IgG Heavy Chain Research Articles

Validation of a MALDI-TOF MS Method for SARS-CoV-2 Detection on the Bruker Biotyper and Nasopharyngeal Swabs: A Brazil—UK Collaborative Study

We developed a MALDI-TOF mass spectrometry method for the detection of the SARS-CoV-2 virus in saliva-gargle samples using Shimadzu MALDI-TOF mass spectrometers in the UK. This was validated in the USA to CLIA-LDT standards for asymptomatic infection detection remotely via sharing protocols, shipping key reagents, video conferencing, and data exchange. In Brazil, more so than in the UK and USA, there is a need to develop non-PCR-dependent, rapid, and affordable SARS-CoV-2 infection screening tests that also identify variant SARS-CoV-2 and other virus infections. In addition, travel restrictions necessitated remote collaboration with validation on the available clinical MALDI-TOF-the Bruker Biotyper (microflex® LT/SH)-and on nasopharyngeal swab samples, as salivary gargle samples were not available. The Bruker Biotyper was shown to be almost log103 more sensitive at the detection of high molecular weight spike proteins. A protocol for saline swab soaks out was developed, and duplicate swab samples collected in Brazil were analyzed by MALDI-TOF MS. The swab collected sample spectra that varied from that of saliva-gargle in three additional mass peaks in the mass region expected for IgG heavy chains and human serum albumin. A subset of clinical samples with additional high mass, probably spike-related proteins, were also found. Further, spectral data comparisons and analysis, subjected to machine learning algorithms in order to resolve RT-qPCR positive from RT-qPCR negative swab samples, showed 56-62% sensitivity, 87-91% specificity, and a 78% agreement with RT-qPCR scoring for SARS-CoV-2 infection.

Abstract 6315: Human granzyme B fusions targeting VEGFR on tumor cells and activated vasculature

Abstract VEGF121 is a naturally-occurring splice variant that binds to VEGF receptors R-1 and R-2, which are over-expressed on the endothelium of tumor vasculature but not normal vasculature, as well as several tumor cells. While anti-VEGF therapy is a key therapeutic in several cancer treatment regimens, none of the current therapeutics (bevacizumab, VEGF-trap, tyrosine kinase inhibitors) are directly cytotoxic against tumor cells. The serine protease Granzyme B (GrB) is a key effector in immune-mediated cell killing by inducing apoptosis through both caspase-dependent and -independent mechanisms. We previously reported the development and characterization of a fusion protein composed of GrB and VEGF121 (GrB/VEGF121). Here, we report on GrB-Fc-VEGF121, a fusion protein that incorporates a human IgG heavy chain Fc fragment for dimerization which increases the molecular weight and improves in vivo circulation and targeting potential. GrB-Fc-VEGF121 was expressed in HEK-293E cells under serum-free conditions and purified from the conditioned medium with a final yield of approximately 40 mg/L. The enzymatic activity of GrB in GrB-Fc-VEGF121 was comparable to that of commercially available human GrB. In vitro studies of GrB-Fc-VEGF121 showed cytotoxicity in the nanomolar range against tumor and endothelial cell lines expressing high levels of VEGFR-1 or VEGFR-2. Receptor-negative cells demonstrated IC50 levels in the high micromolar range. Immunofluorescence and confocal microscopy demonstrated that GrB-Fc-VEGF121 readily internalized into VEGFR-2+ tumor cells and endothelial cells within 2 hours of treatment while untargeted GrB did not internalize, suggesting internalization was receptor-mediated. Treatment of VEGFR-2+ endothelial and tumor cells at the IC50 dose resulted in >50% cell death via apoptosis and/or necrosis within 48 h. Ex vivo serum stability of GrB-Fc-VEGF121 indicated a gradual protein loss of GrB-Fc-VEGF121, with an overall stability of about 50% over 96 hours. No toxicity was observed in BALB/c mice treated with a total 475 mg/kg given IP every other day over 5 doses, indicating that the maximum tolerated dose of GrB-Fc-VEGF121 exceeds this dose level. In vivo efficacy studies in an OVCAR8 tumor xenograft model with GrB-Fc-VEGF121 (100 mg/kg over 5 doses) resulted in significant growth inhibition of established tumors compared to vehicle controls. We observed a significant decrease in the number of CD31+ blood vessels and Ki-67+ proliferating tumor cells in treated tumors compared to controls. Thus, GrB-Fc-VEGF121 has significant anti-tumor effects in vivo with virtually no toxicity against normal tissues at doses 4-5 times higher than therapeutic levels. This tumor- and vascular-targeting agent appears to have significant potential as a new class of targeted therapeutic agents with a unique mechanism of action. Research conducted, in part, by the Clayton Foundation for Research. Citation Format: Khalid A. Mohamedali, Lawrence H. Cheung, Ana Alvarez-Cienfuegos, Walter N. Hittelman, Michael G. Rosenblum. Human granzyme B fusions targeting VEGFR on tumor cells and activated vasculature [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6315.

Abstract 6316: In vitro and in vivo studies of human granzyme B fusion constructs targeting mesothelin

Abstract Mesothelin is highly expressed on tumor cells and promotes tumor growth and drug resistance. Pancreatic, ovarian, mesothelioma, and lung cancers highly express mesothelin and are cancers of unmet needs with ≤16% relative 5-year survival using standard of care. When delivered to tumor cells by immune effector cells, granzyme B (GrB) is a highly cytotoxic serine protease which induces apoptosis through both caspase-dependent and -independent mechanisms. We developed a fusion protein (GrB-Fc-SD1) composed of human GrB and containing SD1, a novel human single-domain antibody that uniquely targets Region III of the glycoprotein, mesothelin. The SD1 and GrB domains are connected by a human IgG heavy chain fragment which enables dimerization. Thus, the increase in total construct molecular weight leads to an increased in vivo circulation time and anti-tumor efficacy. GrB-Fc-SD1 was transiently expressed in HEK-293E cells under serum-free conditions and purified to homogeneity to a final yield of approximately 5 mg/L. Anti-GrB western blot analysis confirmed the expression of GrB-Fc-SD1 exclusively as a dimer. Comparison of GrB-Fc-SD1 with its expected molecular weight suggests significant glycosylation when produced in HEK-293E cells. The enzymatic activity of granzyme B in GrB-Fc-SD1 was comparable to that of commercially available human granzyme B. In vitro cytotoxicity against a limited panel of lung, pancreatic and ovarian cancer cell lines expressing surface mesothelin indicate cytotoxicity in the nanomolar range, while cytotoxicity against mesothelin-negative cells was at micromolar levels. GrB-Fc-SD1 bound specifically to mesothelin extra-cellular domain compared to a non-specific control, as determined by ELISA. Biacore analysis confirmed binding with a dissociation constant between 10−7 to 10−8 M. These data warrant continued investigation of the protein as a therapeutic candidate. Accordingly, ex vivo serum stability studies are underway. In addition, screening of an expanded panel of tumor cell lines for in vitro cytotoxicity is currently in progress and will be reported. In vivo efficacy studies against nude mice bearing Capan-2 xenograft models are currently underway and will also be reported. Research supported by H2Biologics. Research conducted, in part, by the Clayton Foundation for Research. Citation Format: Khalid A. Mohamedali, Lawrence H. Cheung, Louis R. DePalatis, Claire Thuning-Roberson, Michael G. Rosenblum. In vitro and in vivo studies of human granzyme B fusion constructs targeting mesothelin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6316.

Abstract 6314: Novel granzyme B-based fusion constructs targeting FRα on liquid and solid tumors

Abstract Folate receptor α (FRα) has been an attractive therapeutic target for many decades because it is highly overexpressed on several solid tumors and demonstrates low or restricted distribution on healthy tissues. Additionally, high expression of FRα has been associated with tumor progression and thus poor prognosis of cancer patients. Therefore, utilizing FRα to effectively locate tumor cells for targeted therapy using antibody drug conjugates, small molecule drug conjugates, radioimmunoconjugates or chimeric antigen receptor T cells, has been extensively investigated. Although some of the mentioned targeted therapies have shown promising preclinical results, their clinical utility has been limited due to associated toxicities in healthy tissues. We generated two new fusion constructs targeting FRα and containing active human granzyme B (GrB) as the cytotoxic payload. We used as binding domains two different scFvs targeting the FRα from antibodies previously used in clinical trials. These human scFvs (mov003 and mov018) were fused to the human serine protease GrB through an engineered human IgG heavy-chain fragment (designated as Fc) which contains a dimerization domain for longer half-life. The cytotoxicity of the constructs against a panel of cell lines expressing various levels of FRα showed a specific cytotoxic effect with an IC50 in the low nanomolar range (5-100 nM), particularly exhibiting very low IC50 values against breast and ovarian cell lines. FRα negative cells demonstrated IC50 levels in the micromolar range. Immunofluorescence studies demonstrated that the fusion constructs targeting FRα are efficiently internalized and effectively delivered the active serine protease to the cytosol. In vitro stability assays over 96 hours showed that both constructs are highly stable in mouse serum. Additionally, recent publications suggested that FRα is significantly associated with acute myeloid leukemia (AML) and might serve as a therapeutic target. We assessed the cytotoxic efficacy of GrB-based constructs on a panel of FRα+ AML cell lines and have found that these cell lines were very sensitive (IC50 in the low nanomolar range). Furthermore, evaluation of GrB fusion efficacy in two AML cell lines resistant to Bcl-2 inhibitor Venetoclax showed IC50s to be similar or lower than their parental cell lines. These results, therefore, demonstrate no cross-resistance to GrB. The safe and effective profile of these drugs together with their unique mechanism of action makes them ideal candidates for advancing pre-clinical development and may offer a potential new strategy for treatment of solid tumors and particularly Venetoclax relapsed AML patients, ineligible for intensive chemotherapy. In vivo efficacy studies to compare the two constructs are warranted and will help to identify the optimal therapeutic for further clinical consideration. Research conducted, in part, by the Clayton Foundation for Research. Citation Format: Ana Alvarez-Cienfuegos, Lawrence H. Cheung, Khalid A. Mohamedali, Natalia Baran, Marina Konopleva, Michael G. Rosenblum. Novel granzyme B-based fusion constructs targeting FRα on liquid and solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6314.

Development of Porcine Monoclonal Antibodies with In Vitro Neutralizing Activity against Classical Swine Fever Virus from C-Strain E2-Specific Single B Cells.

Neutralizing antibodies (nAbs) can be used before or after infection to prevent or treat viral diseases. However, there are few efficacious nAbs against classical swine fever virus (CSFV) that have been produced, especially the porcine-originated nAbs. In this study, we generated three porcine monoclonal antibodies (mAbs) with in vitro neutralizing activity against CSFV, aiming to facilitate the development of passive antibody vaccines or antiviral drugs against CSFV that offer the advantages of stability and low immunogenicity. Pigs were immunized with the C-strain E2 (CE2) subunit vaccine, KNB-E2. At 42 days post vaccination (DPV), CE2-specific single B cells were isolated via fluorescent-activated cell sorting (FACS) baited by Alexa Fluor™ 647-labeled CE2 (positive), goat anti-porcine IgG (H + L)-FITC antibody (positive), PE mouse anti-pig CD3ε (negative) and PE mouse anti-pig CD8a (negative). The full coding region of IgG heavy (H) chains and light (L) chains was amplified by reverse transcription-polymerase chain reaction (RT-PCR). Overall, we obtained 3 IgG H chains, 9 kappa L chains and 36 lambda L chains, which include three paired chains (two H + κ and one H + λ). CE2-specific mAbs were successfully expressed in 293T cells with the three paired chains. The mAbs exhibit potent neutralizing activity against CSFVs. They can protect ST cells from infections in vitro with potent IC50 values from 14.43 µg/mL to 25.98 µg/mL for the CSFV C-strain, and 27.66 µg/mL to 42.61 µg/mL for the CSFV Alfort strain. This study is the first report to describe the amplification of whole-porcine IgG genes from single B cells of KNB-E2-vaccinated pig. The method is versatile, sensitive, and reliable. The generated natural porcine nAbs can be used to develop long-acting and low-immunogenicity passive antibody vaccine or anti-CSFV agents for CSF control and prevention.

Production and Characterization of Monoclonal Antibodies Against Structural Proteins of Hirame Novirhabdovirus

Hirame novirhabdovirus (HIRRV) is a significant viral pathogen of Japanese flounder (Paralichthys olivaceus). In this study, seven monoclonal antibodies (mAbs) against HIRRV (isolate CA-9703) were produced and characterized. Three mAbs (1B3, 5G6, and 36D3) were able to recognize nucleoprotein (N) (42 kDa) and four mAbs (11-2D9, 15-1G9, 17F11, and 24-1C6) recognized matrix (M) protein (24 kDa) of HIRRV. Western blot, Enzyme-linked immunosorbent assay, and indirect fluorescent antibody technique (IFAT) results indicated that the developed mAbs were specific to HIRRV without any cross-reactivity against other different fish viruses and epithelioma papulosum cyprini cells. All the mAbs comprised IgG1 heavy chain and κ light chain except 5G6, which has a heavy chain of IgG2a class. These mAbs can be very useful in development of immunodiagnosis of HIRRV infection.

Sequencing the B Cell Receptor Repertoires of Antibody-Deficient Individuals With and Without Infection Susceptibility

PurposeMost individuals with antibody deficiency (hypogammaglobulinemia) need immunoglobulin replacement therapy (IgG-RT) from healthy plasma donors to stay clear of infections. However, a small subset of hypogammaglobulinemic patients do not require this substitution therapy. We set out to investigate this clinical conundrum by asking whether the peripheral B cell receptor repertoires differ between antibody-deficient patients who do and do not need IgG-RT.MethodsWe sequenced and analyzed IgG and IgM heavy chain B cell receptor repertoires from peripheral blood mononuclear cells (PBMCs) isolated from patients with low serum IgG concentrations who did or did not require IgG-RT.ResultsCompared to the patients who did not need IgG-RT, those who needed IgG-RT had higher numbers of IgG antibody clones, higher IgM diversity, and less oligoclonal IgG and IgM repertoires. The patient cohorts had different heavy chain variable gene usage, and the patients who needed IgG-RT had elevated frequencies of IgG clones with higher germline identity (i.e., fewer somatic hypermutations).ConclusionAntibody-deficient patients with infection susceptibility who needed IgG-RT had more diverse peripheral antibody repertoires that were less diverged from germline and thus may not be as optimal for targeting pathogens, possibly contributing to infection susceptibility.

Tuftsin-tailored fusion protein inhibits the growth of circulating gastric tumor cells associated with macrophage phagocytosis

Circulating tumor cells (CTCs) are a major cause of tumor metastasis and resistance to anticancer therapies. To date, no effective low-toxicity chemotherapeutic agents or antibodies have exhibited significant clinical activity against CTCs. Macrophages are important mediators of antitumor immunity. Tuftsin (TF), a tetrapeptide located at residues 289–292 of the CH2 domain of the Fc region of the IgG heavy chain, binds to Nrp-1, a receptor on the surface of macrophages that promotes phagocytosis and induces nonspecific activation of the immune system against tumors. Lidamycin (LDM) is an antitumor chemotherapy agent that is strongly cytotoxic to tumors and can dissociate into an apoprotein (LDP) and active enediyne (AE) in vitro. We previously constructed the fusion protein LDP-TF through genetic engineering and inserted the chromophore AE to produce LDM-TF, which can target macrophages to promote their phagocytic and cytotoxic activity against tumor cells. Preliminary experiments confirmed the anti-tumor activity of LDM-TFs. In this study, we found that LDM-TF effectively inhibited the growth of CTCs of gastric cancer origin and enhanced macrophage phagocytosis both in vivo and in vitro. Tumor cell expression of CD47, which helps to evade phagocytosis by macrophages, was substantially downregulated by LDM-TF. Notably, our in vitro experiments demonstrated that the combination of LDM-TF and anti-CD47 antibodies promoted phagocytosis more than either component alone. Our findings demonstrate the significant inhibitory effect of LDM-TF on the growth of CTCs of gastric cancer origin and suggest that the combination of LDM-TF and anti-CD47 antibodies may exhibit synergistic effects, thereby providing a new option for the clinical treatment of patients with advanced tumors that have metastasized.

Preparation of IgE Antibody and Distribution of IgE+ Secretory Cells in the Palatine Tonsil of Bactrian Camel.

Allergic diseases induced by dust have seriously threatened human health, while Bactrian camels can live in a sandy environment for a long time. To prepare rabbit anti-Bactrian camel IgE antibody and explore the distribution characteristics of IgE+ secretory cells in the palatine tonsils, which lays a theoretical foundation for the distribution of local antibodies in the palatal tonsils of Bactrian camel and the study of immune function. In this study, the amino acid sequences of Bactrian camel IgE, IgA, IgM and IgG heavy chain constant regions were compared, and a specific IgE gene fragment were selected (447 bp). The recombinant plasmid pET-28a-IgE was induced in Escherichia coli BL21(DE3) by IPTG and its expression conditions were optimized. The antibody was prepared by immunizing rabbits with purified IgE recombinant protein, its titer and specificity were detected by indirect ELISA and Western blotting. Immunohistochemical and statistical methods investigated the distribution of IgE+ secretory cells in the palatine tonsils. The IgE recombinant protein was expressed in the form of inclusion bodies with a size of 16 kDa. The optimal IPTG induction concentration was 0.7 mmol/L and the induction time was 8 h. The titer of the antibody was 1:16000 by ELISA, and the antibody could specifically bind to the recombinant protein by Western blotting. IgE+ secretory cells were mainly distributed in the subepithelial compartments of reticulated crypt epithelium of the palatine tonsil of the Bactrian camel, followed by the subepithelial compartments of stratified squamous epithelium and occasionally in the extrafollicular region. The rabbit anti-Bactrian camel IgE polyclonal antibody was successfully prepared. It is confirmed that IgE exists in the palatine tonsils of Bactrian camels under normal living conditions. In addition, IgE+ secretory cells are mainly distributed in the subepithelial compartments of reticulated crypt epithelium of the palatine tonsil, which is consistent with the distribution characteristics of IgG+ and sIgA+ secretory cells in the palatal tonsils of the Bactrian camel.

Rheumatoid factor recognizes specific domains of the IgG heavy chain complexed with HLA class II molecules.

We previously reported that RF recognized the IgG heavy chain (IgGH)/RA-susceptible HLA class II molecule complex. In the present study, we investigated the molecular mechanisms underlying HLA binding to and the RF recognition of IgGH. We synthesized various types of IgGH segments, including VH, CH1, CH2 and CH3, and transfected them with or without HLA class II molecules into the Human Embryonic Kidney 293T cell line. IgGH single domains linked with the HLA-Cw3 peptide, which binds to the binding groove of the HLA class II molecule, were also synthesized. The expression of IgGH domains on the cell surface and their recognition by RF were examined using flow cytometry. Flag-tagged IgGH segments containing CH1 (CH1, VH-CH1, CH1-CH2, VH-CH1-CH2, CH1-CH2-CH3 and VH-CH1-CH2-CH3) were clearly presented on the cell surface by HLA-DR4, while segments without the CH1 domain were expressed at a low level, and the CH3 single domain was only weakly detected on the cell surface, even with HLA-DR4. We then transfected IgGH single domains linked to the Cw3 peptide together with HLA-DR4 and showed that RF-containing sera from RA patients only recognized the CH3 domain and none of the other single domains. When various segments without the Cw3 peptide were transfected with HLA-DR4, only the CH1-CH2-CH3 segment and full-length IgGH were detected by the sera of RA patients. The CH1 domain of IgGH binds to the RA-susceptible HLA-DR molecule and is expressed on the cell surface. RF specifically recognizes the CH3 domain of the IgGH/HLA-DR4 complex.

Plasma Protein Levels of Young Healthy Pigs as Indicators of Disease Resilience.

Selection for disease resilience, which refers to the ability of an animal to maintain performance when exposed to disease, can reduce the impact of infectious diseases. However, direct selection for disease resilience is challenging because nucleus herds must maintain a high health status. A possible solution is indirect selection on indicators of disease resilience. To search for such indicators, we conducted phenotypic and genetic quantitative analyses of the abundances of 377 proteins in plasma samples from 912 young and visually healthy pigs and their relationships with performance and subsequent disease resilience after natural exposure to a polymicrobial disease challenge. Abundances of 100 proteins were significantly heritable (false discovery rate (FDR) <0.10). The abundance of some proteins was or tended to be genetically correlated (rg) with disease resilience, including complement system proteins (rg=-0.24, FDR=0.001) and IgG heavy chain proteins (rg=-0.68, FDR=0.22). Gene set enrichment analyses (FDR<0.2) based on phenotypic and genetic associations of protein abundances with subsequent disease resilience revealed many pathways related to the immune system that were unfavorably associated with subsequent disease resilience, especially the innate immune system. It was not possible to determine whether the observed levels of these proteins reflected baseline levels in these young and visually healthy pigs or were the result of response to environmental disturbances that the pigs were exposed to before sample collection. Nevertheless, results show that, under these conditions, the abundances of proteins in some immune related pathways can be used as phenotypic and genetic predictors of disease resilience and have the potential for use in pig breeding and management.

Switching Heavy Chain Constant Domains Denatures the Paratope 3D Architecture of Influenza Monoclonal Antibodies.

Several human monoclonal Abs for treating Influenza have been evaluated in clinical trials with limited success despite demonstrating superiority in preclinical animal models including mice. To conduct efficacy studies in mice, human monoclonal Abs are genetically engineered to contain mouse heavy chain constant domain to facilitate the engagement of Fc-receptors on mouse immune effector cells. Although studies have consistently reported discrepancies in Ab effectiveness following genetic engineering, the structural and mechanistic basis for these inconsistencies remain uncharacterized. Here, we use homology modeling to predict variable region (VR) analogous monoclonal Abs possessing human IgG1, mouse IgG1, and mouse IgG2a heavy chain constant domains. We then examine predicted 3D structures for variations in the spatial location and orientation of corresponding paratope amino acid residues. By structurally aligning crystal structures of Fabs in complex with hemagglutinin (HA), we show that corresponding paratope amino acid residues for VR-analogous human IgG1, mouse IgG1, and mouse IgG2a monoclonal Abs interact differentially with HA suggesting that their epitopes might not be identical. To demonstrate that variations in the paratope 3D fine architecture have implications for Ab specificity and effectiveness, we genetically engineered VR-analogous human IgG1, human IgG4, mouse IgG1, and mouse IgG2a monoclonal Abs and explored their specificity and effectiveness in protecting MDCK cells from infection by pandemic H1N1 and H3N2 Influenza viruses. We found that VR-analogous monoclonal Abs placed on mouse heavy chain constant domains were more efficacious at protecting MDCK cells from Influenza virus infection relative to those on human heavy chain constant domains. Interestingly, mouse but not human heavy chain constant domains increased target breadth in some monoclonal Abs. These data suggest that heavy chain constant domain sequences play a role in shaping Ab repertoires that go beyond class or sub-class differences in immune effector recruitment. This represents a facet of Ab biology that can potentially be exploited to improve the scope and utilization of current therapeutic or prophylactic candidates for influenza.

In-silico selection of appropriate signal peptides for romiplostim secretory production in Escherichia coli

BackgroundThrombocytopenia is a prevalent condition caused by a reduction in platelet production, increased platelet depletion, or a combination of both processes. Romiplostim, the most potent drug and the only thrombopoietin (TPO) receptor agonist, consists of four TPO receptor–activating peptides fused as a fusion protein to the human IgG1 heavy chain segment. Romiplostim could be produced by recombinant DNA technology in Escherichia coli. Extracellular and periplasmic secretion using signal peptides (SPs) of the recombinant proteins could have several advantages in purification and protein recovery. SPs are short amino-acid sequences used in protein translocation through various membranes. The structure of the different SPs identified in various bacteria and their compatibility with the protein could affect the transfer success and efficacy. ObjectiveThis study aimed to identify the best SP for extracellular secretion of romiplostim to facilitate its purification and reduce production costs in E. coli. MethodsThe amino acid sequences of the SPs and the position of their cleavage sites in the proteins were evaluated using the Signal 5.0 server. The solubility of the proteins produced in E. coli was predicted by protein-sol. The physicochemical parameters of the SPs were analyzed by ProtParam using the ExPASy server. Results/ConclusionWe found that SfmC is the most suitable candidate, followed by TorT and bla. However, this result should be confirmed by further experimental assessment. Furthermore, for the successful secretion of a recombinant protein, the optimal growth conditions should be controlled in addition to a suitable SP.

High expression of IGHG1 promotes breast cancer malignant development by activating the AKT pathway

ABSTRACT This study researched the exact function of IgG1 heavy chain (IGHG1) on breast cancer (BC) progression. IGHG1 level within BC and paired normal tissues was acquired in Gene Expression Profiling Interactive Analysis dataset. Meanwhile, this work harvested tumor and paired healthy tissues in 42 BC cases. siRNA targeting IGHG1 was transfected into BC cells. SC79 was used to treat the transfected BC cells. CCK-8 assay, clone formation experiment, BrdU assay, Transwell experiment and flow cytometry were carried out to measure the viability, colony formation, proliferation, invasion, and apoptosis of BC cells. Paclitaxel and cisplatin sensitivity of BC cells was evaluated by MTT assay. Real-time quantitative reverse transcription-polymerase chain reaction and Western-blot were performed for measuring mRNA and protein expression. The overexpressed IGHG1 indicated dismal BC survival. IGHG1 silencing attenuated the viability, invasion, proliferation, epithelial-mesenchymal transition, but enhanced the apoptosis of BC cells. IGHG1 silencing enhanced the paclitaxel and cisplatin sensitivity of BC cells. IGHG1 silencing suppressed the activity of the MEK, AKT, and ERK pathways. AKT agonist partially reversed the inhibition of IGHG1 silencing on BC cell malignant phenotype and resistance to paclitaxel and cisplatin. IGHG1 promotes the malignant development of BC by activating the AKT pathway. It may be an effective target for BC treatment.

External Validation of the Saved Score for Venous Thromboembolism Risk Stratification in Patients with Multiple Myeloma Receiving Immunomodulatory Drugs

Background: High thrombogenicity is a well-established phenomenon in multiple myeloma (MM) and can lead to high morbidity and mortality. The risk of venous thromboembolism (VTE) is particularly elevated in the subset of MM patients treated with immunomodulatory drug (IMiD) containing regimens. The National Comprehensive Cancer Network (NCCN) and International Myeloma Working Group (IMWG) have implemented VTE risk assessment models, and recommend prophylactic anticoagulation based on individual patient, disease, or treatment-related factors specifically for MM patients receiving IMiD. With the recent incorporation of the SAVED (PMID 31319391) scoring system in the NCCN guidelines for VTE risk stratification, we sought to externally validate this risk assessment model and evaluate its predictive ability in our MM patient cohort. Methods: We retrospectively evaluated 501 adult patients with either newly diagnosed or relapsed/refractory MM who were newly initiated on an IMiD-based regimen between 2010 and 2019. Patients already on therapeutic or prophylactic anticoagulation were excluded from the study (aspirin was allowed). The SAVED score was calculated at IMiD initiation according to the following five parameters: surgery within last 90 days (+2), Asian race (-3), prior VTE history (+3), age ≥80 years (+1), dexamethasone use (+2 for high dose [>160 mg/30-day], or +1 for standard dose [120-160 mg/30-day]). Patients with SAVED scores of <2 and ≥2 were considered of low and high risk, respectively, for developing VTE after IMiD initiation. Time to VTE was estimated by Kaplan-Meier failure method. Logistic regression model was used to test components of the SAVED score with 1-year VTE status. Model discrimination was assessed via c statistic by calculating the area under the receiver operating characteristic curve (AUC) with bootstrapped 95% confidence interval (CI). Results: The median age of our cohort at the IMiD initiation was 64 years (range 34-89); 264 (53%) patients were male and 378 (75%) were white. IgG and IgA heavy chain subtypes were present in 280 (56%), and 105 (21%) patients respectively, whereas 110 (22%) patients had light chain disease only. The International Staging System (ISS) was I, II, III, and unknown for 161 (32%), 149 (30%), 136 (27%), and 55 (11%) of patients, respectively. Most patients (94%) received lenalidomide, whereas 14 (3%) and 15 (3%) patients were treated with thalidomide and pomalidomide, respectively. The distribution of SAVED covariates is shown in Table 1. The cumulative incidence of VTE at 12-month after IMiD initiation for the entire cohort was 16% (95% CI: 13-20%). Among the 84 patients who developed new VTE after IMiD initiation, 60 (71%) had lower extremity deep venous thrombosis (DVT), 10 (12%) had pulmonary embolism, 9 (11%) had both DVT and PE, and 5 (6%) had upper extremity DVT. The SAVED score stratified 112 patients (22%) as high-risk and 389 patients (78%) as low-risk (similar proportion as the original derivation study). Cumulative incidence of VTE at 6 and 12 months was 31% and 41% in the high-risk group, versus 6% and 9% in the low-risk group, respectively (Figure 1). Each point of SAVED score increase was associated with significantly higher chance of VTE within 1-year post IMiD initiation with excellent discrimination (odds ratio = 2.10 [95% CI 1.71-2.56], c statistic (AUC) = 0.76 [95% CI 0.70-0.81]). Conclusion: Our results successfully externally validated the SAVED model to predict IMiD-associated VTE in a real-life cohort of patients with MM. Patients with SAVED score of ≥2 had significantly higher incidence for VTE. This indicates that more aggressive pharmacologic thromboprophylaxis is needed in this specific patient population. The SAVED score outperformed several other similar VTE prediction models that were also validated in the current cohort (c statistic=0.68 for IMPEDE [PMID: 33997961] and 0.62 for PRISM [PMID: 35772005]). In contrast to other risk models that focused on all patients with MM, the SAVED score was derived and now validated specifically for patients starting IMiD as this it is the only cancer population with a strong guideline recommendation for ambulatory thromboprophylaxis. Future prospective validation would be useful to determine the predictive nature of SAVED while evaluating safety and efficacy of thromboprophylaxis with direct oral anticoagulant in high-risk patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

P055: Validation of the tumor cell-specific rearranged IgG-encoding circulating cell-free DNA for the treatment response monitoring in patients with classic Hodgkin lymphoma

Table 1: Clinical characteristics of patients Background: Detection and longitudinal monitoring of the cell-free circulating tumor-specific DNA (ctDNA) in plasma of cancer patients is a potent tool for treatment response assessment and detection of the early disease recurrence. There is a number of the next-generation sequencing (NGS) panels for analysis of the commonly mutated genes in tumors of classic Hodgkin lymphoma (cHL) patients, which are being validated for clinical use. The Cleveland Clinic team, in collaboration with Adaptive Biotechnologies, is exploring if longitudinal assessment of ctDNA encoding the patient- and tumor cell-specific rearranged heavy or light (kappa/lambda) chain IgG (r-IgH/r-IgK/L, could be used for the treatment response and early recurrence monitoring in cHL patients. Methods: Enrolled are patients (9–99 y) with newly diagnosed and previously untreated cHL. First, the diagnostic biopsy specimen and the pre-treatment plasma are tested for concordant presence of dominant r-IgH/r- IgK/L ctDNA sequences. Sequence dominance and suitability for tracking is determined according to the Adaptive Biotechnologies’ diagnostic algorithm validated for other B cell neoplasms. If dominant r-IgH/r-IgK/L sequence(s) is detected, the patient plasma is serially tested during and following completion of therapy. Results: Pilot results on first 4 patients are presented. Patients’ clinical characteristics are summarized in Table 1. P1 had a single dominant r-IgH ctDNA sequence; P2 had two dominant r-IgH ctDNA sequences; P3 had two r-IgH and one dominant IgK ctDNA sequences; the latter was chosen for serial tracking; P4 had a single dominant r-IgH ctDNA sequence. In P1 and P2, the concentration of ctDNA precipitously declined below detectable threshold following completion of the first two cycles of therapy, and remained undetectable through the end of treatment. These laboratory changes correlated well with the treatment response assessed by functional imaging. For P2 and P3, the in-treatment samples are in process of acquisition. Conclusion: The preliminary results of this pilot project demonstrate feasibility of establishing trackable ctDNA sequences encoding IgG heavy or light chain, with intra- and interpatient clonotypic heterogeneity of the dominant sequences. Correlation between results of the functional imaging treatment assessment and detectable quantity of ctDNA in plasma was observed. Further testing is underway.

Abstract ND07: JNJ-78306358: A first-in-class bispecific T cell redirecting HLA-G antibody

Abstract JNJ-78306358 is a first-in-class bispecific antibody (bsAb), engineered using the Zymeworks Azymetric™ platform, to treat advanced stage solid tumors. Human leukocyte antigen G (HLA-G) is a non-classical major histocompatibility class I molecule with an immune tolerance role at the maternal-fetal interface. HLA-G has limited normal tissue expression, mainly detected in placenta and pituitary gland. However, HLA-G is expressed in multiple human cancers, with a potential role in cancer immune evasion. Comprehensive immunohistochemistry analysis of patient-derived tumors revealed high prevalence of HLA-G expression in renal cell carcinoma (RCC, 75%), ovarian (61%), colon (64%) and rectal cancers (40%), and moderate HLA-G expression prevalence in lung adenocarcinoma, endometrial, and pancreatic cancer. JNJ-78306358 induces HLA-G-expressing tumor cell killing via T cell redirection. This bsAb features an anti-HLA-G single-chain fragment variable (scFv) domain that binds with high affinity (KD ~ 13 pM) to HLA-G on tumor cells and a Fab domain that binds with weaker affinity (KD ~22 nM) to the epsilon subunit of the cluster of differentiation 3 (CD3ε). The immunoglobulin (Ig)G1 heavy chains feature Fc region mutations that disrupt interaction with Fcγ receptors. JNJ-78306358 demonstrated potent PBMC- and T cell-mediated in vitro cytotoxicity (EC50 10.4 - 442.3 pM) against endogenous membrane HLA-G-expressing tumor cell lines and absence of killing against cancer cells lacking HLA-G membrane expression, highlighting the specificity against antigen-expressing tumor cells. JNJ-78306358 exhibited hallmarks of T cell engagement in vitro, including T cell proliferation and cytokine release. In addition, JNJ-78306358 showed HLA-G-expression-dependent anti-tumor activity in mice (humanized with human donor CD3+ pan-T cells or human umbilical cord-blood-derived CD34+ hematopoietic stem cells [HSCs]) bearing cell line- and patient-derived tumors. In these xenograft models, a dose-dependent increase in CD4+ and CD8+ T cell infiltration into tumors was observed with complete tumor regressions at low doses of JNJ-78306358 (0.03 mg/kg). JNJ-78306358’s safety, tolerability and preliminary anti-tumor activity are currently being evaluated in a first-in-human phase I study in advanced stage solid tumors with high prevalence of HLA-G protein expression (NCT04991740). This antigen-targeting approach may address an unmet medical need in patients with tumors expressing HLA-G. Citation Format: Nataša Obermajer, Adam Zwolak, Kelly Van De Ven, Shana Versmissen, Aleksandra Brajic, Ted Petley, Dan Weinstock, Jason Aligo, Fang Yi, Stephen Jarantow, Keith Schutsky, Ken Tian, Angelilo Lorraine, Diana Alvarez Arias, Kristel Buyens, Vince Torti, Krista Menard, Katharine Rogers, Brian Geist, Marjolein Van Heerden, Gerald Chu, Bie Verbist, Maté Ongenaert, Julien Hasler, Kathryn Packman, Jacintha Shenton, Dirk Brehmer, Josh Lauring, Regina J. Brown, James Greger, Daphne Salick Ryan, Sanjaya Singh, Matthew V. Lorenzi, Laurie Lenox, Sylvie Laquerre. JNJ-78306358: A first-in-class bispecific T cell redirecting HLA-G antibody [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 ND07.

Abstract 337: Development of an immune-oncology agent containing granzyme B to target VEGFR+ ovarian cancer cells and activated tumor vasculature

Abstract Granzyme B is a key effector in immune-mediated cell killing by inducing apoptosis through both caspase-dependent and -independent mechanisms. We developed a fusion protein (GrB-Fc-VEGF121) composed of granzyme B, VEGF121 (a naturally-occurring isoform of the VEGF-A family that binds to VEGF receptors R-1 and R-2), and a human IgG heavy chain fragment (for dimerization and to augment in vivo circulation and anti-tumor efficacy). GrB-Fc-VEGF121 was transiently expressed in HEK-293E cells under serum-free conditions purified with a final yield of approximately 40 mg/L. Comparison of GrB-Fc-VEGF121 with its expected molecular weight suggests significant glycosylation when produced in HEK-293E cells. The enzymatic activity of granzyme B in GrB-Fc-VEGF121 was comparable to that of commercially available human granzyme B. GrB-Fc-VEGF121 readily internalized into VEGFR-2+ cells within 2 hours of treatment while untargeted granzyme B was not internalized, demonstrating that internalization was receptor-mediated. Cytotoxicity studies against a panel of tumor and endothelial cells indicated cytotoxicity in the nanomolar range against cell lines expressing high levels of VEGFR-1 or VEGFR-2 while control (receptor-negative) cells demonstrated IC50 levels in the high micromolar range. Treatment of VEGFR-2+ endothelial and tumor cells with GrB-Fc-VEGF121 resulted in robust cell death via apoptosis and/or necrosis. Ex vivo serum stability of GrB-Fc-VEGF121 indicated a gradual protein loss of GrB-Fc-VEGF121, with an overall loss of about 50% over 96 hours. The maximum tolerated dose of GrB-Fc-VEGF121 was not reached when BALB/c mice were treated with a total 475 mg/kg given every other day over 5 doses. Treatment of mice bearing OVCAR8 tumor xenografts with GrB-Fc-VEGF121 (100 mg/kg over 5 doses) resulted in significant growth inhibition of established tumors compared to vehicle controls. Treated tumors showed a significant decrease in the number of CD31+ blood vessels and Ki-67+ proliferating tumor cells compared to controls, when assessed at the end of the study. These studies clearly indicate that GrB-Fc-VEGF121 has significant anti-tumor effects in vivo with virtually no toxicity against normal tissues at doses 4-5 times higher than therapeutic levels. The tumor- and vascular-targeted agent appears to have significant potential as a new class of targeted therapeutic agents with a unique mechanism of action. Research conducted, in part, by the Clayton Foundation for Research. Citation Format: Khalid A. Mohamedali, Lawrence H. Cheung, Ana Alvarez-Cienfuegos, Walter N. Hittelman, Michael G. Rosenblum. Development of an immune-oncology agent containing granzyme B to target VEGFR+ ovarian cancer cells and activated tumor vasculature [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 337.

Abstract 3439: An internalizing anti-CEA construct for targeted delivery of granzyme B results in significant antitumor activity in human lung carcinoma xenografts

Abstract Carcinoembyronic antigen (CEA) is a cell surface protein normally found at low levels in adult tissues but over-expressed in numerous solid tumors. CEA is constitutively released from tumor cells and reaches detectable levels in peripheral blood, making CEA targeting particularly challenging. Granzyme B is a key effector in immune-mediated killing of target cells, with cell death through both caspase-dependent and -independent mechanisms. We developed a fusion protein composed of granzyme B and a human scFv targeting the juxta-membrane epitope of CEA. The construct also incorporates a fragment of human IgG heavy chain to cause dimerization and provide increased in vivo circulation and antitumor efficacy. The GrB-Fc-CEA construct was cloned into a mammalian expression vector, transiently expressed by HEK-293E suspension cells, and secreted into the culture media. The final yield after purification and activation was 10-15 mg/L. The enzymatic activity of granzyme B in GrB-Fc-CEA was comparable to that of commercially available human granzyme B. In vitro cytotoxicity against CEA-positive cell lines demonstrated cytotoxicity in the nanomolar range, compared to micromolar range cytotoxicity against control (CEA-negative) cell lines. Importantly, GrB-Fc-CEA cytotoxicity against tumor cells in vitro was unaffected by exogenously-added CEA (200 ng/ml) extracellular domain, suggesting that the construct is not competitively absorbed by soluble CEA released by tumors and present in the circulation of patients. GrB-Fc-CEA internalized rapidly (within one hour) into the target cell lines A-431 and HT-1080/CEA whereas internalization was not observed with untargeted granzyme B, clearly demonstrating receptor-mediated internalization. GrB-Fc-CEA induced mitochondrial depolarization and caspase activation, consistent with the well-described granzyme B mechanism of action. Treatment of female nude mice bearing A-549 tumors with GrB-Fc-CEA (80 mg/kg over 5 doses) resulted in a significant growth inhibition of the established tumors compared to vehicle controls (1200 mm3 vs &amp;lt;40 mm3). Complete tumor regression was observed in seven of ten tumors in mice treated with GrB-Fc-CEA. Mouse weight was unaffected in both treated and control groups for the duration of the study, suggesting no apparent toxicity of the construct. Thus, GrB-Fc-CEA appears to have significant potential in targeting a large category of CEA-expressing solid tumors and is a promising candidate for further pre-clinical development. Research conducted, in part, by the Clayton Foundation for Research. Citation Format: Lawrence H. Cheung, Khalid A. Mohamedali, Ana Alvarez-Cienfuegos, Walter N. Hittelman, Michael G. Rosenblum. An internalizing anti-CEA construct for targeted delivery of granzyme B results in significant antitumor activity in human lung carcinoma xenografts [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 3439.

P19: BASELINE CORRELATES OF COMPLETE RESPONSE TO IDECABTAGENE VICLEUCEL (IDE-CEL, BB2121), A BCMA-DIRECTED CAR T CELL THERAPY IN PATIENTS WITH RELAPSED AND REFRACTORY MULTIPLE MYELOMA: SUBANALYSIS OF THE KARMMA TRIAL

Introduction: In the pivotal phase 2 KarMMa trial (NCT03361748), in heavily pretreated RRMM, 33% of patients (pts) who received ide-cel, a B-cell maturation antigen (BCMA)-directed CAR T cell therapy, had complete response (CR) or stringent CR (sCR), with a median duration of response of 21.5 months (Anderson et al. ASCO 2021. Poster 8016). This subanalysis aimed to identify baseline correlates of pts attaining CR/sCR in KarMMa. Methods: Pts with ≥3 prior lines of therapy (including an immunomodulatory agent, proteasome inhibitor, and anti-CD38 antibody), and MM refractory to last regimen per IMWG criteria received ide-cel infusion (target doses 150–450 x 106 CAR+ T cells) after lymphodepletion. Bridging therapy was optional. Endpoints included overall response rate (primary) and CR/sCR rate (key secondary). Baseline characteristics were collected prior to lymphodepletion and for select biomarkers on day of infusion. Univariate and multivariate logistic regression models were used to identify baseline characteristics correlating with the likelihood of achieving CR/sCR. Results: Of 128 pts who received ide-cel (data cutoff Dec 21, 2020), 42 pts achieved CR/sCR and 86 had non-CR/sCR (very good partial response, partial response, or no response). In pts with CR/sCR, 32 (76%) were negative for minimal residual disease (MRD) (sensitivity level of <10-5) and 19 maintained MRD negativity at 12-mo follow-up. Baseline characteristics were generally balanced between pts with CR/sCR and non-CR/sCR; notable exceptions included revised International Staging System (ISS) stage III disease, IgG chain type, CD138+ plasma cell percentage, and β-2-microglobulin levels (Table). Univariate analysis of CR/sCR by baseline characteristics showed that IgG heavy chain versus other heavy chain types (odds ratio [OR]: 0.162, P<0.0001), high sBCMA (OR: 0.646, P=0.0007), β-2-microglobulin (≥5.5 vs <3.5 mg/L; OR: 0.201, P=0.0072), and presence of extramedullary disease (OR: 0.428, P=0.0394) were negatively associated with CR/sCR, whereas high vector copy number in drug product was positively associated with CR/sCR (OR: 1.290, P=0.0287). Multivariate analysis of CR/sCR identified IgG heavy chain versus other heavy chain types (OR: 0.100, P<0.0001), high sBCMA (OR: 0.637, P=0.0110), and elevated prothrombin time-international normalized test (PT-INR) (OR: 0.005, P=0.0365) as negative correlates of CR/sCR, and high vector copy number in drug product (OR: 1.486, P=0.0168) as a positive correlate of CR/sCR. Descriptive analysis demonstrated lower median (range) sBCMA levels at baseline in pts with CR/sCR (191 ng/mL [11–909]) versus non-CR/sCR (340 ng/mL [19–2735]); across both groups, sBCMA levels had increased from screening (CR/sCR, 161 ng/mL [27–689]; non-CR/sCR, 302 ng/mL [24–1490]). Conclusions: In this subanalysis of KarMMa, multivariate analysis identified IgG, sBCMA, and PT-INR as negative correlates of CR/sCR, and vector copy number in drug product a positive correlate. As sBCMA is a tumor burden indicator and can affect BCMA-targeted therapies (Cowan et al. ASH 2019. Abstract 204), selecting pts with lower tumor burden and controlling tumor burden during manufacturing or bridging therapy may aid in achieving CR/sCR with ide-cel. Previously presented and published: Shah N, et al. ASH 2021, Blood 2021; 138:1739. Study support: 2seventy bio, formerly bluebird bio, and Celgene, a Bristol-Myers Squibb Company.