Radiotherapy can elicit systemic immune control of local tumors and distant nonirradiated tumor lesions, known as the abscopal effect. Although this effect is enhanced using checkpoint blockade or costimulatory antibodies, objective responses remain suboptimal. As radiotherapy can induce secretion of VEGF and other stress products in the tumor microenvironment, we hypothesized that targeting immunomodulatory drugs to such products will not only reduce toxicity but also broaden the scope of tumor-targeted immunotherapy. Using an oligonucleotide aptamer platform, we show that radiation-induced VEGF-targeted 4-1BB costimulation potentiated both local tumor control and abscopal responses with equal or greater efficiency than 4-1BB, CTLA-4, or PD1 antibodies alone. Although 4-1BB and CTLA-4 antibodies elicited organ-wide inflammatory responses and tissue damage, VEGF-targeted 4-1BB costimulation produced no observable toxicity. These findings suggest that radiation-induced tumor-targeted immunotherapy can improve the therapeutic index and extend the reach of immunomodulatory agents. .

Our laboratory has developed a process for generating mAbs with selectivity to unique peptides in the context of MHC molecules. Recently, we reported that RL4B, an mAb that we have called a TCR mimic (TCRm) because it recognizes peptide in the context of MHC, has cytotoxic activity in vitro and prevented growth of tumor cells in a prophylactic setting. When presented in the context of HLA-A2, RL4B TCRm recognizes the peptide GVLPALPQV derived from human chorionic gonadotropin (hCG)-β. In this study, we show that RL4B TCRm has strong binding affinity for the GVLPALPQV peptide/HLA-A2 epitope and fine binding specificity for cells that express endogenous hCGβ Ag and HLA-A2. In addition, suppression of tumor growth with RL4B TCRm was observed in orthotopic models for breast cancer. Using two aggressive human tumor cell lines, MDA-MB-231 and MCF-7, we provide evidence that RL4B TCRm significantly retards tumor growth, supporting a possible role for TCRm agents in therapeutic settings. Moreover, tumors in mice responded to RL4B TCRm therapy in a dose-dependent manner, eliminating tumors at the highest dose. RL4B TCRm strongly detects the hCGβ peptide/HLA-A2 epitope in human primary breast tumor tissue, but does not react or reacts weakly with normal breast tissue from the same patient. These results further illustrate the selective nature of TCRm Abs and the clinical relevance of the GVLPALPQV peptide/HLA-A2 epitope expression in tumor cells, because they provide the first evidence that Abs that mimic the TCR can be used to markedly reduce and suppress tumor growth.

mAbs that recognize peptides presented on the cell surface by MHC class I molecules are potential therapeutic agents for cancer therapy. We have previously demonstrated that these Abs, which we termed TCR mimic mAbs (TCRm), reduce tumor growth in models of breast carcinoma. However, mechanisms of TCRm-mediated tumor growth reduction remain largely unknown. In this study, we report that these Abs, in contrast to several mAbs used currently in the clinic, destroy tumor cells independently of immune effector mechanisms such as Ab-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). We found that TCRm-mediated apoptosis of tumor cells was associated with selective and specific binding of these Abs to peptide/HLA class I complexes, which triggered the activation of JNK and intrinsic caspase pathways. This signaling was accompanied by the release of mitochondrial cytochrome c and apoptosis-inducing factor. TCRm-induced apoptosis in tumor cells was completely inhibited by soluble MHC tetramers loaded with relevant peptide as well as with inhibitors for JNK and caspases. Furthermore, mAbs targeting MHC class I, independent of the peptide bound by HLA, did not stimulate apoptosis, suggesting that the Ab-binding site on the MHC/peptide complex determines cytotoxicity. This study suggests the existence of mechanisms, in addition to ADCC and CDC, through which these therapeutic Abs destroy tumor cells. These mechanisms would appear to be of particular importance in severely immunocompromised patients with advanced neoplastic disease, since immune cell-mediated killing of tumor cells through ADCC and CDC is substantially limited in these individuals.

The clinical success of immune checkpoint modulators and the development of next-generation immune-oncology (IO) agents underscore the need for robust preclinical models to evaluate novel IO therapeutics. Human immune system (HIS) mouse models enable in vivo studies in the context of the HIS via a human tumor. The immunodeficient mouse strains NOG (Prkdc scid Il2rg tm1Sug ) and triple-transgenic NOG-EXL [Prkdc scid Il2rg tm1Sug Tg (SV40/HTLV-IL3, CSF2)], which expresses human IL-3 and GM-CSF, allow for human CD34+ hematopoietic stem cell (huCD34+ HSC) engraftment and multilineage immune cell development by 12 to 16 weeks post-transplant and facilitate studies of immunomodulatory agents. A more rapid model of human immune engraftment utilizes peripheral blood mononuclear cells (PBMCs) transplanted into immunodeficient murine hosts, permitting T-cell engraftment within 2 to 3 weeks without outgrowth of other human immune cells. The PBMC-HIS model can be limited due to onset of xenogeneic graft-versus-host disease (xGVHD) within 3 to 5 weeks post-implantation. Host deficiency in MHC class I, as occurs in beta-2 microglobulin knockout in either NOG or NSG mice, results in resistance to xGVHD, which permits a longer therapeutic window. In this article, detailed processes for generating humanized mice by transplantation of HSCs from cord blood-derived huCD34+ HSCs or PBMCs into immunodeficient mouse strains to respectively generate HSC-HIS and PBMC-HIS mouse models are provided. In addition, the co-engraftment and growth kinetics of patient-derived and cell line-derived xenograft tumors in humanized mice and recovery of tumor-infiltrating lymphocytes from growing tumors to evaluate immune cell subsets by flow cytometry are described. © 2020 The Authors.Basic Protocol 1: Establishment of patient-derived xenograft tumors in CD34+ hematopoietic stem cell-humanized mice Basic Protocol 2: Establishment of patient-derived xenograft tumors in peripheral blood mononuclear cell-humanized mice Support Protocol 1: Flow cytometry assessment of humanization in mice Support Protocol 2: Flow cytometry assessment of tumor-infiltrating lymphocytes in tumor-bearing humanized mouse models Keywords: humanized mouse model r immune oncology r patient-derived xenograft (PDX) models r tumor-infiltrating lymphocytes

Summary The human leukocyte antigen (HLA; also called major histocompatibility, or MHC) class I system presents peptides that distinguish healthy from diseased cells. Therefore, the discovery of peptide/MHC class I markers can provide highly specific targets for immunotherapy. Over the course of almost two decades, various strategies have been used, with mixed success, to produce antibodies that have recognition specificity for unique peptide/MHC class I complexes that mark infected and cancerous cells. Using these antibody reagents, novel peptide/MHC class I targets have been directly validated on diseased cells and new insight has been gained into the mechanisms of antigen presentation. More recently, these antibodies have shown promise for clinical applications such as therapeutic targeting of cancerous and infected cells and diagnosis and imaging of diseased cells. In this review, we comprehensively describe the methods used to identify disease-specific peptide/MHC class I epitopes and generate antibodies to these markers. Finally, we offer several examples that illustrate the promise of using these antibodies as anti-cancer agents.

The TCRm RL1B could be a new approach to immunotherapy of Her2-expressing malignancies.

This report describes a novel HLA/peptide complex with potential prognostic and therapeutic roles for invasive breast cancer. Macrophage migration inhibitory factor (MIF) mediates inflammation and immunity, and MIF overexpression is observed in breast cancer. We hypothesized that the HLA class I of cancerous breast epithelial cells would present MIF-derived peptides. Consistent with this hypothesis, the peptide FLSELTQQL (MIF19–27) was eluted from the HLA-A*0201 (HLA-A2) of breast cancer cell lines. We posited that if this MIF19–27/HLA-A2 complex was exclusively found in invasive breast cancer, it could be a useful prognostic indicator. To assess the presentation of MIF peptides by the HLA of various cells and tissues, mice were immunized with the MIF19–27/HLA-A2 complex. The resulting mAb (RL21A) stained invasive ductal carcinoma (IDC) but not ductal carcinoma in situ, fibroadenoma, or normal breast tissues. RL21A did not stain WBCs (total WBCs) or normal tissues from deceased HLA-A2 donors, substantiating the tumor-specific nature of this MIF/HLA complex. As this MIF/HLA complex appeared specific to the surface of IDC, RL21A was tested as an immunotherapeutic for breast cancer in vitro and in vivo. In vitro, RL21A killed the MDA-MB-231 cell line via complement and induction of apoptosis. In an in vivo orthotopic mouse model, administration of RL21A reduced MDA-MB-231 and BT-20 tumor burden by 5-fold and by >2-fold, respectively. In summary, HLA-presented MIF peptides show promise as prognostic cell surface indicators for IDC and as targets for immunotherapeutic intervention.

The identification and validation of new cancer-specific T cell epitopes continues to be a major area of research interest. Nevertheless, challenges remain to develop strategies that can easily discover and validate epitopes expressed in primary cancer cells. Regarded as targets for T cells, peptides presented in the context of the major histocompatibility complex (MHC) are recognized by monoclonal antibodies (mAbs). These mAbs are of special importance as they lend themselves to the detection of epitopes expressed in primary tumor cells. Here, we use an approach that has been successfully utilized in two different infectious disease applications (WNV and influenza). A direct peptide-epitope discovery strategy involving mass spectrometric analysis led to the identification of peptide YLLPAIVHI in the context of MHC A*02 allele (YLL/A2) from human breast carcinoma cell lines. We then generated and characterized an anti-YLL/A2 mAb designated as RL6A TCRm. Subsequently, the TCRm mAb was used to directly validate YLL/A2 epitope expression in human breast cancer tissue, but not in normal control breast tissue. Moreover, mice implanted with human breast cancer cells grew tumors, yet when treated with RL6A TCRm showed a marked reduction in tumor size. These data demonstrate for the first time a coordinated direct discovery and validation strategy that identified a peptide/MHC complex on primary tumor cells for antibody targeting and provide a novel approach to cancer immunotherapy.