Christoph Rader, Ph.D. - US grants

DESCRIPTION (PROVIDED BY APPLICANT): The emerging molecular picture of tumor angiogenesis has inspired new strategies for cancer therapy, which are currently in various stages of preclinical and clinical development. Among these are monoclonal antibodies that target individual molecular components involved in the pathologic process. In the proposed project, we hypothesize that a more specific strategy would result from targeting two rather than one molecular component. In particular, we are testing the hypothesis that selective neutralization of VEGF at the site of tumor angiogenesis is superior to unselective neutralization. For this, we will develop bifunctional antibody constructs that combine a VEGF neutralization activity with a targeting device and compare them to the corresponding monofunctional antibody constructs. Our antibody constructs are designed to facilitate (i) selective VEGF neutralization through Tie-2 or Tie-2/ang-2 complex targeting, (ii) systemic delivery by gene transfer using recombinant adenoviruses, and (iii) preclinical evaluation in syngeneic mouse tumor models. Compared to existing monoclonal antibodies that target tumor angiogenesis, we anticipate our antibody constructs to be superior in terms of efficacy. In addition, they are designed to facilitate a faster transition from preclinical to clinical development based on (i) cross-reactivity with human and mouse antigen and (ii) established antibody humanization strategies.

Our current and future research utilizes antibody technology for the generation of a large nave human Fab library in a newly designed phage display vector. We anticipate that this phage display library will become an important source for the generation of human monoclonal antibodies (mAbs) to a number of targets with relevance for hematologic malignancies. In FY05, we selected human mAb KYK-1 to human NKG2D from this library. NKG2D is expressed on NK cells which mediate the perhaps most important activity mechanism of antibodies, i.e. antibody-dependent cellular cytotoxicity (ADCC). In FY06, we further evolved KYK-1 through sequential affinity maturation based on phage display technology. The matured antibody now binds human NKG2D with subnanomolar affinity. We are currently evaluating KYK-1 (i) as diagnostic and therapeutic targeting device for NK-cell and T-cell lymphoma and leukemia, (ii) for the generation of bispecific antibodies designed to recruit NKG2D+ NK cells and NKG2D+ CD8+ T cells to the tumor site, and (iii) for interfering with autoimmune processes mediated by NK-cells and T-cells. Complementing our efforts for generating human monoclonal antibodies, we are continuing the mining of rabbit antibody repertoires by phage display. In particular, we have generated rabbit monoclonal antibodies selective for all three members of the Nogo receptor (NgR) family. NgR family members are cell surface proteins involved in the development, plasticity, and regeneration of the central nervous system. However, we have found recently that mRNAs of NgR family members are expressed also by peripheral blood mononuclear cells (PBMC). Our rabbit monoclonal antibodies will allow us to detect and define NgR family member expression on the surface of normal and malignant lymphocytes with a particular emphasis on T-cell and B-cell lymphoma and leukemia. Another aspect of our antibody engineering efforts is the development of new antibody conjugation technologies. In this area we are closely collaborating with chemists from the Laboratory of Medicinal Chemistry, CCR, NCI, NIH in Frederick, MD. In particular, we are exploring new technologies at the interface of monoclonal antibodies and small synthetic molecules, resulting in uniquely defined immunoconjugates in which the biological and chemical components are endowed with pharmacological advantages.

Due to its recognized, though insufficiently defined, graft-versus-tumor (GVT) activity, allogeneic hematopoietic stem cell transplantation (alloHSCT) is currently the only curative treatment available for patients with certain hematologic malignancies including B cell chronic lymphocytic leukemia (B-CLL). GVT activity, as well as its counterpart graft-versus-host-disease (GVHD), is believed to be mediated primarily by alloreactive donor T cells. Shifting the focus to another component of the adaptive immune system, we are investigating whether alloreactive antibodies derived from donor B cells may also have a role in GVT activity. Specifically, we are interested in alloHSCT-induced antibodies that selectively recognize tumor cell surface antigens and are capable of mediating antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). In addition to their possible involvement in GVT activity, these antibodies may serve as tools for the discovery of new targets for human monoclonal antibody therapy. We developed a sensitive flow cytometry assay for detection of post-alloHSCT serum antibodies against cell surface antigens. Post-alloHSCT sera collected from B-CLL patients at defined time points after transplantation showed significant binding to B-CLL tumor cells but not normal B cells. Pre-alloHSCT sera, donor sera, and control sera were negative. The alloreactive antibody response correlated with the disappearance of circulating B-CLL tumor cells, suggesting an antigen-driven immune response. In order to identify the alloreactive antibodies and subsequently the cell surface antigens they recognize, we generated a human antibody library from post-alloHSCT peripheral blood mononuclear cells (PBMC) and selected it on primary B-CLL tumor cells by phage display. We are currently screening the selected antibodies for B-CLL cell surface binding. In summary, we have shown that (i) alloHSCT induces an antibody response to tumor cell surface antigens and (ii) this antibody response may be harnessed for human monoclonal antibody drug and target discovery. The identification and characterization of B-CLL-specific antigens could open avenues for the development of more effective and precise immunotherapeutic strategies. Recent gene expression profiling in B-CLL identified several genes that are uniquely expressed by B-CLL cells, although the expression and function of the corresponding proteins, and more importantly their suitability as candidate target antigens remain largely unknown. We have analyzed the cell surface expression of one such protein, receptor tyrosine kinase-like orphan receptor 1 (ROR1), on B-CLL cells. Collectively, our ROR1 protein expression analysis in human B-CLL suggests that, based on its highly restricted, consistent, and constitutive expression, ROR1 protein may be a promising target antigen for monoclonal antibody therapy of B-CLL.

This project generates, validates, and delivers novel antibody-drug conjugates (ADCs) that are designed to selectively and potently eradicate chronic lymphocytic leukemia (CLL), the most common leukemia in the U.S., without affecting healthy cells and tissues. CLL is an indolent yet incurable B-cell malignancy that afflicts more than 150,000 men and women and causes more than 4,500 deaths per year in the U.S. alone. There are currently no treatment options for CLL that allow for selective targeting of malignant B cells and that spare healthy B cells and other healthy cells and tissues. With this Premise, the project is built on the Hypothesis that the Fcµ receptor FCMR, on its own or in combination with other selectively expressed CLL cell surface antigens, can mediate rapid and effective cellular entry of cytotoxic drugs for potent and specific therapeutic intervention. Two independent Specific Aims will be pursued to rigorously test this hypothesis. In Aim 1, a series of molecularly defined ADCs will be generated that deliver and release a highly cytotoxic tubulin inhibitor and a highly cytotoxic DNA-targeting drug, on their own or in combination, via the FCMR internalization and trafficking pathway. These ADCs will be based on the selenomab-drug conjugate platform which utilizes an engineered selenocysteine residue for site-specific drug conjugation. By extensive validation in vitro, ex vivo, and in vivo, a panel of FCMR-targeting selenomab-drug conjugates will be assessed for their stability, specificity, potency, toxicity, and pharmacokinetics. Aim 2 builds on a novel dual variable domain (DVD)-IgG1- based ADC platform that utilizes a unique reactive lysine residue for site-specific drug conjugation. DVD-IgM- based ADCs that can simultaneously engage FCMR and a second CLL cell surface antigen will be built and extensively validated. In addition to a highly modular research strategy that systematically compares different targets, different antibodies, different antibody formats, different linkers, and different drugs, the ex vivo and in vivo experiments in both Specific Aims will be based on peripheral blood mononuclear cells from male and female CLL patients rather than on cell lines to collectively achieve Robust and Unbiased Results toward delivering a candidate for advanced preclinical investigations and eventual clinical translation. Throughout this campaign, conceptually novel biological and chemical components with broad applicability to next-generation ADCs for cancer therapy will be developed.

DESCRIPTION (provided by applicant): The majority of multiple myeloma (MM) patients will initially respond to standard chemotherapy. However, eventually relapse of the disease, associated with a multi-drug resistant phenotype, contributes to poor clinical outcomes. Modulation Therapeutics is dedicated towards developing strategies for targeting cancers like MM that home or metastasize to the bone. Our current lead compound binds a CD44/VLA-4 complex and induces necrotic cell death. The in vivo efficacy of MTI-101 has been demonstrated using two in vivo myeloma models which consider the bone marrow microenvironment when evaluating tumor response. Our lead compound is a cyclic peptide which reduces concerns of proteolytic degradation. However, the efficacy of the compound may still be limited by a short circulating half-life often typical of peptide based therapies. The oveall goal of this proposal is to utilize antibody conjugation strategies designed to increase the therapeutic window of our lead compound. The first goal of this proposal is to determine whether conjugation of MTI-101 to a non-targeting antibody will increase the in vivo efficacy of the compound. The second goal of this proposal is to generate and evaluate a conjugate of MTI-101 and a CD138-targeting antibody to determine if we can increase specificity of the compound and thereby increase the therapeutic window of our lead compound.

DESCRIPTION (provided by applicant): Bispecific antibodies (biAbs) that exert cytotoxicity by binding to tumor cells with one arm and by simultaneously recruiting and activating tumor cell-lysing endogenous immune cells with the other arm are an emerging category of next-generation antibody drugs for cancer therapy. Our research team will develop and deliver conceptually novel chemically programmed biAbs that recognize tumor cells with a variable small molecule component and that recruit and activate T cells and NK cells with a generic antibody component. Chemically programmed biAbs are more versatile than conventional biAbs as they only require the cloning, expression, and purification of a single protein. Further, to target a variety of different tumor cell surface antigens, chemically programmed biAbs can make use of a wealth of small molecules derived from chemical libraries or from structure-based design campaigns, linking advances in both immunology and chemistry for the benefit of cancer patients. The proposed study will rigorously test the hypothesis that chemically programmed biAbs can recruit and activate T cells and NK cells that selectively and potently kill tumor cells n vivo. Specifically, we will develop and deliver two entirely different molecular formats of the generic antibody component and then chemically program biAbs to selectively target folate receptor 1 (FOLR1). FOLR1 was chosen as a prototype as this tumor cell surface antigen is a clinically investigated target for both small molecules and monoclonal antibodies in ovarian and lung cancer and in other devastating solid malignancies. The two molecular formats that will be interrogated are based on the reactive selenocysteine (Sec) and the reactive lysine (Lys) technologies that we developed for molecularly defined chemical programming of antibodies. In Aim 1 we will generate and validate chemically programmed (FOLR1 x CD3) and (FOLR1 x NKG2D) biAbs based on a single antibody module in Fab format with an engineered C-terminal Sec. In Aim 2 we will generate and validate chemically programmed (FOLR1 x CD3) and (FOLR1 x NKG2D) biAbs based on a dual antibody module in DART (Dual-Affinity Re-Targeting) format that displays a single reactive Lys residue. Chemically programmed biAbs in these two molecular formats will be analyzed and compared for their ability to recruit and activate T cells (via CD3) and NK cells (via NKG2D) to direct killing of FOLR1-expressing tumor cells. Finally, in Aim 3, we will test the efficacy and safety of our chemically programmed biAbs in immunocompromised mice engrafted with both human effector and target cells. Collectively, our campaign will deliver both novel concepts and constructs for next-generation antibody drugs that are explicitly designed for broad utility in cancer therapy.

Both the two FDA-approved antibody-drug conjugates (ADCs) (Adcetris® and Kadcyla®) and nearly all ADCs in clinical trials are prepared by randomly conjugating drugs to lysine or cysteine residues in the antibody, affording a heterogeneous ADC mixture. Site-specific conjugation is a major recent improvement to ADC development, yielding homogeneous ADCs with greatly improved therapeutic index. Among the 60+ ADCs currently in clinical development, nearly 50 of them use auristatin and maytansine, the two payloads used in Adcetris® or Kadcyla®, respectively, with the rest using one of only five other drugs. The ADC field is in critical need of new, highly potent, and rapidly acting cytotoxic payloads that are active in many tumor types. We propose in this application to develop new enediyne-based site-specific ADCs for cancer therapy. Specifically, we will focus on tiancimycins (TNMs), which we recently discovered as novel members of the enediyne family of natural products. Our hypotheses are: (i) TNMs are outstanding ADC payload candidates owing to their exquisite potency and validated mode of action, (ii) genetic manipulation of TNM biosynthesis provides outstanding opportunities to produce the most promising TNM analogues to develop the linker chemistry and facilitate site-specific conjugation, (iii) site-specific conjugation of TNMs to engineered thiomabs and selenomabs will generate homogeneous ADCs with defined drug-to-antibody ratios (DARs), and (iv) complementary targeting of HER2 and ROR1 will help evaluate the novel enediyne-based ADCs against current benchmarks and improve therapeutic outcomes for breast cancer patients. The specific aims for this grant are: (i) manipulation of TNM biosynthesis in Streptomyces sp. CB03234 to produce the most promising TNMs for site-specific conjugation, (ii) production and purification of anti-HER2 and anti-ROR1 thiomabs and selenomabs with 2 engineered cysteine (Cys) and 2 or 1 engineered selenocysteine (Sec) residues, respectively, (iii) development of linker chemistry for site-specific conjugation and delivery of a panel of anti- HER2 and anti-ROR1 thiomab-TNM and selenomab-TNM conjugates, and (iv) evaluation of selectivity and potency of the anti-HER2 and anti-ROR1 thiomab-TNM and selenomab-TNM conjugates against HER2+ and HER2?/ROR1+ breast cancers in both in vitro and in vivo models. The outcomes of this application include (i) fundamental contributions to ADCs and (ii) a panel of anti-HER2 and anti-ROR1 thiomab-TNM and selenomab- TNM conjugates as next-generation ADC therapeutics for cancers. The long-term goal of our research is to discover novel microbial natural products and harness their exquisite cytotoxicity as ADC payloads for anticancer drug discovery.

PROJECT SUMMARY In response to funding opportunity announcement PAR-16-176, this joint proposal by Drs. Christoph Rader from The Scripps Research Institute (Jupiter, FL) and Natarajan Muthusamy from The Ohio State University (Columbus, OH) proposes to validate a novel target for immunotherapy of chronic lymphocytic leukemia (CLL). As part of the proposed study, we will develop novel T-cell engaging bispecific antibodies (biAbs) and a novel transgenic mouse model to assess and initiate further preclinical development. Our research team will generate, validate, and deliver novel biAbs that are specifically designed to recruit and activate T cells for selective and potent eradication of malignant B cells in CLL patients. CLL is the most common leukemia in the U.S. with a lifetime risk of 0.6% and an annual mortality of ~5,000. Despite the advent of immunochemotherapy and small molecule kinase inhibitors, there remains a great need for potent and safe treatment options for newly diagnosed, refractory, and relapsed CLL patients. In fact, there are currently no Food and Drug Administration (FDA)- approved therapeutic modalities in CLL that allow for selective targeting of malignant B cells without harming healthy cells and tissues. Notably, allogeneic hematopoietic stem cell transplantation (alloHSCT) has remained the only potentially curative treatment of CLL, underscoring the unmatched capability of immunotherapy. Pursuing potential contributors of the powerful graft-versus-leukemia (GVL) response, we devised a concerted antibody drug and target discovery strategy by generating the first post-alloHSCT antibody library from a cured CLL patient and selecting it by phage display against primary CLL cells. We identified a panel of fully human monoclonal antibodies (mAbs) with a common antigen on primary CLL cells. In still undisclosed work presented for the first time in this proposal, we identified the new target as Siglec-6. In healthy individuals, the expression of Siglec-6 is highly restricted to the placenta, to mast cells, and to certain B-cell subpopulations. Its expression on leukemia cells in CLL has not been reported previously. Our proposed study, which builds on our combined strengths in antibody engineering and transgenic mouse models, on the availability of clinical samples from both untreated and treated CLL patients for ex vivo and in vivo preclinical studies, as well as on our extensive supporting data, has the objective to rigorously test the hypothesis that Siglec-6 can serve as a target for T-cell recruiting and activating biAbs to mediate potent and safe eradication of leukemia cells in CLL patients. The experimental execution of this objective will be based on two Specific Aims. In Aim 1, we will build and test a panel of Siglec-6 x CD3 biAbs for primary CLL cell killing by autologous T-cells ex vivo and in vivo. In Aim 2, we will develop a novel CLL mouse model expressing transgenic human Siglec-6 and employ it for the in vivo evaluation of surrogate Siglec-6 x CD3 biAbs in a native and competent immune system. Collectively, our study explores a unique route for targeted therapy of CLL by interrogating a novel target with novel agents and a novel mouse model to assess and initiate further preclinical development.

PROJECT SUMMARY In response to NCI?s FOA PAR-20-292 for early and conceptual stages of translational cancer research, our NIH R21 grant application seeks the generation and validation of a new format of conditionally active T-cell engaging bispecific antibodies (T-biAbs) designed for proteolytic activation in the tumor microenvironment of solid malignancies. As such, the proposed T-biAb format permits the targeting of tumor-associated antigens (TAAs) that prohibit interrogation by conventional T-biAbs due to their basal expression levels on healthy cells of vital organs. While such conditionally active T-biAbs have broad therapeutic utility, we will focus our proposed studies on rigorously validating the new format in in vitro, in vivo, and ex vivo models of ovarian cancer. This includes experiments with both ovarian cancer cell lines (in vitro and in vivo) and primary tumor cells from ovarian cancer patients (ex vivo). There is an urgent public health need for conceptually new treatments for ovarian cancer. Less than half of the ~235,000 U.S. women currently living with ovarian cancer will survive 5 years. In 2020, ~22,000 U.S. women will be newly diagnosed and ~14,000 will die of ovarian cancer. We will test the hypothesis that conditionally active T-biAbs targeting the TAAs EGFR, HER2, and FOLR1, all of which are overexpressed in ovarian cancer, can mediate potent and safe eradication of tumor cells. With the overall objective of incentivizing advanced preclinical investigations, we will deliver both innovative tools and techniques for probing TAA targeting with conditionally active T-biAbs designed for proteolytic activation in the tumor microenvironment of solid malignancies in general and ovarian cancer in particular.