Chromatography affinity resin with photosynthetically-sourced protein A ligand.

Green, photosynthesizing plants can be proficiently used as cost-effective, single-use, fully biodegradable bioreactors for environmentally-friendly production of a variety of valuable recombinant proteins. Being near-infinitely scalable and most energy-efficient in generating biomass, plants represent profoundly valid alternatives to conventionally used stationary fermenters. To validate this, we produced a plastome-engineered tobacco bioreactor line expressing a recombinant variant of the protein A from Staphylococcus aureus, an affinity ligand widely useful in antibody purification processes, reaching accumulation levels up to ~ 250 mg per 1 kg of fresh leaf biomass. Chromatography resin manufactured from photosynthetically-sourced recombinant protein A ligand conjugated to agarose beads demonstrated the innate pH-driven ability to bind and elute IgG-type antibodies and allowed one-step efficient purification of functional monoclonal antibodies from the supernatants of the producing hybridomas. The results of this study emphasize the versatility of plant-based recombinant protein production and illustrate its vast potential in reducing the cost of diverse biotechnological applications, particularly the downstream processing and purification of monoclonal antibodies.

Anti-IL-8 monoclonal antibodies inhibits the autophagic activity and cancer stem cells maintenance within breast cancer tumor microenvironment.

BACKGROUND: Breast cancer tumor microenvironment (TME) is a promising target for immunotherapy. Autophagy, and cancer stem cells (CSCs) maintenance are essential processes involved in tumorigenesis, tumor survival, invasion, and treatment resistance. Overexpression of angiogenic chemokine interleukin-8 (IL-8) in breast cancer TME is associated with oncogenic signaling pathways, increased tumor growth, metastasis, and poor prognosis. OBJECTIVE: Thus, we aimed to investigate the possible anti-tumor effect of neutralizing antibodies against IL-8 by evaluating its efficacy on autophagic activity and breast CSC maintenance. METHODS: IL-8 monoclonal antibody supplemented tumor tissue culture systems from 15 females undergoing mastectomy were used to evaluate the expression of LC3B as a specific biomarker of autophagy and CD44, CD24 as cell surface markers of breast CSCs using immunofluorescence technique. RESULTS: Our results revealed that anti-IL-8 mAb significantly decreased the level of LC3B in the cultured tumor tissues compared to its non-significant decrease in the normal breast tissues.Anti-IL-8 mAb also significantly decreased the CD44 expression in either breast tumors or normal cultured tissues. While it caused a non-significant decrease in CD24 expression in cultured breast tumor tissue and a significant decrease in its expression in the corresponding normal ones. CONCLUSIONS: Anti-IL-8 monoclonal antibody exhibits promising immunotherapeutic properties through targeting both autophagy and CSCs maintenance within breast cancer TME.

Structure-guided mutagenesis of Henipavirus receptor-binding proteins reveals molecular determinants of receptor usage and antibody-binding epitopes.

Nipah virus (NiV) is a highly lethal, zoonotic Henipavirus (HNV) that causes respiratory and neurological signs and symptoms in humans. Similar to other paramyxoviruses, HNVs mediate entry into host cells through the concerted actions of two surface glycoproteins: a receptor-binding protein (RBP) that mediates attachment and a fusion glycoprotein (F) that triggers fusion in an RBP-dependent manner. NiV uses ephrin-B2 (EFNB2) and ephrin-B3 (EFNB3) as entry receptors. Ghana virus (GhV), a novel HNV identified in a Ghanaian bat, uses EFNB2 but not EFNB3. In this study, we employ a structure-informed approach to identify receptor-interfacing residues and systematically introduce GhV-RBP residues into a NiV-RBP backbone to uncover the molecular determinants of EFNB3 usage. We reveal two regions that severely impair EFNB3 binding by NiV-RBP and EFNB3-mediated entry by NiV pseudotyped viral particles. Further analyses uncovered two-point mutations (NiVN557SGhV and NiVY581TGhV) pivotal for this phenotype. Moreover, we identify NiV interaction with Y120 of EFNB3 as important for the usage of this receptor. Beyond these EFNB3-related findings, we reveal two domains that restrict GhV binding of EFNB2, confirm the HNV-head as an immunodominant target for polyclonal and monoclonal antibodies, and describe putative epitopes for GhV- and NiV-specific monoclonal antibodies. Cumulatively, the work presented here generates useful reagents and tools that shed insight to residues important for NiV usage of EFNB3, reveal regions critical for GhV binding of EFNB2, and describe putative HNV antibody-binding epitopes. IMPORTANCE: Hendra virus and Nipah virus (NiV) are lethal, zoonotic Henipaviruses (HNVs) that cause respiratory and neurological clinical features in humans. Since their initial outbreaks in the 1990s, several novel HNVs have been discovered worldwide, including Ghana virus. Additionally, there is serological evidence of zoonotic transmission, lending way to concerns about future outbreaks. HNV infection of cells is mediated by the receptor-binding protein (RBP) and the Fusion protein (F). The work presented here identifies NiV RBP amino acids important for the usage of ephrin-B3 (EFNB3), a receptor highly expressed in neurons and predicted to be important for neurological clinical features caused by NiV. This study also characterizes epitopes recognized by antibodies against divergent HNV RBPs. Together, this sheds insight to amino acids critical for HNV receptor usage and antibody binding, which is valuable for future studies investigating determinants of viral pathogenesis and developing antibody therapies.

Enhanced targeting: Production of filamentous bacteriophage monoclonal antibodies using the major coat protein pVIII as anchor.

Filamentous bacteriophage display technology has been employed in antibody discovery, drug screening, and protein-protein interaction study across various fields, including food safety, agricultural pollution, and environmental monitoring. Antifilamentous bacteriophage antibodies for identifying filamentous bacteriophage are playing a pivotal role in this technology. However, the existing antifilamentous bacteriophage antibodies lack sensitivity and specificity, and the antibodies preparation methods are cumbersome and hyposensitive. The major coat protein pVIII of filamentous bacteriophage has an advantage in quantification, which is benefit for detecting signal amplification but its full potential remains underutilized. In this study, the partial polypeptide CT21 of the major coat protein pVIII of filamentous bacteriophage was intercepted as the targeted immunogen or coating antigen to prepare antifilamentous bacteriophage antibodies. Six filamentous bacteriophage-specific monoclonal antibodies (mAbs) M5G8, M9A2, P6B5, P6D2, P8E4, and P10D4 were obtained. The limit of detections of the prepared six mAbs for detecting filamentous bacteriophage was 1.0 × 107  pfu mL-1 . These mAbs stayed stable under different pH, temperature, and exhibited high specificity in real application. This study not only provides a new idea for simplifying the preparation of antifilamentous bacteriophage antibodies which could apply in filamentous bacteriophage display, but it also presents a novel strategy for preparing antibodies against protein-specific epitopes with high sensitivity.

Selective Uptake of Macromolecules to the Brain in Microfluidics and Animal Models Using the HAVN1 Peptide as a Blood-Brain Barrier Modulator.

Monoclonal antibodies (mAbs) possess favorable pharmacokinetic properties, high binding specificity and affinity, and minimal off-target effects, making them promising therapeutic agents for central nervous system (CNS) disorders. However, their development as effective therapeutic and diagnostic agents for brain disorders is hindered by their limited ability to efficiently penetrate the blood-brain barrier (BBB). Therefore, it is crucial to develop efficient delivery methods that enhance the penetration of antibodies into the brain. Previous studies have demonstrated the potential of cadherin-derived peptides (i.e., ADTC5, HAVN1 peptides) as BBB modulators (BBBMs) to increase paracellular porosities for penetration of molecules across the BBB. Here, we test the effectiveness of the leading BBBM peptide, HAVN1 (Cyclo(1,6)SHAVSS), in enhancing the permeation of various monoclonal antibodies through the BBB using both in vitro and in vivo systems. In vitro, HAVN1 has been shown to increase the permeability of fluorescently labeled macromolecules, such as a 70 kDa dextran, 50 kDa Fab1, and 150 kDa mAb1, by 4- to 9-fold in a three-dimensional blood-brain barrier (3D-BBB) microfluidics model using a human BBB endothelial cell line (i.e., hCMEC/D3). HAVN1 was selective in modulating the BBB endothelial cell, compared to the pulmonary vascular endothelial (PVE) cell barrier. Co-administration of HAVN1 significantly improved brain depositions of mAb1, mAb2, and Fab1 in C57BL/6 mice after 15 min in the systemic circulation. Furthermore, HAVN1 still significantly enhanced brain deposition of mAb2 when it was administered 24 h after the administration of the mAb. Lastly, we observed that multiple doses of HAVN1 may have a cumulative effect on the brain deposition of mAb2 within a 24-h period. These findings offer promising insights into optimizing HAVN1 and mAb dosing regimens to control or modulate mAb brain deposition for achieving desired mAb dose in the brain to provide its therapeutic effects.

Pathogenic Th17 cell-mediated liver fibrosis contributes to resistance to PD-L1 antibody immunotherapy in hepatocellular carcinoma.

Understanding the mechanisms of resistance of hepatocellular carcinoma (HCC) to targeted therapies and immune checkpoint blockade is critical for the development of new combination therapies and improving patient survival. Here, we found that in HCC, anti-programmed cell death 1 ligand 1 (PD-L1) therapy reduces liver cancer growth, but the tumors eventually become resistant to continued therapy. Experimental analyses shows that the infiltration of pathogenic T helper 17 (pTh17) cells increases in drug-resistant HCC, and pTh17 cells secrete interleukin-17A (IL-17A), which promotes the expression of PD-L1 on the surface of HCC cells and produces resistance to anti-PD-L1 therapy. Anti-IL-17A combined with PD-L1 blockade significantly increased the infiltration of cytotoxic CD8+ T cells expressing high levels of interferon-γ and reduced treatment resistance in HCC. These results support the combination of anti-PD-L1 and anti-IL-17A as a novel strategy to induce effective T cell-mediated anti-tumor immune responses.

GT-00AxIL15, a Novel Tumor-Targeted IL-15-Based Immunocytokine for the Treatment of TA-MUC1-Positive Solid Tumors: Preclinical In Vitro and In Vivo Pharmacodynamics and Biodistribution Studies.

GT-00AxIL15 is a novel interleukin-15-based immunocytokine targeting a tumor-specific, glycosylated epitope of MUC1 (TA-MUC1). We characterized mode of action, pharmacokinetic (PK) and pharmacodynamic (PD) properties and investigated the relevance of TA-MUC1 binding for the concept of delivering IL-15 to solid tumors. In vitro pharmacology was analyzed in binding and cell-based assays. The in vivo PK profile and IL-15-mediated PD effects of GT-00AxIL15 were investigated in tumor-free mice. Tumor accumulation, immune infiltration and anti-tumor activity were assessed in TA-MUC1+ syngeneic and xenogeneic murine tumor models. GT-00AxIL15 was shown to specifically bind TA-MUC1 on tumor cells via its mAb moiety, to IL-15 receptors on immune cells via its IL-15 fusion modules and to FcγRs via its functional Fc-part. In vitro, NK, NKT and CD8+ T cells were activated and proliferated, leading to anti-tumor cytotoxicity and synergism with antibody-dependent cellular cytotoxicity (ADCC)-mediating mAbs. In vivo, GT-00AxIL15 exhibited favorable PK characteristics with a serum half-life of 13 days and specifically accumulated in TA-MUC1+ tumors. In the tumor microenvironment, GT-00AxIL15 induced robust immune activation and expansion and mediated anti-metastatic and anti-tumor effects in syngeneic and xenograft tumor models. These results support the rationale to improve PK and anti-tumor efficacy of IL-15 by increasing local concentrations at the tumor site via conjugation to a TA-MUC1 binding mAb. The tumor-selective expression pattern of TA-MUC1, powerful immune activation and anti-tumor cytotoxicity, long serum half-life and tumor targeting properties, render GT-00AxIL15 a promising candidate for treatment of solid tumors with high medical need, e.g., ovarian, lung and breast cancer.

Host Cell Protein Analysis using Enrichment Beads Coupled with Limited Digestion.

Host cell proteins (HCPs) are impurities that can adversely affect therapeutic proteins, even in small quantities. To evaluate the potential risks associated with drug products, methods have been developed to identify low-abundance HCPs. A crucial approach for developing a sensitive HCP detection method involves enriching HCPs while simultaneously removing monoclonal antibodies (mAbs) before analysis, utilizing liquid chromatography-mass spectrometry (LC-MS). This protocol offers detailed instructions for enriching host cell proteins using commercially available proteome enrichment beads. These beads contain a diverse library of hexapeptide ligands with specific affinities for different proteins. The protocol also incorporates limited digestion and subsequent peptide detection using nano LC-MS/MS. By employing these techniques, HCPs with low abundance can be enriched over 7000-fold, resulting in an impressive detection limit as low as 0.002 ppm. Significantly, this protocol enables the detection of 850 HCPs with a high level of confidence using a NIST mAb. Moreover, it is designed to be user-friendly and includes a video demonstration to assist with its implementation. By following these steps, researchers can effectively enrich and detect HCPs, enhancing the sensitivity and accuracy of risk assessment for drug products.

Analysis of Host Cell Proteins in AAV Products with ProteoMiner Protein Enrichment Technology.

Despite substantial efforts to detect host cell proteins (HCPs) in antibody drugs, information regarding HCPs in gene therapy products remains limited and has not been widely integrated into the host cell engineering or purification processes. Most methods that have successfully detected HCPs in antibody drugs are not applicable to gene therapy products, except for the ProteoMiner enrichment method. Here, we demonstrate that ProteoMiner beads effectively enrich HCPs in adeno-associated virus (AAV) products and simultaneously remove the detergent Pluronic F-68 without a loss of low-abundance HCPs. Following optimization of this technique, there was up to a 34-fold increase in the enrichment of HCPs compared to direct digestion. Moreover, the detection limit was significantly lowered with the ability to detect HCPs at levels as low as 0.1 ng/mL after ProteoMiner treatment. This approach holds promise in AAV HCP analysis and may be adaptable to other gene therapy products. The findings from this study provide valuable insights into HCPs in AAV products and may facilitate process development and host cell line optimization. The high sensitivity of this approach also facilitates detection of critical low-abundance HCPs, thereby contributing to risk assessment of their impact on the safety and quality of the AAV-based gene therapy products.

A PEGylated liposomal formulation of prochlorperazine that limits brain exposure but retains dynamin II activity: A potential adjuvant therapy for cancer patients receiving chemotherapeutic mAbs.

Anti-cancer monoclonal antibodies often fail to provide therapeutic benefit in receptor-positive patients due to rapid endocytosis of antibody-bound cell surface receptors. High dose co-administration of prochlorperazine (PCZ) inhibits endocytosis and sensitises tumours to mAbs by inhibiting dynamin II but can also introduce neurological side effects. We examined the potential to use PEGylated liposomal formulations of PCZ (LPCZ) to retain the anti-cancer effects of PCZ, but limit brain uptake. Uncharged liposomes showed complete drug encapsulation and pH-dependent drug release, but cationic liposomes showed limited drug encapsulation and lacked pH-dependent drug release. Uncharged LPCZ showed comparable inhibition of EGFR internalisation to free PCZ in KJD cells. After IV administration to rats, LPCZ reduced the plasma clearance and brain uptake of PCZ compared to IV PCZ. The results suggest that LPCZ may offer some benefit over PCZ as an adjunct therapy in cancer patients receiving mAb treatment.

Proliferative glomerulonephritis with monoclonal immunoglobulin deposits and atypical pathological findings treated with corticosteroid and rituximab.

A 16-year-old girl with fever that appeared after taking the second COVID-19 vaccine presented to the clinic with a serum creatinine of 0.89 mg/dL and C-reactive protein of 6.9 mg/dL. She had proteinuria and microscopic hematuria, with slowly worsening kidney function. Her kidney biopsy showed fibrocellular crescents in seven of nine glomeruli that were observed under light microscopy. Another glomerulus showed endocapillary hypercellularity and mesangial cell proliferation. Electron-dense deposits were significant in the mesangial area, with monoclonal IgG1-κ and C3 deposition by immunofluorescence. The patient was diagnosed with proliferative glomerulonephritis with monoclonal immunoglobulin deposits (PGNMID) and atypical pathological finding of diffuse crescent formation. The treatment regimen for PGNMID has not yet been established, and the appropriate duration of treatment is unknown. In our case, considering that rituximab acts by binding to CD20 on the surface of B cells through its crystallizable fragment, it was administered in addition to prednisolone, which successfully decreased the proteinuria over time.

Novel immune checkpoint targets: A promising therapy for cancer treatments.

The immune system frequently comprises immunological checkpoints. They serve as a barrier to keep the immune system from overreacting and damaging cells that are robust. Immune checkpoint inhibitors (ICIs) are utilized in immunotherapy to prevent the synergy of partner proteins of checkpoint proteins with auxiliary proteins. Moreover, the T cells may target malignant cells since the "off" signal cannot be conveyed. ICIs, which are mostly composed of monoclonal antibodies (mAbs) against cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) and anti- programmed death-1/programmed ligand 1 (anti-PD-1/PD-L1), might transform the context of cancer therapy. Further, more patients continued to exhibit adaptive resistance, even though several ICIs demonstrated convincing therapeutic benefits in selective tumor types. Immune checkpoint therapy's overall effectiveness is still lacking at this time. A popular area of study involves investigating additional immune checkpoint molecules. Recent research has found a number of fresh immune checkpoint targets, including NKG2A ligands, TIGIT, B7-H6 ligands, Galectin 3, TIM3, and so on. These targets have been focus of the study, and recent investigational approaches have shown encouraging outcomes. In this review article, we covered the development and present level understanding of these recently identified immune checkpoint molecules, its effectiveness and limitations.

Engagement of CD300c by a novel monoclonal antibody induces the differentiation of monocytes to M1 macrophages.

Human CD300c is expressed on various immune or cancer cells and is a novel B7 family member, functioning as an activity modulator on immune cells. To elucidate the function of CD300c, we developed CL7, a human CD300c-specific monoclonal antibody, and assessed its biological activity. The specific binding of CL7 monoclonal antibody against recombinant CD300c antigen was confirmed using enzyme-linked immunosorbent assay and surface plasmon resonance analysis. The binding affinity of CL7 was strong at the sub-nanomolar level. Furthermore, CL7 effectively bound to exogenously expressed CD300c on 293T cells. CL7 antibody differentiated monocytes to M1 macrophages, as evidenced by the upregulated expression of M1-specific cell surface markers and increased secretion of M1-specific cytokines in vitro in THP-1 cells and primary macrophages, as well as the increased population size of M1 macrophages in tumors grafted into mice. Additionally, CL7 treatment upregulated PD-L1 expression on THP-1 cells. We confirmed that the mechanism of M1 macrophage differentiation was through the mitogen-activated protein kinase and NF-κB signaling pathways. CD300c expression on various immune and cancer cells was similar to that of the well-known immune checkpoint PD-L1, suggesting the possibility of CD300c as a novel tumor biomarker. We also confirmed that the tumor size was substantially reduced by CL7 antibody treatment in the CT26 mouse model. Our study supports that CD300c is a potential therapeutic target in immuno-oncology. Overall, the CD300c-specific monoclonal antibody, CL7, is a promising immunotherapeutic agent, and it induces enhanced differentiation of M1 macrophages and/or their infiltration into the tumor microenvironment.

Preclinical Evaluation of 99mTc-MAG3-5-Fab Targeting TREM2 in Lung Cancer Mouse Models: A Comparison with 99mTc-MAG3-5-F(ab')2.

Triggering receptor expressed on myeloid cells-2 (TREM2), which is expressed on the surface of tumor-associated macrophages (TAMs), has been found to play a major role in the diagnosis and treatment of tumors. TREM2 expression is significantly upregulated in tumor tissues, and therefore, targeting TREM2 for tumor imaging may be of value. Previously, we performed TREM2 targeting imaging by using 68Ga-NOTA-COG1410 or a 124I-labeled monoclonal antibody (mAb) and F(ab')2 in mouse models of colon and gastric tumors. However, some of the shortcomings of these probes (i.e., the high uptake of 68Ga-NOTA-COG1410 in the liver, the difficulty of obtaining iodine-124, and the long half-life of iodine-124) have hindered their clinical use. Herein, we sought to synthesize novel molecular probes targeting TREM2 that are more conducive to clinical translation, eliminating the interference of isotope availability and in vivo probe biodistribution issues. Therefore, we established A549 cell lines with negative human TREM2 (hTREM2) expression (GFP tag; hTREM2- A549) or upregulated hTREM2 expression (GFP tag; hTREM2+ A549) using lentiviral transfection and confirmed these with Western blotting and immunocytochemistry. We then prepared a mouse anti-human TREM2 (5-mAb) by immunizing with the hTREM2 antigen. The antibody fragments 5-F(ab')2 and 5-Fab were prepared from 5-mAb, and 99mTc-MAG3-5-F(ab')2 and 99mTc-MAG3-5-Fab were then synthesized with excellent stability and specificity. 99mTc-MAG3-5-F(ab')2 had a slightly higher in vitro affinity than 99mTc-MAG3-5-Fab (Kd = 3.32 ± 0.05 nmol versus 4.62 ± 0.85 nmol). 99mTc-MAG3-5-F(ab')2 and 99mTc-MAG3-5-Fab both showed excellent specificity: after adding a 100-fold precursor, the two probes binding to the cells were almost blocked. In vivo pharmacokinetics showed that the distribution and elimination half-lives of 99mTc-MAG3-5-Fab (T1/2α = 1.25 ± 0.30 min and T1/2ß = 21.98 ± 2.80 min, respectively) were significantly reduced compared to those of 99mTc-MAG3-5-F(ab')2 (T1/2α = 2.64 ± 0.37 min and T1/2ß = 86.55 ± 26.86 min, respectively). In micro single-photon emission computed tomography/computed tomography (micro-SPECT/CT) imaging, the tumor was clearly displayed at 1 h after 99mTc-MAG3-5-Fab injection, while the blood background was extremely low at 3 h, and the probe was mainly excreted through the kidneys and biliary tract. 99mTc-MAG3-5-F(ab')2 uptake was also detected at the tumor site, although the blood background was consistently high. The biodistribution results were consistent with the micro-SPECT/CT imaging results. 99mTc-MAG3-5-Fab could clearly display hTREM2+ A549 tumors in a short time (1 h) with low uptake in nontumor organs and tissues and thus has clinical application prospects.

Screening of Deoxynivalenol Cyclic Heptapeptide Mimotope Antigen.

In this study, phage clones that can bind to DON were selected from the phage cyclohepta peptide library by screening through the principle of solid-phase affinity, and mimotope were synthesized to replace the DON toxin standard to establish a green low toxicity detection system. The author conducted four rounds of screening in the phage cyclic heptapeptide library with DON-10a1a monoclonal antibody as the target molecule. Then 38 phage clones were selected and validated, and the results showed that 35 of them could bind to the DON-10a1a monoclonal antibody and were inhibited by DON toxin. Finally, the DNA was extracted and sequenced to obtain 6 different DNA sequences, which were named D1-D6 respectively. The peptides synthesized according to the corresponding amino acid sequences can replace DON toxin to establish a series of green and low toxicity assays.

Genetic Code Expansion and a Photo-Cross-Linking Reaction Facilitate Ribosome Display Selections for Identifying a Wide Range of Affinity Peptides.

Cell-free molecular display techniques have been utilized to select various affinity peptides from peptide libraries. However, conventional techniques have difficulties associated with the translational termination through in-frame UAG stop codons and the amplification of non-specific peptides, which hinders the desirable selection of low-affinity peptides. To overcome these problems, we established a scheme for ribosome display selection of peptide epitopes bound to monoclonal antibodies and then applied genetic code expansion with synthetic X-tRNAUAG reprogramming of the UAG codons (X = Tyr, Trp, or p-benzoyl-l-phenylalanine (pBzo-Phe)) to the scheme. Based on the assessment of the efficiency of in vitro translation with X-tRNAUAG, we carried out ribosome display selection with genetic code expansion using Trp-tRNAUAG, and we verified that affinity peptides could be identified efficiently regardless of the presence of UAG codons in the peptide coding sequences. Additionally, after evaluating the photo-cross-linking reactions of pBzo-Phe-incorporated peptides, we performed ribosome display selection of low-affinity peptides in combination with genetic code expansion using pBzo-Phe-tRNAUAG and photo-irradiation. The results demonstrated that sub-micromolar low-affinity peptide epitopes could be identified through the formation of photo-induced covalent bonds with monoclonal antibodies. Thus, the developed ribosome display techniques could contribute to the promotion of diverse peptide-based research.

Prognostic and predictive biomarkers for anti-EGFR monoclonal antibody therapy in RAS wild-type metastatic colorectal cancer: a systematic review and meta-analysis.

BACKGROUND: RAS mutations affect prognosis in patients with metastatic colorectal cancer (mCRC) and have been identified as strong negative predictive markers for anti-epidermal growth factor receptor monoclonal antibody (anti-EGFR mAb) therapy, but many tumors containing wild-type RAS genes still do not respond to these therapies. Some additional biomarkers may have prognostic or predictive roles, but conclusions remain controversial. METHODS: We performed a meta-analysis and systematic review of randomized controlled trials comparing anti-EGFR mAb therapy with alternative therapy that investigated the prognostic and predictive impact of additional biomarkers in RAS wild-type (wt) mCRC patients. Hazard ratios (HRs) and 95% confidence intervals (CIs) for progression-free survival (PFS) and overall survival (OS) and odds ratios (ORs) for objective response rate (ORR) were calculated. The prognostic value of biomarkers was investigated by separately pooling HR and OR for different treatment groups in an individual study. The predictive value was assessed by pooling study interactions between treatment effects and biomarker subgroups. RESULTS: Thirty publications reporting on eighteen trials were selected, including a total of 13,507 patients. In prognostic analysis, BRAF mutations were associated with poorer PFS [HRs = 3.76 (2.47-5.73) and 2.69 (1.82-3.98)] and OS [HRs = 2.66 (1.95-3.65) and 2.45 (1.55-3.88)] in both the experimental and control arms; low miR-31-3p expression appeared to have longer PFS and OS. In terms of predictive effect, a lack of response to anti-EGFR therapy was observed in patients with BRAF mutant tumors (Pinteraction < 0.01 for PFS). Patients with tumors with any mutation in the KRAS/NRAS/BRAF/PIK3CA gene also showed similar results compared with all wild-type tumors (Pinteraction for PFS, OS, and ORR were < 0.01, < 0.01 and 0.01, respectively). While low miR-31-3p expression could predict PFS (Pinteraction = 0.01) and OS (Pinteraction = 0.04) benefit. The prognostic and predictive value regarding PIK3CA mutations, PTEN mutations or deletions, EGFR, EREG/AREG, HER2, HER3, and HER4 expression remains uncertain. CONCLUSIONS: In RAS wt mCRC patients receiving EGFR-targeted therapy, BRAF mutation is a powerful prognostic and therapy-predictive biomarker, with no effect found for PIK3CA mutation, PTEN mutation or deletion, but the combined biomarker KRAS/NRAS/BRAF/PIK3CA mutations predict resistance to anti-EGFR therapy. Low miR-31-3p expression may have positive prognostic and therapy predictive effects. Evidence on the prognostic and predictive roles of EGFR and its ligands, and HER2/3/4 is insufficient.

IGF2 Peptide-Based LYTACs for Targeted Degradation of Extracellular and Transmembrane Proteins.

Lysosome-targeting chimeras (LYTACs) have recently been developed to facilitate the lysosomal degradation of specific extracellular and transmembrane molecular targets. However, the LYTAC particles described to date are based on glycopeptide conjugates, which are difficult to prepare and produce on a large scale. Here, we report on the development of pure protein LYTACs based on the non-glycosylated IGF2 peptides, which can be readily produced in virtually any facility capable of monoclonal antibody production. These chimeras utilize the IGF2R/CI-M6PR pathway for lysosomal shuttling and, in our illustrative example, target programmed death ligand 1 (PD-L1), eliciting physiological effects analogous to immune checkpoint blockade. Results from in vitro assays significantly exceed the effects of anti-PD-L1 antibodies alone.

Chimeric antigen receptor T cells to target CD79b in B-cell lymphomas.

BACKGROUND: Chimeric antigen receptor (CAR) T cells targeting CD19 mediate potent and durable effects in B-cell malignancies. However, antigen loss or downregulation is a frequent cause of resistance. Here, we report development of a novel CAR T-cell therapy product to target CD79b, a pan B-cell antigen, widely expressed in most B-cell lymphomas. METHODS: We generated a novel anti-CD79b monoclonal antibody by hybridoma method. The specificity of the antibody was determined by testing against isogenic cell lines with human CD79b knock-in or knock-out. A single-chain variable fragment derived from the monoclonal antibody was used to make a panel of CD79b-targeting CAR molecules containing various hinge, transmembrane, and co-stimulatory domains. These were lentivirally transduced into primary T cells and tested for antitumor activity in in vitro and in vivo B-cell lymphoma models. RESULTS: We found that the novel anti-CD79b monoclonal antibody was highly specific and bound only to human CD79b and no other cell surface protein. In testing the various CD79b-targeting CAR molecules, superior antitumor efficacy in vitro and in vivo was found for a CAR consisting CD8α hinge and transmembrane domains, an OX40 co-stimulatory domain, and a CD3ζ signaling domain. This CD79b CAR specifically recognized human CD79b-expressing lymphoma cell lines but not CD79b knock-out cell lines. CD79b CAR T cells, generated from T cells from either healthy donors or patients with lymphoma, proliferated, produced cytokines, degranulated, and exhibited robust cytotoxic activity in vitro against CD19+ and CD19- lymphoma cell lines and patient-derived lymphoma tumors relapsing after prior CD19 CAR T-cell therapy. Furthermore, CD79b CAR T cells were highly efficient at eradicating pre-established lymphoma tumors in vivo in three aggressive lymphoma xenograft models, including two cell line-derived xenografts and one patient-derived xenograft. Notably, these CAR T cells did not demonstrate any significant tonic signaling activity or markers of exhaustion. CONCLUSION: Our results indicated that this novel CD79b CAR T-cell therapy product has robust antitumor activity against B-cell lymphomas. These results supported initiation of a phase 1 clinical trial to evaluate this product in patients with relapsed or refractory B-cell lymphomas.

Selection, characterization and in vivo evaluation of novel CD44v6-targeting antibodies for targeted molecular radiotherapy.

Molecular radiotherapy combines the advantages of systemic administration of highly specific antibodies or peptides and the localized potency of ionizing radiation. A potential target for molecular radiotherapy is the cell surface antigen CD44v6, which is overexpressed in numerous cancers, with limited expression in normal tissues. The aim of the present study was to generate and characterize a panel of human anti-CD44v6 antibodies and identify a suitable candidate for future use in molecular radiotherapy of CD44v6-expressing cancers. Binders were first isolated from large synthetic phage display libraries containing human scFv and Fab antibody fragments. The antibodies were extensively analyzed through in vitro investigations of binding kinetics, affinity, off-target binding, and cell binding. Lead candidates were further subjected to in vivo biodistribution studies in mice bearing anaplastic thyroid cancer xenografts that express high levels of CD44v6. Additionally, antigen-dependent tumor uptake of the lead candidate was verified in additional xenograft models with varying levels of target expression. Interestingly, although only small differences were observed among the top antibody candidates in vitro, significant differences in tumor uptake and retention were uncovered in in vivo experiments. A high-affinity anti-CD44v6 lead drug candidate was identified, mAb UU-40, which exhibited favorable target binding properties and in vivo distribution. In conclusion, a panel of human anti-CD44v6 antibodies was successfully generated and characterized in this study. Through comprehensive evaluation, mAb UU-40 was identified as a promising lead candidate for future molecular radiotherapy of CD44v6-expressing cancers due to its high affinity, excellent target binding properties, and desirable in vivo distribution characteristics.