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Multiple myeloma is a malignant disorder of plasma cells. Although the prognosis of MM patients has improved significantly with the widespread use of new targeted drugs such as immunomodulators, proteasome inhibitors and autologous hematopoietic stem cell transplantation, the disease will eventually recur and progress. In recent years, immunotherapy has made breakthroughs in the treatment of MM, chimeric antigen receptor T-cell therapy, antibody conjugate drugs and bispecific antibodies have shown significant effectiveness and good safety at different stages of the disease. Bispecific antibodies, which bind both CD3 on T cells and target molecules on the surface of malignant plasma cells, are effective immunotherapeutic agents for patients with relapsed/refractory MM. This article reviews the research progress of bispecific antibodies in the treatment of MM in conjunction with the reports at the 64th American Society of Hematology Annual Meeting.
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@#Objective Establish quality control methods for critical quality attribute of bispecific antibody against programmed cell death protein 1(PD-1)/cytotoxic T-lymphocyte-associated protein 4(CTLA-4).Methods The biological activity of PD-1 target was determined by reporter gene assay,and the competitive binding activity of CTLA-4 target was determined by flow cytometry;The antibody molecular size variants were controlled by reducing/non-reducing capillary electrophoresis-sodium dodecyl sulfonate(CE-SDS) and size exclusion chromatography-high performance liquid chromatography(SEC-HPLC);Charge heterogeneity was determined by imaging capillary isoelectric focusing electrophoresis(iCIEF);Bispecific anti-PD-1/CTLA-4 antibody was identified by peptide map analysis;Glycosylation was analysed by high performance liquid chromatography(HPLC)Results The concentration for 50% of maximal effect(EC_(50)) of PD-1target was(6.91±0.78) nmol/L,and the relative biological potency to the reference was(103.50±13.08)% with the RSD of 12.64%;The EC_(50) of CTLA-4 target activity was(0.35±0.28) nmol/L,and the relative biological potency was(99.30±9.15)% with the RSD of 8.32%.The percentage of peak area of light chain and heavy chain of reducing CE-SDS was(98.86±0.02)%.The main peak area percentage of non-reducing CE-SDS was(93.07±0.13)%,fragment percentage was(4.44±0.13)%,and polymer percentage was(2.49±0.15)%.The peak area percentage of SEC-HPLC monomer and polymer were(97.20±0.01)% and(2.68±0.01)%,respectively.The area percentage of peak A group,peak B group,peak C group and peak D group were(38.43±0.54)%,(43.26±0.32)%,(11.31±0.14)% and(7.00±0.17)%,respectively.Peptide mapping showed the specific spectrum of the bispecific anti-PD-1/CTLA-4 antibody,which could be adopted for identification test.The highest proportion of glycotype was GOF,with a content of(41.06±0.11)%,There were three types of glycan containing sialic acid,namely G2F+G1F-NANA,G2F-NANA and G2F-2NANA,with the content of(12.44±0.12)%,(12.00±0.05)% and(5.37±0.05)%,respectively.The total content of glycan containing sialic acid was(29.80±0.20)%.Conclusion The critical quality attributes of bispecific anti-PD-1/CTLA-4 antibody were studied and the corresponding quality control methods were established to ensure its safety,effectiveness and quality control,which provides a reference for the quality control methods and strategies of this type of monoclonal antibody products.
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@#Objective To prepare bispecific antibody targeting cluster of differentiation 73(CD73) and programmed cell death-ligand 1(PD-L1),and evaluate its binding ability and killing ability in vitro.Methods Using genetic engineering method,PD-L1 single-chain fragment variable(scFv) was inserted into the hinge region of CD73 monoclonal antibody to construct anti-CD73/PD-L1 bispecific antibody(BS-21),which was screened by CHO GS expression system to obtain highly expressed cell line.After purified by Protein A and molecular sieve,the purity of antibody was detected by size exclusion chromatography-high performance liquid chromatography(SEC-HPLC),the binding ability of antibody in vitro was detected by flow cytometry,and the killing ability in vitro was detected by using peripheral blood mononuclear cell(PBMC) to kill Calu 1 lung cancer cells in vitro.Results High-yield cell lines were obtained by pressure screening.A bispecific antibody BS-21 with a purity of 99.6% was obtained by purification,which bound to CD73 and PD-L1 molecules simultaneously.Compared with anti CD73 and anti PD-L1 groups,BS-21 group significantly increased the killing rate of immune cells to Calu 1 tumor cells(F=30.36,each P<0.001).Conclusion Bispecific antibody BS-21 reduced the immunosuppressive effect of CD73 and PD-Ll on immune cells simultaneously,and showed good anti-tumor function.
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Bispecific antibodies (BsAb) are special antibodies that can bind to two different antigenic epitopes at the same time, and their diverse forms provide the basis for different effects. In 2021, amivanamab was approved to treat metastatic or surgically unresectable non-small cell lung cancer patients with epidermal growth factor receptor (EGFR) 20 exon insertion mutation who experience tumor progression during or after platinum doublet chemotherapy. BsAb has made great progress in the treatment of solid tumors, especially lung cancer. This article reviews the structural form, mechanism and research progress of BsAb in the field of lung cancer.
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Blinatumomab, as a novel bispecific antibody targeting CD19 and CD3, can induce T lymphocytes to precisely target CD19 positive B lymphocytes to apoptosis. At present, it is the only bispecific antibody approved for the treatment of hematological malignancies in China. Blinatumomab is effective in the treatment of newly diagnosed, relapsed/refractory, minimal residual disease positive patients with B-cell acute lymphoblastic leukemia (B-ALL) . It can improve the survival of the patients and is well tolerated. The further study of blinatumomab can provide theoretical basis and new ideas for induction therapy, salvage therapy and subsequent hematopoietic stem cell transplantation in patients with B-ALL.
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CD47 is widely expressed on the cell surface, and combines with signal regulatory protein α (SIRPα) to transmit the "don't eat me" signal, which plays a key role in self-recognition and tumor immune escape. Studies have shown that the high expression of CD47 in different hematologic neoplasms is associated with the occurrence, progression and poor prognosis of tumors. As a new immune checkpoint, CD47 is gradually becoming an effective target for tumor immunotherapy. and various related preclinical and clinical studies for hematologic neoplasms are underway. This article summarizes the application of CD47 in hematologic neoplasms, in order to provide references for the treatment.
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The CD19-targeting bispecific T-cell engager blinatumomab has shown remarkable efficacy in patients with relapsed/refractory B-cell precursor acute lymphoblastic leukemia. However, several studies showed that blinatumomab has a short plasma half-life due to its low molecular weight, and thus its clinical use is limited. Furthermore, multiple trials have shown that approximately 30% of blinatumomab-relapsed cases are characterized by CD19 negative leukemic cells. Here, we design and characterize two novel antibodies, A-319 and A-2019. Blinatumomab and A-319 are CD3/CD19 bispecific antibodies with different molecular sizes and structures, and A-2019 is a novel CD3/CD19/CD20 trispecific antibody with an additional anti-CD20 function. Our in vitro, ex vivo, and in vivo experiments demonstrated that A-319 and A-2019 are potent antitumor agents and capable of recruiting CD3 positive T cells, enhancing T-cell function, mediating B-cell depletion, and eventually inhibiting tumor growth in Raji xenograft models. The two molecules are complementary in terms of efficacy and specificity profile. The activity of A-319 demonstrated superior to that of A-2019, whereas A-2019 has an additional capability to target CD20 in cells missing CD19, suggesting its potential function against CD19 weak or negative CD20 positive leukemic cells.
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Humans , Antigens, CD19/therapeutic use , Antineoplastic Agents/pharmacology , Immunotherapy , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , T-LymphocytesABSTRACT
@#[Abstract] Objective: To construct and purify the recombinant bispecific antibody (BsAb) targeting PD-1 and CD19 and evaluate its activity. Methods: With pCAR1 plasmid as the vector, the eukaryotic expression vector of anti-PD-1/CD19 BsAb was constructed by molecular cloning technology, and then transfected into mammalian cell line CHO-S by PEI reagent for transiently expressing antibody. The BsAb was purified by Affinity chromatography and then identified by SDS-PAGE and WB. The blocking activity of BsAb on PD-1/PD-L1 in vitro was detected by Luciferase reporter gene assay. The activity of antibody (BsAb)-dependent cell (PBMC)-mediated cytotoxicity (ADCC) in vitro was evaluated by lactate dehydrogenase (LDH) cytotoxicity assay. Results: The double plasmid eukaryotic expression vector pCAR1-19X3 was successfully constructed, and anti-PD-1/CD19 BsAb was successfully expressed in CHO-S cells, named pCAR1-19X3-TY. pCAR1-19X3-TY could effectively block the binding of PD-1 to its ligand PD-L1 in vitro, and the EC50 based on the dose-response curve was 0.306 μg/ml. ADCC results showed that pCAR1-19X3-TY could mediate the cytotoxicity of PBMC against Raji cells, and the curve showed a linear upward trend; when the effect/target ratio was 50∶1, the target cell lysis rate of pCAR1-19X3-TY was (38.9±0.3)%, which was not significantly different from that of the positive treatment group (46.7±4.9)% (P>0.05), but significantly higher than that of the negative control group (1.2±0.1)% (P<0.05). Conclusion: The recombinant anti-PD-1/CD19 BsAb can effectively block the binding of PD-1 and PD-L1 and activate PBMC mediated cytotoxicity against Raji cells. pCAR1-19X3-TY has the potential application value in the treatment of B-cell malignant tumor.
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M701 is a bispecific CD3/EpCAM T-cell engager antibody for the treatment of malignant ascites. We developed a population pharmacokinetic/pharmacodynamic (PK/PD) model to quantitatively describe and predict the antitumor effect of M701 in human colorectal cancer xenograft mice. We developed the M701 PK model based on plasma concentration data after i.v. administration. A tumor growth model for human colorectal cancer xenograft was developed to evaluate the antitumor effect of M701. We additionally simulated the inhibitory effect of M701 on tumor volume under different dose regimens based on a PK/PD model. A two-compartment model was developed to predict the PK in human colorectal cancer xenograft mice. The relationship between the M701 concentration and tumor growth inhibition was characterized by a combined Simeoni tumor growth/transit compartment model. The estimated pharmacodynamic parameters were related to the tumor growth characteristics λ0 (0.212 d-1) and λ1 (0.044 7 cm3·d-1), to the drug potency k2 (0.071 5 mL·ng-1·d-1), and to the kinetics of tumor cell death k1 (2×10-5 d-1). A model visual predictive check showed that both the PK model and the tumor growth model closely fit the observed data. Simulated tumor growth after administration of M701 (0.5 mg·kg-1 every 6 days and 0.25 mg·kg-1 every 3 days) could be effectively inhibited. This population PK/PD model of M701 provides insight into the antitumor effect of M701 and supports the further therapeutic development of M701.
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Bispecific antibody (BsAb) has two different antigen-binding sites, divided into the "IgG-like" format and the "non-IgG-like" format. Different formats have different characteristics and applications. BsAb has higher sensitivity and specificity than conventional antibodies, with special functions such as recruitment of immune cells and blocking of dual signaling pathways, playing an important role in immune-diagnosis and therapy. With the deterioration of the global environment and the irregular living habits of people, the incidence of tumor is becoming higher and higher. Tumor becomes the most serious fatal disease threatening human health after cardiovascular disease. There are 12 million estimated new tumor cases each year worldwide. The major clinical treatments of tumor are surgical resection, chemoradiotherapy, target therapy. Tumor immunotherapy is a novel approach for tumor treatment in recent years, and activates human immune system to control and kill tumor cells. Although the traditional monoclonal antibodies have already acquired some therapeutic effects in tumor targeted therapy and immunotherapy, they induce drug resistance resulted from the heterogeneity and plasticity of tumors. Binding to two target antigens at the same time, BsAb has been used in the clinical treatment of tumors and obtained promising outcomes. This review elaborates the research progress and applications of bispecific antibody in clinical tumor therapy.
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Humans , Antibodies, Bispecific/therapeutic use , Antibodies, Monoclonal/therapeutic use , Immunotherapy , Neoplasms/therapyABSTRACT
Significant advances have been made in cancer immunotherapy recently, of which, bispecific antibodies (BsAbs), through bridging, redirecting and activating immune effector cells to kill cancer cells, are attracting increasing attention.Since the anti-CD19 and anti-CD3 BsAb, blinatumomab, was approved in 2014 by the FDA for the treatment of acute lymphoblastic leukemia, preclinical and clinical research with immune-cell-redirecting BsAbs have been fast growing in the area of hematologic malignancies. This review summarizes the current scientific and clinical investigation of BsAbs targeting different tumor-associated antigens from B lymphocytes, plasma cells and myeloid cells, covering three most common blood cancers, namely, lymphoma, multiple myeloma and leukemia. Further development for better therapeutic benefits and lower adverse events, are continuously being pursued, in particular, looking for more specific tumor antigens, optimizing antigen-antibody affinities, extending the half-life of BsAbs and redirecting different immune effector cells, whose breakthroughs and opportunities are soon to be delivered for the management of hematologic malignancies.
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@#Tumor immunotherapy is currently the new direction for the treatment of cancer. Bispecific antibody can bind two different antigens, so the development prospect in the field of tumor treatment is very attractive. The most compelling trifunctional antibody and bispecific T-cell engager in bispecific antibodies have been marketed separately, with representative drugs as catumaxomab and blinatumomab, respectively. So far, nearly 100 antitumor bispecific antibody drugs are undergoing clinical trials and in-depth understanding of their mechanisms of action will provide more powerful solutions for cancer treatment. This review summarizes the progress of catumaxomab, blinatumomab and current highly promising bispecific antibody drugs, for the further development and application of tumor therapy.
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With the rapid development of antibody genetic engineering, bispecific antibody technology has been advanced. They are capable of binding two or more different epitopes simultaneously, thus offering specific advantages over natural monoclonal antibodies in immunotherapy. Bispecific antibodies have been successfully used in cancer therapy (e.g. melanoma, Hodgkin's lymphoma, liver cancer, and stomach cancer) and inflammation therapy (e.g. rheumatoid arthritis, psoriasis and Crohn's disease), but are still in their early stage for viral immunotherapy. In this study, we reviewed the research progress of bispecific antibodies for immunotherapy of virus infections, especially those with good effects in vivo and in vitro, to provide references for the research and development of bispecific antibodies for antivirus treatment.
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Humans , Antibodies, Bispecific , Therapeutic Uses , Antibodies, Monoclonal , Epitopes , Immunotherapy , Virus DiseasesABSTRACT
Bispecific antibodies can target two different targets simultaneously with a wide application prospect and rapid development in tumor treatment. The main function is to recruit effector cells selectively in order to kill tumor cells or bind tumor-associated growth factor receptor to inhibit cell proliferation. This paper reviews the current situation of bispecific antibodies in design, mechanism of action and the research progress in the clinical cancer treatment.
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This study aimed to investigate the efficacy of a bispecific antibody mAb04-MICA on human leukemia cell K562 both in vitro and vivo. mAb04-MICA was previously found to posses excellent anti-angiogenic activity, and have the ability to recruit immune surveillance in tumor microenvironment. In this study, the affinity of mAb04-MICA to VEGFR2 and NKG2D was identified by ELISA. CCK8 was used to detect the effect of mAb04-MICA on K562 proliferation. The cross reactivity of mAb04-MICA to murine VEGFR2 was determined by flow cytometry assay. To evaluate the antitumor activity of mAb04-MICA,tumor volume,tumor weight and the survival of K562 tumor-bearing nude mice were analyzed. The anti-angiogenic activity was determined by immunohisto-chemistry. The results indicated that mAb04-MICA could target to VEGFR2 and NKG2D,and inhibit K562 pro-liferation specifically. Besides,mAb04-MICA showed high binding capacity to murine VEGFR2. The bispecific antibody exhibited superior antitumor efficacy to the maternal monoclonal antibody and prolonged the survival of tumor-bearing mice. The expression of Ki-67,p-VEGFR2,VEGF and CD34 in mAb04-MICA treated group was significantly reduced. The results indicated that mAb04-MICA could attenuate the phosphorylation of VEGFR2 and impair angiogenesis of the tumor microenviroment. Therefore,mAb04-MICA could be further developed as a potential tumor targeted immunotherapeutic agent for leukemia.
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We present a bispecific antibody that recognizes an antigen and a hapten and can be applied to various biological assays, including immunoblotting and immunoprecipitation. In immunoblot analysis of serum, an anti-C5 x anti-cotinine bispecific tandem single-chain variable fragment (scFv)-Fc fusion protein and cotinine-conjugated horseradish peroxidase (HRP) generated a clean signal without the high background that was observed in a parallel experiment using HRP-conjugated goat anti-rabbit immunoglobulin G (Fc-specific) antibody. In immunoprecipitation analysis of serum, use of the bispecific tandem scFv-Fc fusion protein and cotinine-crosslinked magnetic beads significantly reduced the amount of protein contaminants compared with a parallel experiment done with protein A agarose beads. In subsequent immunoblot analysis, use of cotinine-HRP as the secondary probe instead of HRP-conjugated goat anti-rabbit IgG (Fc-specific) antibody successfully eliminated the band corresponding to the bispecific tandem scFv-Fc fusion protein.
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Animals , Humans , Rabbits , Antibodies, Bispecific/immunology , HEK293 Cells , Haptens/immunology , Immunoblotting/methods , Immunoprecipitation/methods , Single-Chain Antibodies/immunologyABSTRACT
Objective To construct bispecific antibody BsAb1/17 against both IL-1β and IL-17A,express and purify the biologically active BsAbl/17 protein in prokaryotic system for further studies and applications. Methods VH1VL17-CL and VL1VH17-CH1 gene segments were constructed by overlap-PCR.Restriction enzyme sites Nco Ⅰ and BamH Ⅰ were designed at the 5'and 3' end primers respectively. The products of overlap-PCR were ligated to the Nco Ⅰ/BamH Ⅰ -prepared pET-27b vector. The recombinant plasmids pET-27b-VH1 VL17-CL(petA) and pET-27b-VL1 VH17-CH1 ( petB ) were transformed into E. coliRosetta separately. The expressing products were analyzed by SDS-PAGE and Western blot. Neutralization activity of the bispecific antibody for blocking the induction of IL-18 expression by IL-1β in human T cells was determined by real-time PCR. Neutralization activity of the bispecific antibody for blocking the induction of IL-6 expression by IL-17A in HeLa cells was determined by ELISA assay. Results The structure of the plasmids pET-27b-VH1 VL17-CL(petA) and pET-27b-VL1 VH17-CH1 (petB)was confirmed by DNA sequencing. After induction, the fusion proteins were expressed mainly as inclusion bodies. The purity of the both proteins exceeded 90%. SDS-PAGE analysis suggests the relative molecular mass of both products expressed by petA and petB were approximately 38× 103, which is in accordance with the theoretical value. The results of Western blot and ELISA test demonstrated that BsAb1/17 molecule had binding ability to both IL-1β and IL-17A. The BsAb1/17 could block IL-1β to stimulate human T cell to express IL-18 and block IL-17A to stimulate HeLa cell to express IL-6. Conclusion We successfully constructed a novel bispecific antibody BsAb1/17 against both IL-1 β and IL-17A, and expressed biologically active BsAb1/17 protein in prokaryotic system.
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Cytokine-induced killer(CIK) cells are heterogeneous lymphocytes generated by incubation of human peripheral blood mononuclear cells with various cytokines in vitro,characterized by both the T lymphocyte's powerful cytotoxic activity and the natural killer(NK) cell's non-major histocompatibility-restricted antitumor activity in vitro and in vivo.CIK cells have their peculiar superiority over standard lymphokine activated killer(LAK) cells,tumor-infiltration lymphocytes(TIL) and anti-CD3 antibody induced activated killer(CD3AK) cells in application to the cancer adoptive immunotherapy and have hence received wide attention from researchers.This article updates recent researches on CIK cells,their interaction with dendritic cells,bispecific antibodies and oncolytic viruses,as well as their use in gene transfer.
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Objective:To construct and express anti CD3/anti CD20 Diabody and identify its biological activity.Methods:PCR and overlap PCR were used to construct anti CD3/anti CD20 Diabody.DNA sequence was analyzed by the Terminus of Dideoxy Nucleotide.The product was purified by affinity chromatography and analyzed by both the detection of Western blot and size exclusion chromatography;its antigen binding activity was examined by FACS and rosetting assay.Results:The data of DNA sequence showed that the anti CD3/anti CD20 Diabody was corrected.The Diabody was recovered in high yield(up to 1 mg/ml) after E taq purification and predominantly(90%) as a dimer.The Diabody binded Jurkat cells(CD3 +) and Daudi cells (CD20 +),respectively.Furthermore,the Diabody was capable of simultaneous binding to Jurkat cells and Daudi cells as shown by cellular rosetting.Conclusion:The anti CD3/anti CD20 BsF(ab') 2 was first recast into the Diabody format and succeeded to obtain high level expression.The results of some biological activity experiments indicated that the Diabody could bind to Jurkat cells and Daudi cells.
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Objective:To analyse and prepare bispecific antibody(BsAb, anti human bladder cancer/anti VEGF) which could enhance the effectiveness of tumor cell killing activity.Methods:Monoclonal antibodies against human bladder cancer and VEGF were used to prepare bispecific antibody.Results:IC50 of BsAb was 10 -9.5 ,while those of antibodies against human bladder cancer and VEGF were 10 -8.9 and 10 -8.3 .Conclusion:The effectiveness of BsAb was significantly higher than that of monoclonal antibodies. [