PGT121 Antibody Engineering: Enhanced Infected Cell Killing and Drug-Like Properties

Introduction Methods

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1. Pancera et al. Nature 2014. 514(7523):455-461.2. Bruner et al. Trends in Microbiology. 2015. 23(4):192-203.3. Julien et al. PLoS Pathog. 2013. 9(5):e1003342.4 Sok et al. PLoS Pathog. 2013. 9(11):e1003754.5. Richman et al. PNAS. 2003. 100(7):4144-9.6. Baker and Jones. Curr Opin Drug Devel. 2007. 10(2):219-227. 7. von Horsten et al. Glycobiology 2010. 20(12):1607-1618.8. Barouch et al. Nature. 2013. 503(7475):224-228.

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Poster # 329

Results

Conclusions

Acknowledgements

References

M Balakrishnan; ND Thomsen; CS Pace; X Zhang; M Hung; MR Nagel; BA Carr; YE Hu, H Yu, JA Corbin

CROI, Seattle, Washington, Feb 13-16, 2017

Latency reversing agent

InfectedCD4+ T cell

NK cells (ADCC)Ab-dependent

cellular cytotoxicity

Macrophages (ADCP)Ab-dependent

cellular phagocytosis

Complement (CDC)Complement-dependent

cytotoxicity

HIV provirus

HIV Envelope(gp120/gp41)

Anti-HIV EnvbNAbs

+

FcγRIIIA

FcγRIIA

C1q

PGT121v ∆Fuc

A therapeutic agent that mediates the selective destruction of cells expressing latent HIV may sustainably suppress viremia in the absence of antiretroviral therapy (ART) and offers the potential for a sterilizing cure. Anti-HIV-1 envelope (Env) antibodies from elite neutralizers can neutralize a high percentage of viral strains across clades. Some of these broadly neutralizing antibodies (bNAbs) can recognize Env expressed on the surface of infected cells and mediate killing via effector function.

A set of patient-derived bNAbs targeting different Env epitopes were tested for natural killer (NK) cell mediated killing of HIV-infected CD4+ T cells to identify a bNAb for further optimization. Fragment antigen-binding (Fab) variants were generated via genetic engineering, while enhancement of effector function was achieved through glycoengineering of the fragment crystallizable (Fc).

1 - Screen bNAbs in primary HIV infected ADCC assay

2 - In-silico and biophysical characterization of PGT121

3 - Design and screen ~30 PGT121 Fab point mutants

4 - Design and screen ~30 combinatorial variants

5 - Fc engineering

3 Fab Glycosylation SitesMultiple T-cell Epitopes

No Fab glycosylation sitesFewer T-cell EpitopesEnhanced low pH stability

Aggregation at low pH

No Fab glycosylation sitesFewer T-cell EpitopesEnhanced low pH stabilityEnhanced ADCC

Figure 3. PGT121 showed the highest ADCC activity among a panel of 28 mAbs targeting diverse epitopes. A panel of anti-Env antibodies was screened for ADCC activity using primary resting CD4+ T cells infected with four different HIV isolates. The mAbs exhibited significant differences in killing efficacy (Emax) and potency (not shown). All antibodies have an identical IgG1 framework.

Figure 4. PGT121 contains numerous sequence liabilities. (a) Primary sequence analysis of PGT121 reveals multiple liabilities including poor match to the human germline (GL) precursor4, three consensus glycosylation sites in the heavy chain and multiple T-cell epitopes identified via a peptide mapping Epi-Screen (50 donors). (b) The liabilities in (a) mapped onto a structural model of the PGT121-gp120 complex based on PDB IDs 4TVP and 4JY41,4.

a

b

Figure 6. Top combinatorial variants have median potencies and breadth comparable to PGT121. PGT121 combinatorial variants were profiled for potency and breadth against a panel of 142 patient envelopes in the Monogram PhenoSense neutralization assay5 as a high throughput and robust approach for detecting compromises in Env recognition. Most variants retained the Env recognition profile of PGT121. PGT121 IC50 = 0.0163 μg/ml, 66% breadth; IC50 of top variants ~0.01 μg/ml, 66-67% breadth.

Figure 5. Several single point mutants retain activity comparable to PGT121. Single point mutants designed in the first phase of Fab engineering were screened for HIV neutralization activity against a set of sensitive isolates. Point mutants showing potencies within 2-fold of PGT121 (dashed lines) were next incorporated into a focused set of combinatorial variants.

Figure 8. Afucosylation of PGT121v enhanced FcγR binding and ADCC activity. (a) Fc glycoengineering7 increased apparent binding affinity to both alleles of FcγRIIIA ~10-fold in an FcγR ELISA. (b) Fc glycoengineered variant exhibited enhanced ADCC activity. Representative ADCC plots shown for cells infected with two different HIV patient isolates.

Figure 7. Engineered PGT121 variants (PGT121v) have reduced risk of immunogenicity. Select PGT121 combinatorial variants were tested in a primary T-cell activation assay (50 donors) alongside PGT121 and control mAbs shown. Removal of T-cell epitopes reduced ex vivo T-cell activation rate from 32% (PGT121) to