TRBC1-targeting antibody-drug conjugates for the treatment of T cell cancers.

Antibody and chimeric antigen receptor (CAR) T cell-mediated targeted therapies have improved survival in patients with solid and haematologic malignancies1-9. Adults with T cell leukaemias and lymphomas, collectively called T cell cancers, have short survival10,11 and lack such targeted therapies. Thus, T cell cancers particularly warrant the development of CAR T cells and antibodies to improve patient outcomes. Preclinical studies showed that targeting T cell receptor ß-chain constant region 1 (TRBC1) can kill cancerous T cells while preserving sufficient healthy T cells to maintain immunity12, making TRBC1 an attractive target to treat T cell cancers. However, the first-in-human clinical trial of anti-TRBC1 CAR T cells reported a low response rate and unexplained loss of anti-TRBC1 CAR T cells13,14. Here we demonstrate that CAR T cells are lost due to killing by the patient's normal T cells, reducing their efficacy. To circumvent this issue, we developed an antibody-drug conjugate that could kill TRBC1+ cancer cells in vitro and cure human T cell cancers in mouse models. The anti-TRBC1 antibody-drug conjugate may provide an optimal format for TRBC1 targeting and produce superior responses in patients with T cell cancers.

Hydrophobic interactions dominate the recognition of a KRAS G12V neoantigen.

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 KRASG12V 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.

Targeting public neoantigens for cancer immunotherapy.

Several current immunotherapy approaches target private neoantigens derived from mutations that are unique to individual patients' tumors. However, immunotherapeutic agents can also be developed against public neoantigens derived from recurrent mutations in cancer driver genes. The latter approaches target proteins that are indispensable for tumor growth, and each therapeutic agent can be applied to numerous patients. Here we review the opportunities and challenges involved in the identification of suitable public neoantigen targets and the development of therapeutic agents targeting them.

Structural engineering of chimeric antigen receptors targeting HLA-restricted neoantigens.

Chimeric antigen receptor (CAR) T cells have emerged as a promising class of therapeutic agents, generating remarkable responses in the clinic for a subset of human cancers. One major challenge precluding the wider implementation of CAR therapy is the paucity of tumor-specific antigens. Here, we describe the development of a CAR targeting the tumor-specific isocitrate dehydrogenase 2 (IDH2) with R140Q mutation presented on the cell surface in complex with a common human leukocyte antigen allele, HLA-B*07:02. Engineering of the hinge domain of the CAR, as well as crystal structure-guided optimization of the IDH2R140Q-HLA-B*07:02-targeting moiety, enhances the sensitivity and specificity of CARs to enable targeting of this HLA-restricted neoantigen. This approach thus holds promise for the development and optimization of immunotherapies specific to other cancer driver mutations that are difficult to target by conventional means.

Targeting loss of heterozygosity for cancer-specific immunotherapy.

Developing therapeutic agents with potent antitumor activity that spare normal tissues remains a significant challenge. Clonal loss of heterozygosity (LOH) is a widespread and irreversible genetic alteration that is exquisitely specific to cancer cells. We hypothesized that LOH events can be therapeutically targeted by "inverting" the loss of an allele in cancer cells into an activating signal. Here we describe a proof-of-concept approach utilizing engineered T cells approximating NOT-gate Boolean logic to target counterexpressed antigens resulting from LOH events in cancer. The NOT gate comprises a chimeric antigen receptor (CAR) targeting the allele of human leukocyte antigen (HLA) that is retained in the cancer cells and an inhibitory CAR (iCAR) targeting the HLA allele that is lost in the cancer cells. We demonstrate that engineered T cells incorporating such NOT-gate logic can be activated in a genetically predictable manner in vitro and in mice to kill relevant cancer cells. This therapeutic approach, termed NASCAR (Neoplasm-targeting Allele-Sensing CAR), could, in theory, be extended to LOH of other polymorphic genes that result in altered cell surface antigens in cancers.

Bispecific antibodies targeting mutant RAS neoantigens.

Mutations in the RAS oncogenes occur in multiple cancers, and ways to target these mutations has been the subject of intense research for decades. Most of these efforts are focused on conventional small-molecule drugs rather than antibody-based therapies because the RAS proteins are intracellular. Peptides derived from recurrent RAS mutations, G12V and Q61H/L/R, are presented on cancer cells in the context of two common human leukocyte antigen (HLA) alleles, HLA-A3 and HLA-A1, respectively. Using phage display, we isolated single-chain variable fragments (scFvs) specific for each of these mutant peptide-HLA complexes. The scFvs did not recognize the peptides derived from the wild-type form of RAS proteins or other related peptides. We then sought to develop an immunotherapeutic agent that was capable of killing cells presenting very low levels of these RAS-derived peptide-HLA complexes. Among many variations of bispecific antibodies tested, one particular format, the single-chain diabody (scDb), exhibited superior reactivity to cells expressing low levels of neoantigens. We converted the scFvs to this scDb format and demonstrated that they were capable of inducing T cell activation and killing of target cancer cells expressing endogenous levels of the mutant RAS proteins and cognate HLA alleles. CRISPR-mediated alterations of the HLA and RAS genes provided strong genetic evidence for the specificity of the scDbs. Thus, this approach could be applied to other common oncogenic mutations that are difficult to target by conventional means, allowing for more specific anticancer therapeutics.

Targeting a neoantigen derived from a common TP53 mutation.

TP53 (tumor protein p53) is the most commonly mutated cancer driver gene, but drugs that target mutant tumor suppressor genes, such as TP53, are not yet available. Here, we describe the identification of an antibody highly specific to the most common TP53 mutation (R175H, in which arginine at position 175 is replaced with histidine) in complex with a common human leukocyte antigen-A (HLA-A) allele on the cell surface. We describe the structural basis of this specificity and its conversion into an immunotherapeutic agent: a bispecific single-chain diabody. Despite the extremely low p53 peptide-HLA complex density on the cancer cell surface, the bispecific antibody effectively activated T cells to lyse cancer cells that presented the neoantigen in vitro and in mice. This approach could in theory be used to target cancers containing mutations that are difficult to target in conventional ways.

TCR ß chain-directed bispecific antibodies for the treatment of T cell cancers.

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 ß chain generated from 1 of 30 TCR ß 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.

Antiretroviral Therapy Administration in Healthy Rhesus Macaques Is Associated with Transient Shifts in Intestinal Bacterial Diversity and Modest Immunological Perturbations.

Gastrointestinal (GI) immune system competency is dependent upon interactions with commensal microbiota, which can be influenced by wide-ranging pharmacologic interventions. In simian immunodeficiency virus (SIV)-infected Asian macaque models of human immunodeficiency virus (HIV) infection, we previously noted that initiation of antiretroviral therapy (ART) is associated with a specific imbalance (dysbiosis) of the composition of the intestinal bacteriome. To determine if ART itself might contribute to dysbiosis or immune dysfunction, we treated healthy rhesus macaques with protease, integrase, or reverse transcriptase inhibitors for 1 to 2 or for 5 to 6 weeks and evaluated intestinal immune function and the composition of the fecal bacterial microbiome. We observed that individual antiretrovirals (ARVs) modestly altered intestinal T-cell proinflammatory responses without disturbing total or activated T-cell frequencies. Moreover, we observed transient disruptions in bacterial diversity coupled with perturbations in the relative frequencies of bacterial communities. Shifts in specific bacterial frequencies were not persistent posttreatment, however, with individual taxa showing only isolated associations with T-cell proinflammatory responses. Our findings suggest that intestinal bacterial instability and modest immunological alterations can result from ART itself. These data could lead to therapeutic interventions which stabilize the microbiome in individuals prescribed ART.IMPORTANCE 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.

Hallmarks of primate lentiviral immunodeficiency infection recapitulate loss of innate lymphoid cells.

Innate lymphoid cells (ILCs) play critical roles in mucosal barrier defense and tissue homeostasis. While ILCs are depleted in HIV-1 infection, this phenomenon is not a generalized feature of all viral infections. Here we show in untreated SIV-infected rhesus macaques (RMs) that ILC3s are lost rapidly in mesenteric lymph nodes (MLNs), yet preserved in SIV+ RMs with pharmacologic or natural control of viremia. In healthy uninfected RMs, experimental depletion of CD4+ T cells in combination with dextran sodium sulfate (DSS) is sufficient to reduce ILC frequencies in the MLN. In this setting and in chronic SIV+ RMs, IL-7Rα chain expression diminishes on ILC3s in contrast to the IL-18Rα chain expression which remains stable. In HIV-uninfected patients with durable CD4+ T cell deficiency (deemed idiopathic CD4+ lymphopenia), similar ILC deficiencies in blood were observed, collectively identifying determinants of ILC homeostasis in primates and potential mechanisms underlying their depletion in HIV/SIV infection.

Experimental microbial dysbiosis does not promote disease progression in SIV-infected macaques.

Intestinal microbial dysbiosis has been described in individuals with an HIV-1 infection and may underlie persistent inflammation in chronic infection, thereby contributing to disease progression. Herein, we induced an HIV-1-like intestinal dysbiosis in rhesus macaques (Macaca mulatta) with vancomycin treatment and assessed the contribution of dysbiosis to SIV disease progression. Dysbiotic and control animals had similar disease progression, indicating that intestinal microbial dysbiosis similar to that observed in individuals with HIV is not sufficient to accelerate untreated lentiviral disease progression.

Tissue-resident macrophages can contain replication-competent virus in antiretroviral-naive, SIV-infected Asian macaques.

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.

Microbial Translocation and Inflammation Occur in Hyperacute Immunodeficiency Virus Infection and Compromise Host Control of Virus Replication.

Within the first three weeks of human immunodeficiency virus (HIV) infection, virus replication peaks in peripheral blood. Despite the critical, causal role of virus replication in determining transmissibility and kinetics of progression to acquired immune deficiency syndrome (AIDS), there is limited understanding of the conditions required to transform the small localized transmitted founder virus population into a large and heterogeneous systemic infection. Here we show that during the hyperacute "pre-peak" phase of simian immunodeficiency virus (SIV) infection in macaques, high levels of microbial DNA transiently translocate into peripheral blood. This, heretofore unappreciated, hyperacute-phase microbial translocation was accompanied by sustained reduction of lipopolysaccharide (LPS)-specific antibody titer, intestinal permeability, increased abundance of CD4+CCR5+ T cell targets of virus replication, and T cell activation. To test whether increasing gastrointestinal permeability to cause microbial translocation would amplify viremia, we treated two SIV-infected macaque 'elite controllers' with a short-course of dextran sulfate sodium (DSS)-stimulating a transient increase in microbial translocation and a prolonged recrudescent viremia. Altogether, our data implicates translocating microbes as amplifiers of immunodeficiency virus replication that effectively undermine the host's capacity to contain infection.

Macrophages in Progressive Human Immunodeficiency Virus/Simian Immunodeficiency Virus Infections.

The cells that are targeted by primate lentiviruses (HIV and simian immunodeficiency virus [SIV]) are of intense interest given the renewed effort to identify potential cures for HIV. These viruses have been reported to infect multiple cell lineages of hematopoietic origin, including all phenotypic and functional CD4 T cell subsets. The two most commonly reported cell types that become infected in vivo are memory CD4 T cells and tissue-resident macrophages. Though viral infection of CD4 T cells is routinely detected in both HIV-infected humans and SIV-infected Asian macaques, significant viral infection of macrophages is only routinely observed in animal models wherein CD4 T cells are almost entirely depleted. Here we review the roles of macrophages in lentiviral disease progression, the evidence that macrophages support viral replication in vivo, the animal models where macrophage-mediated replication of SIV is thought to occur, how the virus can interact with macrophages in vivo, pathologies thought to be attributed to viral replication within macrophages, how viral replication in macrophages might contribute to the asymptomatic phase of HIV/SIV infection, and whether macrophages represent a long-lived reservoir for the virus.

Interleukin-2 Therapy Induces CD4 Downregulation, Which Decreases Circulating CD4 T Cell Counts, in African Green Monkeys.

UNLABELLED: African green monkeys (AGMs) are natural hosts of simian immunodeficiency virus (SIVAGM). Because these animals do not develop simian AIDS despite maintaining high viral loads, there is considerable interest in determining how these animals have evolved to avoid SIV disease progression. Unlike nonnatural hosts of SIV, adult AGMs maintain low levels of CD4(+) T cells at steady states and also have a large population of virus-resistant CD8αα T cells that lack CD4 expression despite maintaining T helper cell functionalities. In recent work, we have shown that homeostatic cytokines can induce CD4 downregulation in AGM T cells in vitro Through administering therapeutic doses of recombinant human interleukin-2 (IL-2) to AGMs, we show here that this mechanism is operative in vivo IL-2 therapy induced transient yet robust proliferation in all major T cell subsets. Within the CD4(+) T cell population, those that were induced into cycle by IL-2 exhibited characteristics of CD4-to-CD8αα conversion. In all animals receiving IL-2, circulating CD4(+) T cell counts and proportions tended to be lower and CD4(-) CD8αα(+) T cell counts tended to be higher. Despite reductions in circulating target cells, the viral load was unaffected over the course of study. IMPORTANCE: The data in this study identify that homeostatic cytokines can downregulate CD4 in vivo and, when given therapeutically, can induce AGMs to sustain very low levels of circulating CD4(+) T cells without showing signs of immunodeficiency.

Macrophages Are Phenotypically and Functionally Diverse across Tissues in Simian Immunodeficiency Virus-Infected and Uninfected Asian Macaques.

UNLABELLED: Macrophages regulate tissue immunity, orchestrating the initiation and resolution of antimicrobial immune responses and repair of damaged tissue architecture. Their dysfunction can, thus, manifest in either pro- and anti-inflammatory responses. Indeed, despite the importance of macrophage function in health and disease, the role of tissue-resident macrophages in human immunodeficiency virus (HIV) disease progression remains incompletely defined. Here, we use flow cytometry to assess the phenotypes and functions of macrophages isolated from the spleens, axillary lymph nodes, colons, jejuna, and livers of healthy and chronically simian immunodeficiency virus (SIV)-infected Asian macaques, the prominent nonhuman primate model for HIV infection. Our data demonstrate that macrophages from healthy animals exhibit considerable phenotypic and functional heterogeneity across tissues and across a variety of stimuli. Further, our analysis reveals changes in the lipopolysaccharide (LPS) responsiveness of macrophages isolated from SIV-infected animals. We anticipate that our findings will inform future research into macrophage-directed immunity across a variety of primate diseases. IMPORTANCE: These findings highlight the functional and phenotypic heterogeneity of tissue macrophages in different anatomic sites and as a result of SIV infection. We believe that our data will lead to novel therapeutic interventions aimed at altering the proinflammatory capacity of tissue macrophages in progressively HIV-infected individuals.