Alternate Antibody-Based Therapeutic Strategies To Purge the HIV Cell Reservoir Giuseppe Pantaleo, M.D. Professor of Medicine Head, Division of Immunology and Allergy Executive Director, Swiss Vaccine Research Institute Lausanne University Hospital, Lausanne, Switzerland
Tfh Cells and CXCR5 - PD-1 + CD4 T-Cells Are Enriched in CD4 T-Cells Containing HIV DNA DNA extraction Taqman RT-PCR HIV-1 gag gene
Tfh Cells and CXCR5 - PD-1 + CD4 T-Cells Are the Most Efficient in Supporting Production of HIV Anti- CD3/CD28 Collection of SNs p24 detection D0 D2 D5 High viremia (>15000 HIV RNA copies/ml)
HIV Isolation from Patients with Low (<1000 HIV RNA copies/ml of plasma) in Different CD4 T-Cell Populations CD8-depleted PBMCs + Coated CD3/28 Collection of SNs p24 detection D0 D3 D7 Low viremia (<1000 HIV RNA copies/ml) Tfh and CXCR5 - PD-1 + but not CXCR5 - PD-1 - and CXCR5 + PD-1 - CD4 T-cells efficiently support virus isolation and production in patients with low viremia levels
The Percentage of Tfh Cells Correlates with HIV Viremia Levels
Conclusions Tfh Cells Serve as the Major CD4 T-Cell Compartment for HIV Infection, Replication and Production (Perreau et al. JEM 2013) Tfh Cells and CXCR5 - PD-1 + Cells May Represent the Primary Obstacle for Achieving Functional HIV Cure/Eradication HIV infected Tfh cells reside in a privileged anatomic site, i.e. germinal centers, with limited accessed to HIVspecific cytotoxic CD8 T-cells
Regulation of T-Cell Entry Within GCs Loss of CCR7 expression Expression of CXCR5 Production of CXCL13 (ligand for CXCR5) by FDCs Therefore, access of cells to GCs is highly selective CD8 T-cells have very limited accessed to GCs, e.g. small proportion exppress CXCR5
Low CXCR5 expression on HIV-specific CD8 T-cells Untreated HIV-infected donor #CNA 2066 (HIV RNA 32250 copies/ml) Gated in memory CD8 T cells A*03 Gag RLRPGGKKK Total memory CD8 PD-1 Tetramer PD-1 50 CXCR5 * 110 Viability dye **** 100 CXCR5 *** p< 0.05 * p< 0.0005 *** p< 0.0001 **** Percentage of CXCR5 + memory CD8 T cells 40 30 20 10 Percentage of PD-1 + memory CD8 T cells 100 90 80 Percentage of memory CD8 T cells 50 **** *** Total memory CD8 T cells HIV-specific CD8 T cells 0 Total HIV-specific 70 Total HIV-specific 0 PD-1 CXCR-5 - + - + - - + +
Conventional Approaches to Purge the HIV Latent Reservoir Therapeutic approaches that have been tested and currently being developed: Therapeutic vaccines have shown limited/no efficacy. Potential explanation for the lack of efficacy include: a) limited immunogenicity in potentiating CD8 T-cell responses, b) latent infected cells are invisible to the immune response and c) the few cells with active virus replication reside in lymphoid tissues and may not be reached by vaccine-induced HIV-specific CD8 T-cells Reactivation of the HIV latent reservoir with HDAC inhibitors which may lead to: a) killing of infected CD4 T-cells after virus reactivation by HIV-specific CD8 T-cells and b) killing of infected CD4 T-cells as a result of direct virus cythopaticity Potent broad neutralizing Abs through passive immunization or gene therapy
A Shift in Paradigm for Clearing the HIV Cell Reservoir Rather than targeting the HIV latent cell reservoir in longterm ART virus suppressed patients, Develop Strategies to Target the HIV CD4 T Cell Reservoir Serving As the Majoir Compartment for Infection, Replication and Production, e.g. PD1+ (PD1+CXCR5+ and PD1+CXCR5-) Cell Populations, in Viremic Patients
Antigen Selective Targeting of Relevant CD4 T Cell Populations Antigens to selectively target Tfh (CD4+PD-1+CXCR5+) and CD4+PD-1+CXCR5- may include: 1. PD-1: targeting PD-1 would allow to eliminate both latent and productive HIV infected cells 2. HIV env: targeting HIV env would allow to eliminate selectively productively HIV infected cells
Proprietary Anti-PD-1 Abs Platform Luminex binding assay: PD-1 Fc fusion, PD-1 his, PD-1/PD-L1 complex Competitive binding studies with PD-L1 blocking and non-blocking commercial antibodies Specific binding to cell surface PD-1 (FACS): Binding affinity to activated PBMCs Absence of non-specific binding Competitive binding with commercial antibodies Sequencing of heavy chain CDR Phylogenic analysis and clustering into related families 240 hybridoma clones Epitope 1 Epitope 2 Epitope 3 Epitope mapping based on: mab competition Related CDR sequences Weak binding to cellular PD-1 and/or non-specific binding Focus on both affinity and diversity to identify less prevalent clones binding to unique epitopes 13
Profiling antibody supernatants for 240 hybridoma clones FACS analysis of mab binding to cell surface PD-1 on stimulated CD4 T cells Low binding to cell surface PD-1 Increase binding to cell surface PD-1 mab supernatant Anti-PD-1 control Negative control 219 out of 240 mabs supernatants bound to cell surface PD-1 At the concentrations tested, 55% of mab supernatants have equivalent or improved detection of cell surface PD-1 compared to the control anti-pd-1 antibody Specificity studies showed that 5% of mab supernatants bound non-specifically to unstimulated cells 14
Epitope mapping and relative affinities of antibody supernatants 40 35 30 <1.5 Fold MFI 1.5 to 2.0 fold MFI 2.0 to 2.5 fold MFI >2.5 fold MFI Number of mabs out of 240 profiled 25 20 15 10 5 0 Non-binding to No cell binding surface to PD1 cell surface PD-1 mab EH12 competitive mab1 competitive Class 1 mab J116 competitive mab2 competitive Class 2 mab EH12 & mab J116 1 & 2 competitive Class 3 Non-competitve with Non-competitive mab EH12 with & mab J116 1 & 2 Class 4 Luminex competitive binding assays distinguishes four different classes of mabs mab1 blocks PD-1 interaction with PD-L1 mab2 binds PD-1 but do not block the interaction with PD-L1 mab1 / mab2 competitive and non-competitive antibodies bind distinct sites on PD-1 High affinity antibodies are represented in each of the four binding classes 15
Functional Assay:HIV-Specific CD8 T Cell Recovery from Exhaustion PBMCs from an HIV infected individual were stained with CFSE and stimulated with an HIV peptide in the absence and presence of an anti-pd-1 antibody The control antibody MK3475 relieved PD-1 induced exhaustion of the CD8 T cells resulting in cellular proliferation Ten proprietary mab supernatants are clear PD-1 antagonists and stimulate proliferation Antagonists from all four binding classes were identified Two mab supernatants acting as PD-1 agonists and suppressed T cell proliferation compared to the peptide control Results will be confirmed with the purified antibody from these hybridomas CD8 Peptide alone 1.54% 2.97% CFSE CD8 Peptide + anti-pd-1 mab CFSE mabs supernatants with clear activity Peptide + MK3475 control Peptide control 16
Mapping the clonal diversity of Anti-PD-1 producing hybridomas The heavy chain CDRs were sequenced for hybridoma clones producing PD-1 binding mabs Phylogenic analysis of >90 clones allowed the division of antibodies into ~30 separate families Antibodies from distinct phylogenic branches and with different binding properties were evaluate in functional assays (e.g. impact on HIV specific CD8 T cells in a CFSE cellular proliferation assays) 20 antibody clones were selected for scaled up expression and purification Selection criteria included: Functional activity Binding to divergent sites on PD-1 Distinct CDR sequences Binding affinity Phylogenic Tree Distinct binding properties Class 1 Class 2 Class 3 Class 4 17 108 sequences from 91 clones
Panel of anti-env Monoclonal Antibodies Antibody Env binding site Neutralization of circulating HIV-1 strains VRC01 CD4bs 90% VRC03 CD4bs ~50% VRC07 CD4bs ~90% PGV04 CD4bs 85-90% b12 CD4bs ~33% PG9 Glycan and V1V2 70-80% PGT145 Glycan and V1V2 75-80% PGT121 V3 N-glycan 65-70% 3BC176 CD4i/V3 60-70% PGT128 Glycan V3 B-strand 70-75% HK20 gp41 Non-neutralizing 10E8 gp41 98% 4E10 gp41 96-98% HGW46 gp41 Non-neutralizing HGD161 gp41 Non-neutralizing HGN36 gp41 Non-neutralizing F240 gp41 Non-neutralizing gp41 Adapted from: Corti D. Annu Rev Immunol. 2013;31:705 gp120 18
anti-gp120 Ab Detection of Cell Surface HIV Env on Unstimulated and TNF-α Stimulated ACH-2 T-Cells VRC01 (CD4bs) VRC07 (CD4bs) PG9 (glycan &V1V2) anti-gp41 Ab HK20 HGW46 HGD161 HGN36 Abs to different Env epitopes bind specifically to the surface of virus producing T cells
Detection of Cell Surface HIV Env on U1-HIV Infected Promonocyte Cells anti-gp120 mab VRC01 (CD4bs) VRC07 (CD4bs) PG9 (glycan &V1V2) anti-gp41 mab HK20 HGW46 HGD161 HGN36 HK20, HGD161 and HGN36 Abs detect Env on U1-HIV infected promonocyte cells
Cell Surface Staining of Env in HIV Infected PBMCs: Gated on CD4+ T-Cells at day 2, 3, 4 and 7 Post-Infection Day 2 Day 3 Day 4 Day 7 29.9% 0.7% Env negative control p24 - FITC 24.2% 13.7% HGD161 (gp41) 28.8% 0.9% VRC07 (CD4bs) Anti-human IgG (AF647) Detection of p24+cd4+ cells with anti-gp41 HGD161 Ab
Cell Surface Staining of Env in HIV Infected PBMCs: Gated on CD3+CD4-CD8- T-Cells at day 2, 3, 4 and 7 Post-Infection Day 2 Day 3 Day 4 Day 7 29.7% 0.2% Env negative control p24 - FITC 17.8% 19.8% HGD161 (gp41) 11.2% 19.1% VRC07 (CD4bs) Anti-human IgG (AF647) Detection of p24+cd4+ cells with anti-gp41 HGD161 and gp120 VRC07 Abs
Analysis of Cell Surface Staining of Env in p24 Positive T Cells Day 2 Day 3 Day 4 Day 7 32.2% 0.2% 17.8% 0.6% 0.6% 0.8% Env negative control 23.9% 11.3% 9.8% 10.9% 1.1% 3.0% CD4 HGD161 (gp41) 19.3% 33.1% 48.8% 32.8% 1.4% 17.7% 1.3% 0.6% 2.3% VRC07 (CD4bs) 41.6% 62.2% 69.3% Anti-human IgG (AF647) Detection of p24+ infected cells by anti-gp120 Abs is improved in CD4-CD8- T cells
17.8% Env negative control Analysis of Cell Surface Staining of Env in p24 Positive T Cells PG9 0.5% 18.5% 0.8% anti-gp120 18.4% VRC01 0.8% VRC07 17.7% 1.3% CD4 CD4 3.0% 57.5% 42.6% 62.2% Anti- human IgG (AF647) Anti-human IgG (AF647) HK20 HGW46 anti-gp41 HGN36 HGD161 16.8% 1.6% 15.5% 4.7% 12.6% 7.6% 9.8% 10.9% CD4 5.9% 16.4% 21.3% 33.1% Anti-human IgG (AF647) Anti-gp120 mabs detect infected cells primarily after CD4 down-modulation Anti-gp41 mabs detect infected cells in both CD4+ and CD4 - cells
Analysis of Cell Surface Staining of Env in p24 Positive Infected T Cells Detection of p24+ cells at Day 4 post-infection Relative detection of CD4+ or CD4- infected cells % Env +ve 70 60 50 40 30 20 CD4+ve CD4-ve All T cells % Env +ve 80 CD4 pos 70 60 50 40 30 20 CD4 neg 10 10 0 0 anti-gp120 anti-gp41 anti-gp120 anti-gp41
Analysis of Cell Surface Staining of Env in p24 Positive T cells Relative detection of CD4 +ve or CD4 -ve infected cells 80.0 70.0 CD4 pos 60.0 % Env +ve 50.0 40.0 30.0 20.0 10.0 0.0 anti-gp120 PGT121 and PGT128 may potentially detect lower percentage of CD4+ and CD4- infected T cells as compared to PG9 and VRC07 anti-gp41 Tested in a separate infection study where p24 + cells were 47% CD4+ and 52% CD4- For the remaining antibodies, the p24 + cells were 30% CD4+ and 68% CD4-
Standard Approach Using Anti-PD-1 Abs in HIV Therapy Identify PD-1 antagonists that act to relieve immune exhaustion Strong evidence from using this approach from humanized mouse and non-human primate models of HIV infection In house functional assays will be used select optimal mab candidates with properties equivalent or improved properties compared to anti-pd-1 mabs in the clinic Anti-PD-1 antagonist PD-1 Exhausted HIV specific CD8 T cell PD-1 blockade Specific APC presented peptide Recovery from exhaustion (increased proliferation and killing) CTL killing of HIV infected cells (or tumor cells) TCR MHC I HIV specific CTL HIV infected cell Cytotoxic granules
Alternate Potential Applications of Anti-PD-1 Abs Targeted elimination of HIV infected PD-1 positive T-cells Either agonist or antagonist anti-pd-1 antibodies could be used PD-1 PD-1 HIV infected cells could be eliminated by: Antibody dependent cellular cytotoxicity (ADCC) Chemical link of Abs with a toxin - i.e. Antibody drug conjugate (ADC) Antibody Dependent Cellular Cytotoxicity Cytotoxic granules PD-1 Env Antibody drug conjugate PD-1 Env NK cell Fcγ receptor PD-1 Toxin PD-1 Env 28
Alternate Potential Applications of anti-pd-1 mabs Targeted elimination of HIV infected PD-1 positive T-cells Either agonist or antagonist anti-pd-1 antibodies could be used HIV Env PD-1 Increased selectivity for infected cells using a bi-specific mab targeting both PD-1 and HIV Env HIV infected cells could be eliminated by: Antibody dependent cellular cytotoxicity (ADCC) Chemical link of mabs with a toxin - i.e. Antibody drug conjugate (ADC) Antibody Dependent Cellular Cytotoxicity CrossMab CH1-CL Cytotoxic granules Env Antibody drug conjugate Env NK cell Fcγ receptor PD-1 Toxin PD-1 29
When To Target Tfh and CD4+PD-1+ Cells? Viremic patients naïve to ART Three scenarios 1. Ab targeting alone: monospecific PD-1 or bispecific PD-1/Env Abs 2. Sequential ART/Ab targeting: 2 weeks of ART to block efficiently virus spreading but still leaving alive a large population of CD4 T-cells actively replicating HIV 3. Ab targeting in combination with therapeutic vaccination and/or HDAC inhibitors
Therapeutic Vaccine i.m. Multivalent Biological Approach Potentiation HDACi Nef Nef Nef Nef Activation of HIV Replication PD-1 Nef HIV Nef Reactivation/Boosting HIV Replication CD4 i.v. Potentiation ADC Bi-specific Abs:PD-1/HIV env PD-1 monospecific Generation / Boosting of HIV-specific CD8 T-cells Killing of HIV infected CD4 T-cells
Acknowledgments Service of Immunology and Allergy Lausanne University Hospital Swiss Vaccine Research Institute Lausanne, Switzerland Craig Fenwick Celine Pellaton Gonzalo Tapia Alex FARINA Alexander Rockinger Line LEUENBERGER Elita Idrizi Biomedicine Research Institute Bellinzone, Switzerland Davide Corti Antonio Lanzavecchia Vaccine Research Center NIAID, NIH Bethesda, USA John Mascola Barney Graham Supported by: Swiss Vaccine Research Institute Lausanne University Hospital Secretariat of Education, Research and Innovation (SERI), Deapartment of Economy, Bern, Switzerland