Anti-tumor Antigen Antibodies Research Articles

Generation of dual specific bivalent BiTEs (dbBIspecific T-cell engaging antibodies) for cellular immunotherapy

BackgroundBispecific T-cell engaging antibodies (BiTES), comprising dual anti-CD3 and anti-tumor antigen scFv fragments, are important therapeutic agents for the treatment of cancer. The dual scFv construct for BiTES requires proper protein folding while their small molecular size leads to rapid kidney clearance.MethodsAn intact (150 kDa) anti-tumor antigen antibody to CEA was joined in high yield (ca. 30%) to intact (150 kDa) anti-murine and anti-human CD3 antibodies using hinge region specific Click chemistry to form dual-specific, bivalent BiTES (dbBiTES, 300 kDa). dbBiTEs were tested in vitro by EM, flow cytometry and cell cytoxicity and in vivo by PET tumor imaging and redirected T-cell therapy.ResultsThe interlocked hinge regions are compatible with a structural model that fits the electron micrographs of 300 kDa particles. Compared to intact anti-CEA antibody, dbBiTES exhibit high in vitro cytotoxicity, high in vivo tumor targeting as demonstrated by PET imaging, and redirected dbBiTE coated T-cells (1 microgram/10 million cells) that kill CEA+ target cells in vivo in CEA transgenic mice.ConclusiondbBiTE redirected T-cell therapy is a promising, efficient approach for targeting and killing cancer cells.

Dissecting FcγR Regulation through a Multivalent Binding Model.

Many immune receptors transduce activation across the plasma membrane through their clustering. With Fcγ receptors (FcγRs), this clustering is driven by binding to antibodies of differing affinities that are in turn bound to multivalent antigen. As a consequence of this activation mechanism, accounting for and rationally manipulating immunoglobulin (Ig)G effector function is complicated by, among other factors, differing affinities between FcγR species and changes in the valency of antigen binding. In this study, we show that a model of multivalent receptor-ligand binding can effectively account for the contribution of IgG-FcγR affinity and immune complex valency. This model in turn enables us to make specific predictions about the effect of immune complexes of defined composition. In total, these results enable both rational immune complex design for a desired IgG effector function and the deconvolution of effector function by immune complexes.

An Innovative in Vitro Potency Assay Designed to Predict the Fate of Chimeric Antigen Receptors Modified T Cell Therapy Post Infusion in ALL

Background: Chimeric antigen receptors modified T cells (CARTs) are T cells engineered to express tumor CARs that consist of T cell activation domains coupled to anti-tumor antigen single-chain variable fragments. Upon binding of tumor antigen to anti-tumor antigen antibody fragments on CART cells, the intracellular T-cell receptor CD3-zeta chain signaling domain induces T cell activation. The activated T cells undergo proliferation, cytokines release and tumor cells killing in an antigen-dependent manner. A number of CART-cell immunotherapies are now advancing to pivotal trials, however, little is known about how to characterize these cellular products before administration; one such gap relates to cell therapy product potency. Methods: Within this context, we developed a novel potency assay that quantifies the in vitro proliferative capacity of CART cell therapy products. We tested this assay using CART cell therapy products from patients with pediatric acute lymphoblastic leukemia (pedALL) and diffuse large B cell lymphoma (DLBCL). Results: In 40 CART products from pedALL patients evaluated for proliferation, and 37 products demonstrated significant (20% higher than background) proliferation. Overall, in vitro proliferation was correlated the expansion of the cell product in the patient after infusion. 23 CART products from DLBCL patients were analyzed, and 20 demonstrated significant specific in vitro proliferation. A strong correlation between in vitro proliferation and cell growth has been observed for the DLBCL patient samples. Conclusions: These data suggest that in vitro proliferation is a highly informative potency assay that may help predict product efficacy and safety of CAR T cell therapies. DisclosuresWang:Novartis: Employment. Dudley:Novartis: Employment. Choquette:Novartis: Employment. Jeschke:Novartis: Employment. Rutjens:Novartis: Employment. Kassim:Novartis: Employment.

Abstract IA25: Synergistic innate and adaptive integrin-targeted immunotherapy

Abstract We have determined that antitumor antibodies, when combined with extended systemic exposure to IL-2, strongly polarize an inflammatory tumor microenvironment and generate a vaccinal effect that significantly potentiates adoptive T cell therapies (1). We have broadened this approach by delivering activating Fc domains to tumors via a small peptide that targets RGD-binding integrins (i.e. alpha V beta 3 and alpha 5 beta1). Combining integrin-targeted Fc-mediated effector functions with extended IL-2 exposure leads to ablation of established syngeneic tumors, with formation of protective immunity against subsequent tumor rechallenge. The therapeutic index of this protocol is excellent in wild-type mice, with mild transient reversible toxicities attributable primarily to IL-2 exposure. These results provide a strong case for using tumor-opsonizing agents such as monoclonal antibodies or Fc fusions to synergize with T-cell directed immunotherapies.Reference1. Zhu EF, Gai SA, Opel CF, Kwan BH, Surana R, Mihm MC, Kauke MJ, Moynihan KD, Angelini A, Williams RT, Stephan MT, Kim JS, Yaffe MB, Irvine DJ, Weiner LM, Dranoff G, Wittrup KD. Synergistic innate and adaptive immune response to combination immunotherapy with anti-tumor antigen antibodies and extended serum half-life IL-2. Cancer Cell 2015;27(4):489-501. Citation Format: Karl Dane Wittrup. Synergistic innate and adaptive integrin-targeted immunotherapy [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr IA25.

Abstract B120: Characterization and combination immunotherapy treatment of an inducible autochthonous murine lung cancer model expressing human carcinoembryonic antigen (CEA) as a tumor-associated self-antigen

Abstract Previous work from our lab has demonstrated that a combination of anti-tumor antibody and an IL-2 fusion protein that exhibits extended serum half-life elicits an immune response that can effectively control a wide variety of tumor models. We have since then combined this therapeutic regimen with a vaccine exhibiting efficient lymph node trafficking that can generate an impressive population of tumor-antigen specific CD8+ T-cells but by itself does not provide good anti-tumor efficacy. The efficacy of this combination immunotherapy is further boosted by immune checkpoint blockade, leading to a robust four-component therapy: 1) anti-tumor antigen antibody; 2) IL-2 fusion protein; 3) anti-tumor antigen vaccine; 4) anti-PD-1 antibody. Although this four-pronged approach is demonstrably effective in the syngeneic subcutaneous melanoma model B16F10, we wish to test its efficacy in a more physiological model. To this end, we have turned to a model developed by the Jacks Lab, known as the KP model: an inducible lung tumor model where lentivirus-driven integration and expression of Cre is able to activate oncogenic Kras and completely remove p53 function. Because our therapeutic regimen requires a targetable tumor-associated antigen with respect to both the antibody and vaccine, we chose to induce expression of human carcinoembryonic antigen (CEA) in these tumors, as CEA has a well-studied structure and biology, and frequently expresses aberrantly in many forms of human adenocarcinomas. Additionally, our lab has previously engineered an antibody targeting CEA possessing picomolar affinity. Finally, to remove any endogenous immunological response against human CEA as a foreign antigen in our mouse system, we have crossed the KP model with a mouse model transgenic for human CEA, which in the literature has been described to have identical spatiotemporal expression of CEA as found in humans and should allow for central tolerance of this antigen. In the course of this work, we have successfully introduced human CEA into our lentivirus constructs and shown tumorigenesis by these constructs in the KP model coincides with expression of tumor-associated CEA, as detected by qPCR. On the therapeutic side, we have tailored the vaccine to successfully drive an anti-CEA CD8+ T-cell response. Performing preliminary therapeutic experiments in a transplant model of the KP tumor with our four-component therapy, we saw tumor control compared to untreated tumors. Upon interrogating the CD8+ T-cell response against CEA, we found 1-15% of CD8+ T-cells in the blood respond to CEA stimulation by intracellular cytokine staining. With regards to the lung tumor model, in the course of establishing the system we have also observed that the growth kinetics of tumors expressing CEA lags behind those tumors without CEA, even in the transgenic background. Preliminary immunophenotyping work by flow cytometry suggests that tumors with CEA seem to have a reduced myeloid-derived suppressor cell (MDSC) population and a higher CD8a+ dendritic cell (DC) population compared to tumors without CEA, suggesting that the former may have a less immunosuppressive tumor microenvironment that is better able to prime an anti-tumor CD8+ T-cell response. We will be planning to conduct therapeutic trials in the more physiological lung tumor KP model in the near future, as well as investigate the differences in the immune response with tumors expressing or lacking CEA. Citation Format: Eric F. Zhu, Kavya Rakhra, Naveen Mehta, Kelly D. Moynihan, Cary F. Opel, Darrell J. Irvine, K. Dane Wittrup. Characterization and combination immunotherapy treatment of an inducible autochthonous murine lung cancer model expressing human carcinoembryonic antigen (CEA) as a tumor-associated self-antigen. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B120.

A bispecific antibody targeting CD47 and CD20 selectively binds and eliminates dual antigen expressing lymphoma cells

Agents that block the anti-phagocytic signal CD47 can synergize with pro-phagocytic anti-tumor antigen antibodies to potently eliminate tumors. While CD47 is overexpressed on cancer cells, its expression in many normal tissues may create an ‘antigen sink’ that could minimize the therapeutic efficacy of CD47 blocking agents. Here, we report development of bispecific antibodies (BsAbs) that co-target CD47 and CD20, a therapeutic target for non-Hodgkin lymphoma (NHL), that have reduced affinity for CD47 relative to the parental antibody, but retain strong binding to CD20. These characteristics facilitate selective binding of BsAbs to tumor cells, leading to phagocytosis. Treatment of human NHL-engrafted mice with BsAbs reduced lymphoma burden and extended survival while recapitulating the synergistic efficacy of anti-CD47 and anti-CD20 combination therapy. These findings serve as proof of principle for BsAb targeting of CD47 with tumor-associated antigens as a viable strategy to induce selective phagocytosis of tumor cells and recapitulate the synergy of combination antibody therapy. This approach may be broadly applied to cancer to add a CD47 blocking component to existing antibody therapies.

Synergistic Innate and Adaptive Immune Response to Combination Immunotherapy with Anti-Tumor Antigen Antibodies and Extended Serum Half-Life IL-2

Cancer immunotherapies under development have generally focused on either stimulating Tcell immunity or driving antibody-directed effector functions of the innate immune system such as antibody-dependent cell-mediated cytotoxicity (ADCC). We find that a combination of an anti-tumor antigen antibody and an untargeted IL-2 fusion protein with delayed systemic clearance induces significant tumor control in aggressive isogenic tumor models via a concerted innate and adaptive response involving neutrophils, NK cells, macrophages, and CD8(+) Tcells. This combination therapy induces an intratumoral "cytokine storm" and extensive lymphocyte infiltration. Adoptive transfer of anti-tumor Tcells together with this combination therapy leads to robust cures of established tumors and development of immunological memory.

Isolation of scFv antibody fragments against HER2 and CEA tumor antigens from combinatorial antibody libraries derived from cancer patients

Tumor cells expressing HER-2/neu and CEA antigens are potentially ideal targets for antibody-targeted therapy. In this study, two large human combinatorial libraries have been generated from the lymph nodes of breast cancer patients that express HER2 and CEA antigens in their tumors. These ‘immune’ libraries have been constructed in two different formats of scFv, differing in the length of the peptide linker connecting the two variable VH and VL domains. Libraries derived from these patients may contain a larger pool of anti-tumor antigen antibodies and are useful repertoire for isolating scFvs against any tumor markers. The results of this study showed that we were successful in obtaining human scFvs against HER-2/neu and CEA. For HER-2, cell-panning strategy was performed and resulted in two scFv binders that detected the complete HER-2 receptor on the cell membrane and internalized to the cells. Also, preliminary ELISA data showed that several anti-CEA scFv binders were isolated by panning.

Cancer biomarkers detection using 3D microstructured protein chip: Implementation of customized multiplex immunoassay

Protein chips have demonstrated to be a sensitive and low cost solution to identify and detect tumor markers. However, efficient multiparametric analysis remains a challenge due to protein variability. Crucial parameters are the design of stable and reproducible surfaces which maintain biological activity of immobilized proteins. These parameters relate to surface chemistry and to immobilization conditions (printing buffers, washing etc.). In this study, we have developed and characterized various surface chemistries for the immobilization of anti-tumor antigen antibodies onto microstructured glass slides. The effect of surface properties and antibody immobilization conditions were evaluated for the detection of tumor antigens involved in colorectal cancer. Experimental results demonstrated that the biological activities of the immobilized antibodies were dependent on the surface chemistry and on the immobilization procedure. Optimal immobilization conditions were different for each antibody. Limit of detection in tumor antigen as low as 10 pM can reach under optimized conditions. Our 3D microstructured chip offers the possibility to implement a customized multiplex immunoassay combining optimal immobilization condition for each antibody-antigen system on the same chip.

Cancer Biomarkers Detection using Microstructured Protein Chip: Implementation of Customized Multiplex Immunoassay

Protein chips have demonstrated to be a sensitive and low cost solution to identify and detect tumor markers. However, efficient multiparametric analysis remains a challenge due to protein variability. Crucial parameters are the design of stable and reproducible surfaces which maintain biological activity of immobilized proteins, and immobilization conditions (buffer, pH, concentration). We have developed and characterized various surface chemistries for the immobilization of anti-tumor antigen antibodies onto microstructured glass slides. The effect of surface properties and antibody immobilization conditions was evaluated on the detection of tumor antigens involved in colorectal cancer. Experimental results demonstrated that each antibody displays variable biological activities depending on the surface chemistry and on the immobilization procedure. Under optimized conditions, we can reach a limit of detection in tumor antigen as low as 10 pM. Our microstructured chip offers the possibility to implement a customized multiplex immunoassay combining optimal immobilization condition for each antibody on the same chip.

Induced fluorescence of medullary carcinoma of the thyroid: A technology with potential to improve visualization of malignant tissue at the time of surgical resection

15503 Background: Surgery for medullary carcinoma of the thyroid can at times be technically challenging to the surgeon. Inducing the cancer cells to be fluorescent would have the potential to improve the surgeon’s ability to quickly and accurately identify and excise all of the malignant tissue. We have previously demonstrated the feasibility of induced tumor fluorescence with fluorophor-tagged anti-tumor antigen antibodies using human colon and breast cancer cell lines. We present here our results using a human medullary carcinoma of the thyroid cell line in the nude mouse model. Methods: A human medullary carcinoma of the thyroid cell line that was demonstrated to express CA 15–3 was used. Thyroid carcinoma cells were subcutaneously implanted in 4 nude mice (3 study mice and 1 control mice). Three weeks after injection, tumor nodules were easily detectable. Using the tail vein method, 3 study mice were injected with fluorophore-tagged anti-CA 15–3 and 1 control mouse with fluorophore-tagged IgG. Mice were examined using a small animal imaging system with a 470 nm light source and appropriate filters. They were also examined using a simple blue LED flashlight fitted with a fixed 470 nm band pass filter for illumination and were observed through filtered goggles. Results: Fluorescence of tumor nodules in the study mice could be seen through the skin. On dissection and exposure of the tumor nodules, this fluorescence was intense and clearly distinguishable from the surrounding normal tissue using either the imaging system or the blue LED. The control mouse injected with fluorophore-tagged IgG and examined in a similar manner revealed no tumor fluorescence. Conclusions: When tumor antigens are known, fluorophore-tagged antibody induced fluorescence is simple, easy to perform, requires no technically complex equipment or operator expertise and could be adapted to thyroid cancer surgery in the academic or community hospital setting. This technology would be indicated in those patients undergoing initial resection of medullary carcinoma of the thyroid as well as in those patients undergoing resection of recurrent disease where accurate identification of tumor tissue may be more difficult and time consuming. No significant financial relationships to disclose.

Induced fluorescence of breast cancer in the mouse model: A simple technology to improve visualization of malignant tissue during breast cancer surgery

10557 Background: Accurate identification of all malignant tissue at the time of lumpectomy or mastectomy can at times be challenging. When positive margins are discovered, significant additional time and effort may be required by the surgeon to complete the initial procedure. Inducing the cancer cells to be fluorescent offers the potential to improve the surgeon’s ability to rapidly and accurately identify and excise the malignant tissue at the time of surgery. We have previously demonstrated the feasibility of inducing tumor fluorescence with fluorophore-tagged anti-tumor antigen antibodies using a human colon cancer cell line. We present here our recent results using a human breast cancer cell line in the nude mouse. Methods: A human breast cancer cell line, MDA-MB-468 known to express CA 15–3 was used. Breast cancer cells were subcutaneously implanted in 14 nude mice. Three to five weeks after injection, tumor nodules were easily visible and palpable. Using the tail vein method, mice were injected with fluorophore-tagged anti-CA 15–3 and were examined using a small animal imaging system with a 470 nm light source. Mice were also examined using a simple blue LED flashlight fitted with a fixed 470 nm band-pass filter and were observed through filtered goggles. Results: Tumor nodules could be seen to fluoresce through the skin when illuminated with a 470 nm light source. When tumor nodules were exposed and examined with white light, the nodules could be seen but when illuminated with the 470 nm light source, tumor fluorescence allowed clear identification of nearby small tumor nodules and clear identification of tumor margins easily distinguishable from the normal tissue. Conclusions: For this technology to work, tumor antigens must be known and antibodies to these antigens available. It is easy to perform, requires no complex equipment or operator expertise and could be easily adapted to surgical oncology in any hospital setting. It has the potential to improve surgical accuracy in women undergoing initial lumpectomy or mastectomy as well as axillary lymph node dissection or sentinel node biopsy. In addition it could aid in the surgery of patients undergoing resection of isolated recurrent hepatic or pulmonary metastases. No significant financial relationships to disclose.

271. Full-Length Antibody Therapy Mediated by Adenovirus in Rodent and Non-Primate Animal Models

Therapeutic monoclonal antibody is increasingly applied in clinic with great clinical success, while its widespread application is limited by antibody manufacturing capability. Adenovirus is considered ad the one of the most proper vector for pre-clinical and clinical application, however, the application of adenoviral vector is restricted by CAR expression on the target cells. Thus adenovirus mediating anti-tumor antigen antibody expression in non-tumor cells or tumor cells is a novel strategy for cancer gene therapy. Herein we present an adenoviral delivery system that allows in vivo sufficient and sustained full-length antibody expression.

1116 POSTER Induced tumor fluorescence using fluorophore-tagged anti-tumor antigen antibodies in the nude mouse model—an example for use in humans

1116 POSTER Induced tumor fluorescence using fluorophore-tagged anti-tumor antigen antibodies in the nude mouse model—an example for use in humans

Immune-promoted tumor cell invasion and metastasis. New considerations in cancer therapy.

Available information shows that the immune system does not spontaneously reject malignant tumors. In addition, medical maneuvers to stimulate an immune response against established tumors do not show repetitive good results. Apparently, malignant tumor cells escape immune recognition and attack through multiple molecular mechanisms. Laboratory experiments show that properly stimulated specific CD8+ T cells, some CD4+ T cells, NK cells, and macrophages are capable of killing tumor cells. But, even in experimentally controlled conditions, a regular, complete, reliable and applicable tumor immune rejection has not been achieved yet. The birth of monoclonal antibodies, easy to produce in the laboratory and with very restricted target recognition, initiated a wave of expectation in the anti-cancer immune therapy believers. The “magic bullet” concept became popular, many new tumor antigens were discovered and new anti-tumor antigen antibodies developed. Thirty years after monoclonal antibodies introduction, few if any of those antibodies have shown a reliable anticancer curative effect. A question is raised looking at this defeating scenario: Is the immune system prepared to reject tumors? The plain answer to this question is another question: Why not? The immune system recognizes damaged cells and eliminates them in a continuous process of tissue repair. If tumor cells are identified as damaged cells the immune system should eliminate them. Nevertheless, in normally differentiated tissues, after elimination of damaged cells, a process of cell substitution and remodeling takes place based on recruitment of reserve cells and stem cells. Normal tissue repair involves, among other features, immune system cells and blood cells. Bone marrow derived scavenger cells remove cell debris. A controlled inflammatory cellular infiltration guaranties local replacement of connective matrix, cell motility/translocation, and neoangiogenesis. Apparently, tumor tissue replacement doesn’t need stem cells while tumor cells can divide infinitely. Therefore, the elimination of tumor cells is a mater of imbalance: if the elimination process is faster the tumor disappear, but if the replacement process is faster the tumor grows. In addition, the particular performance of tumor cells disturbs the repair process and, in consequence, tumor connective matrix replacement, cell relocation, and neoangiogenesis are in chaos.

A mixed hemadsorption assay for detection of cell surface binding anti-tumor antibodies in human sera

A practical mixed hemadsorption assay (MHA) was developed for the detection of cell surface binding anti-tumor antigen antibodies (anti-TA Ab) in the human sera. The assay was compared to enzyme-linked immunosorbent assay (ELISA) and dot ELISA in the detection of the antibodies developed against GA733-2 antigen. SW1116 cell line and recombinant GA733-2E protein were used as targets in the MHA and solid phase immunoassays (SPIA), respectively. Sera were obtained from healthy donors and vaccinated-colon carcinoma patients. The sensitivity level of the MHA was very high for detection of anti-TA Ab in sera.