SIV DNA can be detected in lymphoid tissue–resident macrophages of chronically SIV-infected Asian macaques. These macrophages also contain evidence of recently phagocytosed SIV-infected CD4+ T cells. Here, we examine whether these macrophages contain replication-competent virus, whether viral DNA can be detected in tissue-resident macrophages from antiretroviral (ARV) therapy–treated animals and humans, and how the viral sequences amplified from macrophages and contemporaneous CD4+ T cells compare. In ARV-naive animals, we find that lymphoid tissue–resident macrophages contain replication-competent virus if they also contain viral DNA in ARV-naive Asian macaques. The genetic sequence of the virus within these macrophages is similar to those within CD4+ T cells from the same anatomic sites. In ARV-treated animals, we find that viral DNA can be amplified from lymphoid tissue–resident macrophages of SIV-infected Asian macaques that were treated with ARVs for at least 5 months, but we could not detect replication-competent virus from macrophages of animals treated with ARVs. Finally, we could not detect viral DNA in alveolar macrophages from HIV-infected individuals who received ARVs for 3 years and had undetectable viral loads. These data demonstrate that macrophages can contain replication-competent virus, but may not represent a significant reservoir for HIV in vivo.
Dysbiosis of the fecal microbiome is a common feature observed in ARV-treated people living with HIV. The degree to which HIV infection itself causes this dysbiosis remains unclear. Here, we demonstrate that medications used to treat HIV infection can influence the composition of the GI tract immune responses and its microbiome in the nonhuman primate SIV model.
Abstract Mutation-associated neoantigens (MANAs) are exquisitely cancer-specific therapeutic targets. However, MANAs are present at ultra-low densities on the cancer cell surface (as few as 1-2 copies per cell), leading to the challenge of eliciting a sufficiently robust therapeutic effect. We combined components of both T cell receptors (TCRs) and chimeric antigen receptors (CARs) to create a new receptor with improved potency against an ultra-low-density MANA. From CARs, we utilized the antibody-based antigen recognition domain (i.e. the single chain variable fragment, scFv) and the integrated co-stimulation that amplifies T cell activation. From TCRs, we utilized the multi-chain signaling platform that facilitates high antigen sensitivity. This new receptor, termed a TCR Embedded ScFv for Long-term Activation (TESLA), showed promising characteristics when tested with the H2-scFv which targets the p53 R175H mutation presented on HLA-A*02:01 (R175H/A2). Using CRISPR-based homology directed repair in primary human T cells, we tested 15 configurations of appending the H2-scFv to subunits of the TCR complex to identify a design that maximized T cell cytotoxicity and interferon gamma release in co-cultures with cancer cells expressing endogenous levels of the R175H/A2 antigen. In this system, we showed that the optimal TCR-embedded configuration of the H2-scFv produced similar levels of cytotoxicity and interferon gamma secretion as patient-derived TCRs targeting the same R175H/A2 MANA, while conventional H2-CARs were unable to produce any T-cell activation. We then used a multiple stimulation co-culture system to identify a co-stimulation domain combination (MyD88 and CD40) that improved serial cytotoxicity and proliferation of H2-TESLAs when incorporated on the intracellular side of the TCRbeta chain. Finally, we compared the H2-TESLA receptor to patient-derived TCRs modified with the same MyD88 and CD40 co-stimulation domains. In vivo, H2-TESLAs cured all mice in a tumor model, while co-stimulation-modified TCRs produced only temporary tumor control. Moreover, in vivo, H2-TESLAs elicited 100-fold greater T cell expansion than co-stimulation-modified TCRs. In conclusion, we demonstrated that by combining aspects of both CARs and TCRs, the TESLA receptor improved T cell reactivity against an ultra-low-density neoantigen compared to conventional CARs and patient-derived TCRs. Citation Format: Brian J. Mog, Sarah R. DiNapoli, Michael S. Hwang, Tushar D. Nichakawade, Jacqueline Douglass, Emily Han-Chung Hsiue, Katharine M. Wright, Alexander H. Pearlman, Maximilian F. Konig, Suman Paul, Nicolas Wyhs, Nikita Marcou, Stephanie Glavaris, Jiaxin Ge, Michelle S. Miller, P. Aitana Azurmendi, Evangeline Watson, Drew M. Pardoll, Sandra B. Gabelli, Chetan Bettegowda, Nickolas Papadopoulos, Kenneth W. Kinzler, Bert Vogelstein, Shibin Zhou. Hybrid TCR-CAR design surpasses conventional CARs and patient-derived TCRs in targeting an ultra-low-density neoantigen [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB095.
The emergence of immunotherapy as an important tool in the fight against cancer takes advantage of the exquisite specificity of antibodies.Targets, however, have been limited to those on the cell surface, despite most oncogenic driver mutations occurring in genes which encode intracellular proteins; known as "undruggable" targets.To overcome this limitation, antibodies can be selectively engineered to target mutation-derived neoantigens, peptides derived from mutant proteins that are presented on the cell surface by the Major Histocompatibility Complex Class I (MHC-I), and not their wild-type peptide-MHC counterparts.One such "undruggable" target is tumor-suppressor protein TP53, one of the most commonly mutated driver genes in all cancers.Here, we describe the identification and structural basis of a T cell receptor (TCR)-mimic antibody against the HLA-A*02:01-restricted TP53 R175H epitope, highlighting its specificity.We have developed a TCR-mimic antibody through phage display, selecting the antibody that showed specificity and selectivity toward the mutant peptide TP53 R175H over the wild-type peptide.The TP53 R175H specific antibody was expressed in mammalian cells to generate an optimal protein fold and the antibody Fab fragments were generated using enzymatic cleavage techniques.We performed both structural and biophysical characterization.Structural studies of the Fab/TP53 R175H peptide-MHC complex were carried out using X-ray crystallography to fully understand the interaction between the antibody and TP53 peptide.Furthermore, biophysical characterization included carrying out binding kinetics experiments using Surface Plasmon Resonance (SPR).Determination of the structure revealed the TCR-mimic antibody forms a cage-like configuration around the C-terminal of the TP53 R175H neoantigen, trapping residue H175 into an exposed position by providing a stable interaction.Specifically, all CDRs of the variable heavy chain interact with the displayed TP53 R175H neoantigen.In contrast, only the third CDR of the variable light chain contributes to peptide interaction.Interestingly, the TCR-mimic antibody interaction employs a non-canonical parallel binding mode, different from the typical diagonal orientation utilized by most TCRs and other TCR-mimics.It is possible this new mode of binding contributes to the observed antibody specificity and measured affinity (KD = 86 nM).Exploitation of our detailed structural understanding of the mechanisms of specificity is essential for the development of more potent and selective antibodies.The exquisite selectivity and specificity achievable with antibodies provides the added benefit of distinguishing between wild-type and mutant proteins-the foundation for developing effective treatments with minimal adverse effects to patients.Our study provides a new immunotherapeutic approach to treat cancers with "undruggable" driver alterations.
Immunotherapies such as chimeric antigen receptor (CAR) T cells and bispecific antibodies redirect healthy T cells to kill cancer cells expressing the target antigen. The pan-B cell antigen-targeting immunotherapies have been remarkably successful in treating B cell malignancies. Such therapies also result in the near-complete loss of healthy B cells, but this depletion is well tolerated by patients. Although analogous targeting of pan-T cell markers could, in theory, help control T cell cancers, the concomitant healthy T cell depletion would result in severe and unacceptable immunosuppression. Thus, therapies directed against T cell cancers require more selective targeting. Here, we describe an approach to target T cell cancers through T cell receptor (TCR) antigens. Each T cell, normal or malignant, expresses a unique TCR β chain generated from 1 of 30 TCR β chain variable gene families (TRBV1 to TRBV30). We hypothesized that bispecific antibodies targeting a single TRBV family member expressed in malignant T cells could promote killing of these cancer cells, while preserving healthy T cells that express any of the other 29 possible TRBV family members. We addressed this hypothesis by demonstrating that bispecific antibodies targeting TRBV5-5 (α-V5) or TRBV12 (α-V12) specifically lyse relevant malignant T cell lines and patient-derived T cell leukemias in vitro. Treatment with these antibodies also resulted in major tumor regressions in mouse models of human T cell cancers. This approach provides an off-the-shelf, T cell cancer selective targeting approach that preserves enough healthy T cells to maintain cellular immunity.
Targeting mutation-associated neoantigens (MANAs) is a highly-cancer specific strategy to selectively eliminate cells harboring common driver mutations in genes encoding intracellular proteins such as Ras and p53.1, 2 T cell-engaging bispecific antibodies targeting MANAs redirect T cells to kill cancer cells presenting mutant peptides on human leukocyte antigens (pHLA). However, T cell-redirecting therapies' efficacy can be limited by the low antigen density of these MANAs on the cell surface. Here, we investigate whether increasing the affinity of a T cell-engaging bispecific antibody (clone H2) targeting the p53 R175H MANA (HMTEVVRHC) presented on HLA-A*02:01 (R175H/A2) improves its efficacy in vitro and in vivo.
Methods
To identify higher affinity variants, we screened a phage display library consisting of 1159 single-amino acid variants at 61 sites in the six complementarity determining regions of the H2 single chain variable fragment targeting R175H/A2. Variants retaining R175H/A2 specificity were selected over multiple rounds of panning followed by affinity enrichment via thiocyanate elution. Selected variants were compared to the original H2 bispecific antibody in co-cultures with primary human T cells and cancer cell lines expressing endogenous levels of HLA-A*02:01 and the mutant p53-R175H protein or isogenic control cell lines. For in vivo comparison, NSG mice inoculated with KMS26 or Nalm6 cells and human T cells were treated with a continuous infusion of bispecific antibody for 14 days.
Results
Three variant bispecific antibodies were identified with higher affinity and retained specificity for R175H/A2 (KD of 12.9 nM, 6.8 nM, 3.3 nM vs. the original KD of 29.5 nM). The highest affinity variant was a double mutant incorporating two top single variants. Each higher affinity variant elicited greater T cell activation as measured by interferon gamma release and cytotoxicity in co-cultures with cell lines expressing endogenous levels of the R175H/A2 pHLA. In vivo testing demonstrated that the higher affinity bispecific antibodies had improved tumor control in xenograft models compared to the lower affinity bispecific, particularly at a lower treatment dose (0.075 mg/kg/d).
Conclusions
Increasing affinity for the p53 R175H/A2 pHLA to the low nanomolar range yields increased T-cell activation and cancer cell killing without sacrificing specificity for the R175H mutation.
References
Hsiue EH, Wright KM, Douglass J, Hwang MS, Mog BJ, Pearlman AH, Paul S, DiNapoli SR, Konig MF, Wang Q, Schaefer A, Miller MS, Skora AD, Azurmendi PA, Murphy MB, Liu Q, Watson E, Li Y, Pardoll DM, Bettegowda C, Papadopoulos N, Kinzler KW, Vogelstein B, Gabelli SB, Zhou S. Targeting a neoantigen derived from a common TP53 mutation. Science 2021;371:eabc8697. Douglass J, Hsiue EH, Mog BJ, Hwang MS, DiNapoli SR, Pearlman AH, Miller MS, Wright KM, Azurmendi PA, Wang Q, Paul S, Schaefer A, Skora AD, Molin MD, Konig MF, Liu Q, Watson E, Li Y, Murphy MB, Pardoll DM, Bettegowda C, Papadopoulos N, Gabelli SB, Kinzler KW, Vogelstein B & Zhou S. Bispecific antibodies targeting mutant RAS neoantigens. Sci Immunol 2021;6:eabd5515
Ethics Approval
Animal studies were approved by the Johns Hopkins University Animal Care and Use Committee, #MO18M79.
Abstract Specificity remains a major challenge to current therapeutic strategies for cancer. Mutation associated neoantigens (MANAs) are products of genetic alterations, making them highly specific therapeutic targets. MANAs are HLA-presented (pHLA) peptides derived from intracellular mutant proteins that are otherwise inaccessible to antibody-based therapeutics. Here, we describe the cryo-EM structure of an antibody-MANA pHLA complex. Specifically, we determine a TCR mimic (TCRm) antibody bound to its MANA target, the KRAS G12V peptide presented by HLA-A*03:01. Hydrophobic residues appear to account for the specificity of the mutant G12V residue. We also determine the structure of the wild-type G12 peptide bound to HLA-A*03:01, using X-ray crystallography. Based on these structures, we perform screens to validate the key residues required for peptide specificity. These experiments led us to a model for discrimination between the mutant and the wild-type peptides presented on HLA-A*03:01 based exclusively on hydrophobic interactions.
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