== Neutralization breadth and potency of trispecific antibodies.aNeutralization breadth of the parental, bispecific and trispecific antibodies was tested against a panel of 208 viral strains. for the receptor-binding site, a conserved Env glycan patch and the Env membrane proximal region with nearly pan-isolate neutralization breadth and high potency. == Introduction == Recent advances in the discovery of broadly neutralizing antibodies (bNAbs) targeting the HIV-1 envelope glycoproteins (Env) have awakened great interest in their use as pre-exposure prophylaxis for prevention and as therapeutic agents, particularly in combination with antiretroviral treatment (ART) for HIV remission and eradication13. bNAb isolation and characterization has been accelerated via the integration of emerging functional and structural information and new technologies of single B cell sorting and cloning49. bNAbs are therapeutically beneficial as they possess high capacity for viral neutralization. Additionally, bNAbs can facilitate fragment crystallizable (Fc)-mediated effector functions that promote cell lysis and/or clearance of infected cells that express HIV-1 Env on the cell surface via antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity10. The characterization of HIV-1 bNAbs and their cognate epitopes on the Env spikes has identified five conserved Env sites of vulnerability including the CD4-binding site (CD4bs), the V1/V2-glycan region, the V3-glycan region, the gp41 membrane proximal external region (MPER), and the gp120gp41 interface11. Passive immunization with bNAbs is being SB 525334 explored as a means for prevention in healthy individuals and as treatment for HIV infected patients. Passive immunization in humans has proven highly effective in treating many infections such as hepatitis A, hepatitis B, rabies, and respiratory syncytial viruses12, but these viruses have much lower genetic diversity1216than do circulating HIV-1 isolates17,18, which greatly confounds the clinical outcome of passive immunization for HIV-1 treatment. Administration of a single bNAb as a therapeutic agent has successfully cleared phase I safety clinical trials, demonstrating temporary HIV-1 viremia suppression in the majority of patients19,20. Unfortunately, the HIV virus rapidly develops resistance mutations under pressure from a single bNAb, suggesting that passive treatment with a single bNAb is unlikely to result in long-term viremia suppression19,2123. Some of the Env mutations associated with bNAb resistance can significantly reduce viral fitness. Therefore, simultaneously targeting different Env epitopes SB 525334 may completely compromise viral replication, as mutations that confer resistance to each bNAb often accumulate to severely reduce viral fitness2427. In addition, treatment of simian/human immunodeficiency virus infection in non-human primate models demonstrated that passive immunotherapy with bNAb cocktails prevent mother to child transmission, suppress viremia and, in contrast to combinatorial antiviral therapy (cART) treatments, facilitate CD8+ T-cell immunity for durable suppression of virus replication28,29. Preliminary data on bNAb cocktails suggest significant advantages over either cART or single bNAb treatments for the management of HIV-1 infection. While antibody cocktails demonstrated improved efficacy in preclinical studies, multispecific single agents are desirable for manufacturing purposes30as well as SB 525334 for improved avidity that may result in enhanced neutralization breadth and potency31. Bi-NAbs with two Env-epitope binding sites have been PRSS10 generated using CrossMab formats32,33with up to 97% virus coverage34,35. Their neutralization breadth could be further extended, however, and truly bivalent binding has yet to be experimentally demonstrated. Most recently, one study showed that swapping the IgG1 hinge for a more flexibly IgG3 hinge lacking disulfide bonds (denoted as IgG3C-) greatly improved the potency of anti-HIV CrossMabs34. While both the CrossMab and IgG3C- designs have significantly improved the potency and breadth of antibodies against HIV, they only target two epitopes, one corresponding to each antigen-binding Fragment (Fab) arm. This.