A Humanized, Bispecific Immunoadhesin-Antibody that Retargets CD3+ Effectors to Kill HIV-1-Infected Cells

Abstract

We have developed a humanized, bispecific immunoadhesin-antibody (BsIAb) that targets and kills HIV-infected cells. Comprised of CD4-IgG and humanized anti-CD3-IgG, this BsIAb is bifunctional. First, in targeting, it exploits the natural affinity of CD4 for gp120 to target the BsIAb to HIV-infected cells, and second, it recruits and activates, through its anti-CD3 moiety, cytotoxic T lymphocytes (CTL) to lyse target cells in a non-MHC restricted manner. To produce purified BsIAb from supernantants of transfected mammalian cells, we designed a three-step recovery scheme based on the structural elements of this heterotrimeric protein. The ability of purified BsIAb to specifically lyse HIV-infected target cells was demonstrated using CTL from two different sources: whole peripheral blood lymphocyte (PBL) fractions and pure CTL preparations. In contrast, a human anti-gp120 antibody mediated lysis of HIV-infected target cells only with PBL fractions and not with purified CTL. Moreover, lysis observed in the presence of the human anti-gp120 antibody was completely blocked in the presence of human serum (which competes for Fc gamma receptor binding), whereas BsIAb-mediated lysis of target cells was not affected. We measured the monovalent affinities of BsIAb for HIV-gp120 on infected cells and for CD3 epsilon on CTL. Relative to the bivalent parent molecules, CD4/gp120 affinity in the BsIAb is unchanged, whereas anti-CD3/CD3 is substantially decreased. We further demonstrated by fluorescence microscopy that physical association of CD3+ cells with gp120-expressing cells occurs only in the presence of BsIAb. Thus, the cytocidal activity of BsIAb in the presence of serum reflects its unique ability to recruit CTL as effector cells and highlights a potentially important advantage of this type of construct over antibodies for HIV-directed therapy.

T-cell activation and cytokine production via a bispecific single-chain antibody fragment targeted to blood-stage malaria parasites

“…[7][8][9] An application of this mechanism for the control of infectious diseases is the use of a biscFv composed of OKT3 scFv and CD4 to target and kill HIV-infected cells. 10,11 These immune-activating properties of biscFv may provide a means for enhancing biscFv therapy via several distinct mechanisms. In addition to the current example of 5.2-OKT3 biscFv as an immunotherapeutic drug, we have recently constructed an immunotoxin composed of 5.2 scFv and an antimalaria toxin.…”

“…[7][8][9] In addition, a biscFv composed of CD4 and anti-CD3 also has been reported to retarget T cells against gp120-expressing HIV-infected cells. [10][11][12] Thus, application of biscFvs appears to be a promising avenue for malaria immunotherapy because biscFvs do not rely on the induction of antibody production but rather entail the stimulation and targeting of cell-mediated mechanisms. Moreover, unlike antibodies, which are often strain specific, biscFvs would act upon P falciparum strains of diverse genetic makeup.…”

Engineering broadly neutralizing antibodies for HIV prevention and therapy

“…Historically, bispecific molecules to target HIV latent reservoir cells paired soluble versions of the CD4 receptor to anti-CD3 T cell engaging antibody fragments in a variety of formats [283–285]. Previous attempts to employ anti-HIV mAb specificities in bispecific formats with anti-CD3 demonstrated limited efficacy, likely due to limited breadth [284–286].…”

Secretion of glycosylation site mutants can be rescued by the signal/pro sequence of tissue plasminogen activator

“…Secreted TNFR-IgG was purified from conditioned culture media by immobilized S. aureus protein A affinity chromatography via a modification of a method described previously [Chamow et al, 1994]. Four TNFR-IgG1 variants were purified: NNNN, NNQQ, NSNQ, and QSNQ.…”