Immunosympathectomy as the first phenotypic knockout with antibodies

Abstract

In a PNAS Classic Article published in 1960, Rita Levi-Montalcini offered formal and conclusive proof that endogenous NGF was responsible for the survival of sympathetic neurons in vivo. Thus ended an experimental tour de force lasting a decade, starting with the demonstration that a humoral factor, produced from a tumor transplanted in a chicken embryo, was responsible for stimulating outgrowth of nerve fibers from sympathetic and sensory neurons. From a more general methodological point of view, this work provided a breakthrough in the quest to achieve targeted loss of function and experimentally validate the function of biological molecules. Finally, this work provided an example of the ablation of a specific neuronal subpopulation in an otherwise intact nervous system, an immunological knife of unsurpassed effectiveness and precision. The novelty and the importance of the PNAS Classic Article is discussed here, collocating it within the context of the particular moment of the NGF discovery saga, of Rita Levi-Montalcini's scientific and academic career, and of the general scientific context of those years. This seminal work, involving the use of antibodies for phenotypic knockout in vivo, planted seeds that were to bear new fruit many years later with the advent of monoclonal antibodies and recombinant antibody technologies.

Fig. 1.

Immunosympathectomy and cell ablation. NGF deprivation by anti-NGF antibodies leads to the selective ablation of sympathetic neurons (immunosympathectomy). (A) Immunosympathectomy (2) is the first example of growth factor starvation of target cells. (B) In cancer therapy, tumor angiogenesis is inhibited by antibodies against VEGF (41, 42). (C) Antibodies can be used as genes rather than as proteins. In the neuroantibody approach (46), transgenic mice ectopically express genes specific for an antibody to a given target protein. In the AD11 mouse model (47, 48), an anti-NGF antibody selectively recognizing mature NGF, with respect to its precursor proNGF, is expressed in transgenic mice, creating an NGF/proNGF imbalance that results in a progressive neurodegeneration of the Alzheimer's type. (D) Cell ablation can be achieved by immunotoxins or by cell-type specific expression of gene products toxic for the cell. (E) Silencing of neurons, rather than their ablation, can be obtained by interfering with their excitability, such as for instance with optogenetic proteins. Neuronal silencing can be obtained by expressing channelrhodopsin in inhibitory neurons (I) or halorhodopsin in excitatory neurons (E) and stimulating with light of the appropriate wavelength.

Fig. 2.

Protein silencing with antibodies. Antibodies can be expressed as intracellular proteins (intrabodies) (52). (A) Intrabodies are simpler antibody forms, made only of the variable regions, without the Fc constant portions. (B) Intrabodies, selected from suitable intrabody libraries (60, 63) can be targeted to different subcellular compartments by the use of suitable autonomous and dominant targeting sequences. Intrabodies can interfere with cellular proteins and induce protein knockdown or silencing, by different modes of actions: intrinsically neutralizing, retargeting to a different subcellular compartment, (C) physical inactivation by chromofore assisted laser inactivation (CALI) or fluorophore assisted laser inactivation (FALI), or (D) escorting the target protein to degradation by proteasomes (SIT, or silencing intrabody technology) (65). (E) Intrabodies can mediate antigen-dependent cell-killing: intrabodies fused to caspase-3, upon binding a dimeric protein, cause caspase dimerization by proximity and activate it, inducing cell death (64). (F) Intrabodies allow selective interference with oligomeric vs. monomeric forms of a target antigen, such as for instance Alzheimer's Aβ peptide oligomers (61). (G) The isolation of intrabodies against protein–protein interaction sites allows edge-specific silencing in an intracellular protein network, with selective interference as opposed to “node removal.” (H) In principle, isolating intrabodies against posttranslational modifications of cellular proteins (PTM) would allow PTM-selective protein silencing.