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Jain R.,Texas Tech University Health Sciences Center | Jain R.,Receptor Logic Inc | Rawat A.,Texas Tech University Health Sciences Center | Rawat A.,Receptor Logic Inc | And 5 more authors.
Journal of the National Cancer Institute | Year: 2013

Background Applications of trastuzumab are limited to breast cancer patients with high Her2-expressing tumors. We developed a T-cell receptor mimic (TCRm) monoclonal antibody (hereafter called RL1B) that targets the Her2-E75 peptide (residues 369-377)-HLA-A2 complex and examined its effects in Her2-expressing cancer cells. Methods RL1B binding affinity was determined by surface plasmon resonance and specificity was demonstrated using Her2 antigen-positive and negative tumor cell lines. Immunohistochemistry was used to assess binding to frozen sections of human carcinomas (n = 3). Antitumor activity mediated by RL1B and trastuzumab against Her2+ tumor cell lines was evaluated using the WST-1 cell viability assay and caspase-3 and poly(ADP-ribose) polymerase cleavage assays. A xenograft mouse model (n = 6 per group) was used to assess RL1B antitumor activity. Mechanisms of RL1B-mediated cytotoxicity were evaluated with confocal microscopy, flow cytometry, and histology. All statistical tests were two-sided. Results RL1B bound with high specificity and affinity to the E75 peptide-HLA-A2 complex in all Her2 + and HLA-A2+ cancer cell lines and human carcinomas. Compared with control antibody, RL1B suppressed growth of low Her2-expressing breast tumors in mice (mean volume, RL1B vs control = 241mm3 vs 1531mm3; P =. 0109) and statistically significantly increased mouse survival (P =. 0098). It reduced viability compared to control monoclonal antibody-treated cells and statistically significantly increased caspase 3 activation of all Her2+ carcinoma cell lines tested, whereas trastuzumab induced apoptosis only in high Her2-expressing cancer cells. Mechanisms of RL1B cytotoxicity were associated with antibody internalization and intracellular signaling. Conclusion The TCRm RL1B could be a new approach to immunotherapy of Her2-expressing malignancies. © 2012 The Author.


Kim S.,University of Washington | Li J.,University of Washington | McMurtrey C.P.,The University of Oklahoma Health Sciences Center | Hildebrand W.H.,The University of Oklahoma Health Sciences Center | And 5 more authors.
Journal of Immunology | Year: 2010

The generation of a robust CD8+ T cell response is an ongoing challenge for the development of DNA vaccines. One problem encountered with classical DNA plasmid immunization is that peptides produced are noncovalently and transiently associated with MHC class I molecules and thus may not durably stimulate CD8+ T cell responses. To address this and enhance the expression and presentation of the antigenic peptide/MHC complexes, we generated single-chain trimers (SCTs) composed of a single polypeptide chain with a linear composition of antigenic peptide, β2-microglobulin, and H chain connected by flexible linkers. In this study, we test whether the preassembled nature of the SCT makes them effective for eliciting protective CD8+ T cell responses against pathogens. A DNA plasmid was constructed encoding an SCT incorporating the human MHC class I molecule HLA-A2 and the immunodominant peptide SVG9 derived from the envelope protein of West Nile virus (WNV). HLA-A2 transgenic mice vaccinated with the DNA encoding the SVG9/HLA-A2 SCT generated a robust epitope-specific CD8+ T cell response and showed enhanced survival rate and lower viral burden in the brain after lethal WNV challenge. Inclusion of a CD4+ Th cell epitope within the SCT did not increase the frequency of SVG9-specific CD8+ T cells, but did enhance protection against WNV challenge. Overall, these findings demonstrate that the SCT platform can induce protective CD8+ T cell responses against lethal virus infection and may be paired with immunogens that elicit robust neutralizing Ab responses to generate vaccines that optimally activate all facets of adaptive immunity. Copyright © 2010 by The American Association of Immunologists, Inc.


Kim S.,University of Washington | Pinto A.K.,University of Washington | Myers N.B.,University of Washington | Hawkins O.,Receptor Logic Inc | And 12 more authors.
European Journal of Immunology | Year: 2014

We used a newly generated T-cell receptor mimic monoclonal antibody (TCRm MAb) that recognizes a known nonself immunodominant peptide epitope from West Nile virus (WNV) NS4B protein to investigate epitope presentation after virus infection in C57BL/6 mice. Previous studies suggested that peptides of different length, either SSVWNATTAI (10-mer) or SSVWNATTA (9-mer) in complex with class I MHC antigen H-2Db, were immunodominant after WNV infection. Our data establish that both peptides are presented on the cell surface after WNV infection and that CD8+ T cells can detect 10- and 9-mer length variants similarly. This result varies from the idea that a given T-cell receptor (TCR) prefers a single peptide length bound to its cognate class I MHC. In separate WNV infection studies with the TCRm MAb, we show that in vivo the 10-mer was presented on the surface of uninfected and infected CD8α+CD11c+ dendritic cells, which suggests the use of direct and cross-presentation pathways. In contrast, CD11b+CD11c- cells bound the TCRm MAb only when they were infected. Our study demonstrates that TCR recognition of peptides is not limited to certain peptide lengths and that TCRm MAbs can be used to dissect the cell-type specific mechanisms of antigen presentation in vivo. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.


Verma B.,Texas Tech University | Neethling F.A.,Receptor Logic Inc. | Caseltine S.,Receptor Logic Inc. | Fabrizio G.,Receptor Logic Inc. | And 5 more authors.
Journal of Immunology | Year: 2010

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. Copyright © 2010 by The American Association of Immunologists, Inc.


Verma B.,Texas Tech University Health Sciences Center | Hawkins O.E.,The University of Oklahoma Health Sciences Center | Neethling F.A.,Receptor Logic Inc | Caseltine S.L.,Receptor Logic Inc | And 4 more authors.
Cancer Immunology, Immunotherapy | Year: 2010

The identification and validation of new cancerspecific 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.


Weidanz J.A.,Receptor Logic Inc. | Hildebrand W.H.,The University of Oklahoma Health Sciences Center
International Reviews of Immunology | Year: 2011

The development of immunotherapies offers significant promise for clinical applications in cancer and infectious diseases. Here the authors describe a novel, integrated approach to immunotherapy that combines novel technologies to discover and target disease-specific peptide/HLA class I complexes. This unique class of markers makes the entire proteome accessible to antibody reagents and offers unsurpassed specificity for targeting cancerous and infected cells. Arm one of the three-armed approach uses an innovative technology for the efficient, direct discovery of new peptide/HLA class I markers. Arm two applies a powerful and inventive strategy to generate T-cell receptor mimics (TCRms), which are antibodies with exquisite binding specificity for peptide/HLA class I markers, and uses TCRms to validate the specific expression of markers on cancerous and infected cells. The third arm uses TCRms to target and kill diseased cells with high sensitivity and specificity. In summary, the combination of two pioneering technologies expands the repertoire of disease-specific markers that can be targeted by therapeutic antibodies and enables a powerful, integrated approach to HLA-based immunotherapy. © 2011 Informa Healthcare USA, Inc.


Patent
Receptor Logic Inc. | Date: 2013-10-14

Methods of assaying potency of a vaccine composition are provided. Said methods utilize a T cell receptor mimic that is reactive against a specific peptide/MHC complex. The potency of the vaccine is determined based upon the measured density of specific peptide/MHC complex present on the surface of the vaccine-treated antigen presenting cell.


A methodology of producing and utilizing antibodies that recognize peptides associated with a tumorigenic or disease state, wherein the peptides are displayed in the context of HLA molecules, is disclosed. These antibodies may be utilized in therapeutic methods of mediating cell lysis.


Patent
Receptor Logic LLC | Date: 2015-05-27

The present invention relates to a methodology of producing antibodies that recognize peptides associated with a tumorigenic or disease state, wherein the peptides are displayed in the context of HLA molecules. These antibodies will mimic the specificity of a T cell receptor (TCR) but will have higher binding affinity such that the molecules may be used as therapeutic, diagnostic and research reagents. The method of producing a T-cell receptor mimic of the present invention includes identifying a peptide of interest, wherein the peptide of interest is capable of being presented by an MHC molecule. Then, an immunogen comprising at least one peptide/MHC complex is formed, wherein the peptide of the peptide/MHC complex is the peptide of interest. An effective amount of the immunogen is then administered to a host for eliciting an immune response, and serum collected from the host is assayed to determine if desired antibodies that recognize a three-dimensional presentation of the peptide in the binding groove of the MHC molecule are being produced. The desired antibodies can differentiate the peptide/MHC complex from the MHC molecule alone, the peptide alone, and a complex of MHC and irrelevant peptide. Finally, the desired antibodies are isolated.

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