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#monoclonal antibodies

9 public questions tagged with this topic.

Immunological Applications

Immunotoxins are composed of:

Immunotoxins constructed to overcome lack of selectivity of conventional chemotherapy achieve tumor specific delivery of ultrapotent protein toxins that cannot enter mammalian cells unaided. Design consists of targeting moiety typically single-chain variable fragment scFv or disulfide stabilized Fab derived from murine or humanized antibody recognizing tumor antigen CD22 on hairy cell leukemia, CD25 on adult T cell leukemia, mesothelin on mesothelioma, linked via flexible glycine-serine peptide or reducible disulfide bond formed between engineered cysteines to effector toxin devoid of native r

Ref: Pastan et al Nature Rev Cancer 2006 6:559 immunotoxins PE38; Kreitman Clin Cancer Res 2009 fusion toxin de-immunized.

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Immunological Applications

Monoclonal antibodies bind:

Monoclonal antibody originates from unique genetic event where one developing B cell successfully completed VDJ recombination on heavy chain locus chromosome 14 mediated by RAG1/2 recognition of recombination signal sequences, non-homologous end joining with TdT adding N nucleotides creating unique complementarity determining region three loop that dominates antigen contact, plus VJ recombination on light chain locus producing paired variable domains. All progeny via mitosis carry identical nucleotide sequence encoding paratope whose three-dimensional shape, electrostatic surface, and hydropho

Ref: Abbas Cellular Molecular Immunology 10th ed monoclonal single epitope uniform affinity; Roitt Essential Immunology specificity.

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Immunological Applications

Myeloma cells used in hybridoma technology are selected to be:

Establishing stringent selection requires myeloma background auxotrophic for purine salvage, achieved by exposure to 8-azaguanine, purine analogue structurally similar to guanine. Salvage competent cells convert 8-azaguanine via HGPRT to 8-azaguanosine monophosphate incorporated into mRNA and rRNA causing miscoding, translational errors, and lethal proteotoxicity triggering unfolded protein response. Spontaneous HPRT1 mutants survive because they cannot metabolize analogue, accumulating no toxic nucleotides. Surviving subclones harbor frameshift, nonsense, or missense mutations in coding exons

Ref: Szybalski & Szybalska 1962 HGPRT negative mutants 8-azaguanine; ATCC SP2/0 Ag14 datasheet selection mechanism.

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Immunological Applications

Hybridoma cells survive in HAT medium because they:

Survival in HAT environment demands functional purine and pyrimidine salvage enzymes inherited from normal B parent. B lymphocytes express HPRT1 locus on X chromosome encoding 24 kDa hypoxanthine-guanine phosphoribosyltransferase that catalyzes phosphoribosyl transfer from PRPP to hypoxanthine yielding IMP plus pyrophosphate and to guanine yielding GMP, bypassing de novo steps requiring folate. They also express cytosolic thymidine kinase 1 cell-cycle regulated peaking in S phase and mitochondrial kinase 2 constitutive, phosphorylating thymidine supplied in medium to TMP using ATP. Hybridoma c

Ref: Janeway Immunobiology salvage pathway HGPRT; Freshney Animal Cell Culture hybrids survive HAT via HGPRT TK from B cell.

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Immunological Applications

Aminopterin in HAT medium functions by:

Aminopterin classifies as antifolate antimetabolite structurally analogous to folic acid with pteridine ring substitution preventing enzymatic reduction. Dihydrofolate reductase normally catalyzes NADPH dependent reduction of dihydrofolate to tetrahydrofolate, central one-carbon carrier crucial for biosynthesis. Tetrahydrofolate derivatives required at two distinct steps: 10-formyl tetrahydrofolate donates formyl groups to glycinamide ribonucleotide transformylase and aminoimidazole carboxamide ribonucleotide transformylase in de novo purine pathway synthesizing inosine monophosphate precursor

Ref: Goodman & Gilman Pharmacol 13th ed DHFR antifolate; Lodish MBoC 9th ed Fig 7-32 aminopterin blocks de novo purine thymidylate.

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Immunological Applications

Immunotoxins are composed of:

Immunotoxins chimeric proteins integrating targeting domain derived monoclonal antibody and cytotoxic domain protein toxin to achieve picomolar potency against malignant cells expressing defined surface antigen. Antibody moiety typically Fab prime 50 kDa scFv 25 kDa composed heavy variable light variable domains connected glycine serine linker disulfide-stabilized Fv dsFv recognizing tumor antigens CD22 135 kDa hairy cell leukemia CD25 IL-2 receptor alpha cutaneous T-cell lymphoma mesothelin 40 kDa GPI-anchored mesothelioma pancreatic adenocarcinoma HER2 with affinity 1-10 nM. Toxin component

Ref: Pastan Annu Rev Med 2007 Immunotoxin Construction Principles Review; FDA Lumoxiti Moxetumomab Design Label; Janeway Immunotoxin Antibody Toxin Fusion Mechanism.

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Immunological Applications

Rituximab targets which antigen?

Rituximab first chimeric mouse-human IgG1 kappa mAb targeting CD20 tetra-spanning membrane protein 33-37 kDa encoded MS4A1 11q12 four hydrophobic transmembrane domains intracellular N C termini extracellular small large loops 44 aa large loop epitope. CD20 forms homodimers tetramers lipid rafts regulating calcium flux store-operated entry Orai1 STIM1 and BCR signaling amplifying PLC gamma. Expression pre-B mature B lymphocytes but absent plasma cells HSC no shedding soluble form slow internalization excellent therapeutic target. Binding rituximab large loop crosslinks CD20 redistribution lipid

Ref: FDA Rituxan Rituximab CD20 MOA Label; NEJM Rituximab Lymphoma 1997 Maloney; Janeway CD20 Depletion ADCC CDC Mechanism Chapter 5.

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Immunological Applications

Monoclonal antibodies bind:

Bevacizumab recombinant humanized IgG1 mAb 149 kDa neutralizes soluble VEGF-A isoforms 121 165 189 206 secreted tumor stromal inflammatory cells hypoxia HIF1alpha binding hypoxia response elements driving transcription. VEGF-A binds VEGFR1 FLT1 and R2 KDR tyrosine kinases endothelial cells dimerization autophosphorylation Tyr1175 recruiting PLC gamma generating IP3 DAG increasing calcium PKC stimulating proliferation permeability via VE-cadherin internalization gaps survival Akt phosphorylation caspase-9. Binding epitope Arg82 Lys84 affinity 0.5 nM overlapping receptor binding interface preven

Ref: Ferrara NEJM Bevacizumab VEGF Targeted Therapy 2004; FDA Avastin Mechanism Label VEGF Neutralization; Lodish Angiogenesis VEGF Signaling Pathway Chapter 23.

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Basic Applied Biology

Hybridoma technology produces:

Hybridoma technology developed by Georges Köhler and César Milstein in 1975 and awarded Nobel 1984 created paradigm shift by immortalizing antibody-producing B lymphocytes through somatic cell fusion. Mice immunized with antigen develop germinal center reaction where B cells undergo VDJ recombination, somatic hypermutation and class switching to generate high affinity plasma cells. Splenic lymphocytes harvested shortly after boost are fused with HGPRT-deficient myeloma partner such as SP2/0-Ag14 or NS0 using polyethylene glycol 1500 that perturbs lipid bilayers promoting membrane fusion formin

Ref: Köhler & Milstein Nature 1975 Continuous cultures of fused cells; Abbas Cellular and Molecular Immunology 10th ed. monoclonal antibody generation.

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