Therefore, the development of medicines, such as MARV serological diagnosis, vaccines, and therapeutic antibody drugs, urgently needs a safe, convenient, and biosafety level 2 detection method to measure the neutralizing activity of MARV antibodies. To this end, we report a neutralization assay relying on a Rabies virus (RABV) reverse genetic operating system. We constructed infectious clones carrying the eGFP reporter gene and the full length of the original unmodified MARV GP gene. Based on the critical parameters of phylogenetic analysis, recombinant viruses targeting representative strains in the two major MARV lineages were successfully rescued. These pseudoviruses are safe in mice, and their inability to infect cells after being neutralized by antibodies can be visualized under a fluorescence microscope. We tested the system using the neutralizing antibody MR191. MR191 can significantly block the infection of BSR cells with pseudovirus. We compared it with the traditional lentivirus-type pseudovirus system to verify the system’s credibility and obtained the same results as reported in the literature. In general, we have established a safe and visualized method for evaluating the neutralizing activity of MARV antibodies. Compared with traditional methods, it has the advantages of convenient operation, short cycle, and low cost. It is a candidate method that can replace actual viruses for a neutralization assay.

Purification and determination of antibody drugs in bio-samples by EGFR/cell membrane chromatography method

With the rapid development of therapeutic monoclonal antibody drugs, it is increasingly difficult to meet clinical needs using traditional antibody purification techniques. In this study, epidermal growth factor receptor (EGFR)-SNAP-tag was expressed in HEK293 cells. Then we captured the EGFR-SNAP-tag from the cell membrane suspension onto a O6-benzylguanine-modified silica gel to prepare a new EGFR stationary phase as a bioactive material, which could specifically recognize its antibody in bio-samples. The EGFR stationary phase was systematically characterized via scanning electron microscopy, transmission electron microscopy, X-ray photoelectron spectroscopy, and fourier transform infrared spectroscopy. Then we used EGFR stationary phase to establish a new EGFR cell membrane chromatography (CMC) model. The EGFR/CMC-online-ion exchange chromatography (IEC)/high performance liquid chromatography (HPLC) was established for the efficient purification and specific identification of cetuximab, nituzumab, and panizumab from cell culture medium and human serum. The results show that the EGFR stationary phase prepared by one-step immobilized technique can maintain biological activity and stability like EGFR in cell membrane. The EGFR/CMC-online-IEC/HPLC method has a high specificity, accuracy and sensitivity. Therefore, it will present a valuable method for the purification, identification, and analysis of monoclonal antibody drugs.

Development of a T cell-redirecting bispecific antibody targeting B-cell maturation antigen for the suppression of multiple myeloma cell growth.

BackgroundMultiple myeloma (MM) is the second most common hematological malignancy. It has emerged as one of the next possible hematological diseases amenable to immunotherapy. B-cell maturation antigen (BCMA), a member of the tumor necrosis factor receptor superfamily, is highly expressed in MM cells and is one target with the most potential for developing MM-targeting immunotherapy. Other than the FDA-approved BCMA-targeting CAR T-cell therapy, such as Abecma and CARVYKTI, T cell-engaging multi-specific antibody is another promising therapeutic modality for BCMA-targeting MM treatment. We develop a T-cell redirecting BCMA-targeting bispecific antibody (bsAb) and evaluate its anti-MM activity.MethodsWe first generated several clones of mouse anti-human BCMA monoclonal antibodies using DNA immunization. One of the anti-BCMA antibodies was then used to design and produce a T cell-redirecting BCMA × CD3 bsAb in CHO cells. Finally, we examined the effect of the bsAb on MM cell growth both in vitro and in vivo.ResultsThe BCMA × CD3 bsAb was designed in a FabscFv format and produced in CHO cells with good yield and purity. Moreover, the bsAb can trigger robust T cell proliferation and activation and induce efficient T cell-mediated MM cell killing in vitro. Using a MM xenograft mouse model, we demonstrate that the bsAb can effectively suppress MM cell growth in vivo.ConclusionsOur results suggest that the BCMA × CD3 bsAb in the FabscFv format can efficiently inhibit MM cell growth and have promising potential to be developed into a therapeutic antibody drug for the treatment of MM.