January 15, 2017 (Vol. 37, No. 2)

New Immunotherapeutic Strategies Include Bispecific Antibodies, Engineered Adenoviruses, and Specifically Formulated Vaccines against Cancer

Sometimes standing up to a bully requires digging in and strengthening self-defense strategies. Although the immune system is well-equipped to handle most assaults, a bully, such as an opportunistic pathogen or a crafty cancer cell, may overtake immunity.

Scientists are trying to ramp up the immune system in these cases or, in other instances, to quell an over-exuberant immune response that contributes to diseases such as autoimmunity. New immunotherapeutic strategies include constructing bispecific antibodies for connecting targets to effector cells, arming therapeutically engineered adenoviruses that replicate only inside tumors, and creating specifically formulated vaccines against cancer. Additionally, a new animal model seeks to more faithfully recapitulate human responses, especially for mobilizing natural killer T-cells that affect a vast array of diseases.

Aiming DARTs at Cancer

The concept of creating recombinant bispecific immunoglobulin (IgG)-like antibodies as a therapeutic strategy emerged more than 20 years ago, yet languished due to technical hurdles. However, advances in molecular biology and recombinant protein engineering and production have reignited interest. Scientists at MacroGenics are utilizing their DART® platform, based on a diabody approach.

“A DART molecule is a disulfide-linked heterodimer comprising the variable domains from each of two different antibodies, thus providing dual specificity,” reports Paul Moore, Ph.D., vp, cell biology and immunology.

One application of the DART technology is to connect a target cell with an effector cell to support redirected immune cell killing of tumors. “Importantly, the DART technology is created as a plug-and-play system to leverage different targeting specificities. Once an antibody specificity has been obtained and the desired variable region identified, it’s easily cloned, expressed, and produced as a DART. This format has very favorable manufacturability and stability. It also has a great deal of design flexibility, including the ability to extend pharmacokinetic properties and to tailor the desired binding avidity to the application needs,” Dr. Moore explains.

Six DART molecules are currently in clinical trials. Five are designed to redirect T-cell targeting of tumor or leukemic cells, including two that are being developed by Pfizer and Janssen. One therapeutic DART molecule, MGD006, for the treatment of acute myelogenous leukemia (AML) targets CD3 and CD123. Dr. Moore notes: “CD123, the alpha chain of the interleukin-3 receptor, is overexpressed in many hematological cancers including AML. Co-engagement of MGD006 with CD3 on cytotoxic T-cells and CD123 on AML cells culminates in the redirected T-cell killing of the AML population, including the CD123-positive leukemic stem cell population.”

According to James Karrels, CFO, there are many applications for the DART platform. “In addition to redirected T-cell activation and killing, DART molecules can modulate receptor signaling, simultaneously target multiple co-inhibitory receptors or checkpoints, and can target multiple epitopes on a pathogen to enhance its neutralization and/or clearance. At present we are focusing on both oncology and autoimmune disease applications.”

MacroGenics’ DART® and TRIDENT™ platforms, comprising recombinant bi- or tri-specific antibody-like molecules, respectively, have multiple therapeutic applications.

Tumor-Localized Immunotherapy

The fabled story of the Trojan horse describes how the Greeks conquered Troy by hiding a group of their elite soldiers in a hollowed-out wooden horse that was gifted to the residents. However, the soldiers crept out at night and opened the gates allowing the Greek army to enter and conquer the city. Similarly, scientists at PsiOxus Therapeutics have created a platform technology that arms an oncolytic virus with encoded immunotherapeutics, delivers it systemically to tumors that subsequently express the treatment payload.

“Although the recent successes with immunotherapeutics such as checkpoint inhibitors is a major step forward for cancer patients, combining such therapies can present a number of challenges, including increased side effect profiles and high costs,” explains Brian Champion, Ph.D., CSO. “Our platform employs enadenotucirev (EnAd), a chimeric Ad11p/Ad3 replication competent oncolytic adenovirus that selectively infects and replicates inside tumor cells. This virus is also stable in blood, which allows it to be dosed systemically for delivery to tumors. A wide range of modifications can be introduced to arm EnAd, including the expression of one or more antibodies, cytokines, or other immunomodulatory proteins, enabling combinations of therapeutic agents to be produced locally within the tumor where they are required.”

Dr. Champion also indicated that armed EnAd uses endogenous viral elements to drive transgene expression, thus maintaining tumor selective production despite systemic dosing. “Unlike traditional adenoviruses used for oncolytic immunotherapies, we developed our chimeric adenovirus by natural selection from a library to have the desired properties of potency, tumor selectivity, and blood stability,” Dr. Champion reports.

The company is conducting clinical trials that target epithelial carcinomas with their technology. According to Dr. Champion, “We have finished two clinical trials evaluating safety, dose escalation, and mechanism of action, dosing EnAd to more than 90 patients with colorectal, bladder, lung, or renal carcinoma. We are currently conducting a combination trial with EnAd and the checkpoint inhibitor Nivolumab (in collaboration with Bristol-Myers Squibb) and also EnAd in combination with chemotherapy in ovarian cancer.  The first armed EnAd virus (NG-348), which is designed to activate tumor-infiltrating T-cells, is scheduled for clinical studies at the end of next year.”

In the future, Dr. Champion notes they will be designing combination immunotherapies that synergistically target different immune mechanisms within the tumor microenvironment.

Cancer Vaccine Complexity

While so-called “checkpoint blockade” treatment of cancers can provide a remarkable clinical benefit, it does not work uniformly in all cancers. “Checkpoints refer to immunological T-cell signals that may be excitatory or inhibitory,” explains Willem Overwijk, Ph.D., associate professor, melanoma medical oncology, MD Anderson Cancer Center, University of Texas. “Their overall purpose is to guard against the possible development of autoimmune reactions. Immune checkpoint blockade therapies take the brakes off and promote T-cell attack of cancer cells.”

Contemporary anticancer immune therapies often employ anti-CTLA-4 (for early checkpoint blockade) and anti-PD-1/PDL1 (for later stage blockade). According to Dr. Overwijk, “For some patients, these treatments shrink tumors and prolong survival. For others, little or no substantial effect occurs, probably because of weak spontaneous T-cell antitumor responses. The million dollar question is why?”

New preclinical work is tackling that problem. One approach utilizes vaccination that synergizes with checkpoint blockade therapies. “There are a number of vaccines in development that aim to ramp up the immune response. Such vaccines employ viruses with tumor antigens, peptides, whole tumor cells, or DNA- and RNA-encoding antigens. Most vaccines incorporating an adjuvant will make a good antibody response; but to amplify cytotoxic CD8+ T cells, the bar is higher and a number of different signals have to come together. My lab is studying how to improve T-cell responses,” Dr. Overwijk comments.

Dr. Overwijk and colleagues recently investigated the antitumor activity and mechanism of action of a novel, injectable, tissue-retained TLR 7/8 agonist, 3M-052 (developed by 3M drug delivery systems division). They found that intratumoral administration of 3M-052 in mice generated systemic antitumor immunity, and suppressed both injected and uninjected distant melanomas.

He points out, “In this study, we could take advantage of a drug available from a pharmaceutical company. However, very few companies allow their new drugs to be combined with those of other companies in the kinds of multidrug combinations that we think are necessary for robust therapeutic benefit. This is a huge bottleneck to making progress in cancer vaccines.”

To help solve that challenge, Dr. Overwijk is working to develop his own drugs and examine the effects of specific cocktails. “To create the best cancer vaccines, more potent adjuvants delivering multiple signals through multiple drugs will be required. It’s a complicated picture but the hope is that new cancer vaccines used alone or in combination with checkpoint blockade will enhance anticancer responses.”

LEAPS Vaccines

T cells drive several types of immunity including Th1, Th2, and regulatory responses that influence diseases ranging from autoimmunity to infection and cancer. Disease-targeting T-cell therapies seek to activate and control these responses. One such approach is an immunomodulating and immunoprotective small-peptide vaccine technology called LEAPS (ligand epitope antigen presentation system) patented by CEL-SCI.

“For LEAPS vaccines, a T-cell immunogenic peptide is covalently linked to an immune cell binding ligand (ICBL) to direct the nature of the subsequent immune response,” says developer Ken S. Rosenthal, Ph.D., professor and director, microbiology and immunology, Roseman University of Health Sciences College of Medicine, working in collaboration with the inventor of the technology, Daniel H. Zimmerman, Ph.D., senior vp, CEL-SCI.

How does it work? A T-cell immunogenic peptide as small as eight amino acids is coupled via a triglycine linker to another peptide, an ICBL, that targets an antigen-presenting cell such as a dendritic cell or a T cell. “The key is to decide which antigen and which immune response will work. For herpes simplex virus (HSV) infection, a Th1 response is protective. Therefore, mice were immunized with vaccines consisting of the J-ICBL (a peptide from β-2-microglobulin) coupled to a T-cell immunogenic HSV peptide. J-LEAPS vaccines promote maturation of mouse and human bone marrow DC precursors that promote Th1 T-cell responses. J-LEAPS vaccines elicit immune responses to tuberculosis and protection or therapy in mouse models of HSV and influenza infection, HER2/neu breast cancer and autoimmune diseases,” Dr. Rosenthal explains.

The derG-ICBL (a peptide from the β-chain of MHC II) targets CD4+ T cells and elicits Th2 responses to an antigenic peptide. This approach is being used for a treatment of rheumatoid arthritis in collaboration with colleagues at Rush University Medical Center. “Depending upon the T-cell response and antigen driving the patient’s rheumatoid arthritis, a LEAPS vaccine can provide focused and specific therapy. CEL-SCI would like to take this product into clinical trials as soon as possible,” Dr. Rosenthal says.

Dr. Rosenthal reiterated that the overall goal of the LEAPS platform is to modulate, rather than ablate, appropriate responses. He concludes, “This could be a very powerful approach for personalized treatment of many types of diseases.”

CEL-SCI’s LEAPS vaccines consist of an immune-cell-binding ligand (J or DerG) attached to a disease-related antigenic peptide to promote T-cell responses that provide immunoprotection and immunomodulation of disease.

Mobilizing NKT Cell Responses

Natural killer T (NKT) cells are a group of unconventional T cells that co-express the T-cell receptor (TCR) and NK cell markers and recognize lipid antigens presented by CD1d, a MHC class I-type molecule. The best-known subset, called invariant natural killer T (iNKT) cells, expresses the invariant TCR α-chain, Vα24Jα18 in humans. These cells modulate the activation and phenotype of other immune cells and hence affect cellular responses in a vast array of diseases, including infection, autoimmunity, and cancer.

“There was a great deal of excitement in the field about 20 years ago with the discovery of potent antitumor functions of the prototypic glycolipid ligand of iNKT cells, α-galactosylceramide (α-GalCer),” reports Weiming Yuan, Ph.D., associate professor of microbiology and immunology, University of Southern California.

However, while extensive studies showed a potent antitumor function of α-GalCer in mice, the compound showed very limited efficacy in over 30 anticancer clinical trials. “It turns out that the lipid, α-GalCer, interacts with CD1d and iNKT TCR quite differently in human versus mouse systems. In addition, the substantially different composition of iNKT cells in human versus mouse may have a significant impact on the overall immune responses to lipid ligands. To more reliably test the new α-GalCer analogs being developed for human use, we decided to build a better mouse model that can more faithfully predict the human immune responses toward the glycolipid drug candidates,” Dr. Yuan explains.

To do this, Dr. Yuan’s team created the first human CD1d-knockin mouse. “To further improve the system, we next introduced the human NKT TCRα-chain (Vα24Jα18). Tests of several new α-GalCer analogs using this highly humanized mouse model have now demonstrated a more human-like response with a strong Th1-biased cytokine profile and potent antitumor effects.”

Similar to many animal models, more refinement is needed. “There are lots of technical details we are still pursuing. For example, it is important that the iNKT TCR is not expressed as a transgene, but rather demonstrates a more natural expression pattern,” Dr. Yuan adds.

The model has already garnered the interest of other scientists as well as pharmaceutical companies. “There are many more applications than I initially envisioned. This model can serve in the testing of any NKT cell-targeting immunotherapy such as for cancers, vaccines, autoimmunity, diabetes, asthma, etc. There are more than 6,000 papers on this group of T cells. It could be a great model in general to study the modulation of NKT cells,” Dr. Yuan summarizes.

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