Infiltrating Solid Tumor Strongholds

Cancer immunotherapy developers are mapping the tumor microenvironment in greater detail and planning multiple lines of attack

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When malignant threats arise, cancer immunotherapy can rally elements of the immune system, mobilizing regular troops and special forces alike. Cancer immunotherapy, however, must achieve its objectives without inflicting collateral damage. Cancer patients are not free-fire zones.

Fortunately, cancer immunotherapy developers are finding that they can fire on cancer in highly controlled ways, as the conference programs for an upcoming PEGS Boston event indicate. This event, which was scheduled for May 4–8 (but may be postponed or canceled because of the coronavirus pandemic), includes a conference program entitled “Improving Immunotherapy Efficacy and Safety.” Several developers in the program—Incysus Therapeutics, Gritstone Oncology, Integral Molecular, and Retrogenix—have contributed their views to this article. Another developer—PACT Pharma—is represented here, too. (This company has a presentation in a PEGS Boston program entitled “CAR Ts, TCRs, and TILS.”) In most cases, the company representatives quoted here are also on the PEGS Boston speakers’ list.

T cells ally with chemo

A small subset of T cells bridges the early innate and the adaptive immune responses. This subset, which consists of γδ T cells, does not require priming. The γδ T cells, unlike αβ T cells, react immediately to pathogens and transformed cells. Moreover, γδ T cells respond to upregulated ligands for the natural killer group 2 member D (NKG2D) receptor and kill either by direct NKG2DR:NKG2DL recognition like a natural killer cell or by NKG2DL ligation via the γδ T-cell receptor. In addition, once activated, they can take on
additional roles, such as phagocytosis, cross-presentation of antigens, and cytokine secretion.

“The main function of γδ T cells is to distinguish between self and foreign, that is, between safe and dangerous,” says William Ho, president and CEO of Incysus Therapeutics. “They do not see transplant antigens or drive graft-versus-host disease, and they can go from donor to patient without gene editing. So, they are well suited for allogeneic, off-the-shelf cell therapies for solid tumor cancers.

“These cells are capable of recognizing and lysing diverse cancers in a major histocompatibility complex–unrestricted manner, highlighting their potential for pan-population immunotherapy. Several studies have demonstrated a correlation between γδ T cells and lower relapse rates and better survival outcomes.”

Incysus’ Drug Resistant Immunotherapy (DRI) platform appropriates the tumor’s own resistance mechanism to chemotherapy and genetically engineers it into γδ T cells so they can survive simultaneous combinations at doses that would typically kill white blood cells. Chemotherapy shrinks the tumor toward minimal residual disease and amplifies an immune signal, and then a functional immune response is overlaid in a tumor-independent manner.

Drug Resistant Immunotherapy (DRI) illustration
Incysus Therapeutics is developing a technology for gene-modified T cells that can be used in combination anticancer  therapies. The technology, called Drug Resistant Immunotherapy (DRI), engineers γδ T cells to resist chemotherapy. While
chemotherapy upregulates the DNA damage response pathway, amplifying an immune signal that allows directed killing of
cancer cells, the γδ T cells sustain an MHC-independent immune response. The company has initiated a Phase I study that is evaluating a combination of DRI and temozolomide in patients with newly diagnosed glioblastoma.

“Teaching nature how to do something new can be very difficult,” Ho stresses. “We needed something to amplify in a tumor that the T cell already knew how to effectively target and eliminate. That biologically conserved signal is the DNA damage response. The problem we solved was allowing the lymphocytes to survive and attack while the signal is present.”

Two clinical trials are active. The first, NCT03533816, is evaluating the safety of donor-derived γδ T cells and their ability to reduce relapse in leukemia/lymphoma patients undergoing allogeneic stem cell transplantation. The second, NCT04165941, is the first to evaluate genetically modified γδ T cells for use against solid tumors, specifically glioblastomas. In this trial, the cells are genetically modified to express O6-methylguanine DNA methyltransferase, a DNA repair protein that conveys chemoresistance or protection against alkylating agent chemotherapy.

Adoptive cell therapies

Different patients’ immune systems respond to malignancies and cancer immunotherapies in different ways. For example, among patients with solid cancers, the degree to which immune systems are activated by checkpoint inhibitors varies. A greater degree of activation appears to correlate with tumor mutational burden.

This correlation suggests that in patients that respond well to checkpoint inhibitors, the immune system likely targets neoantigens (antigens or mutated proteins that are unique to the tumor), and that the tumor harbors a large collection of unique mutations. In responsive patients, then, the immune system is better able to recognize neoepitopes, which are peptides that are derived from neoantigens and presented to the immune system on the human leukocyte antigen (HLA) receptors on the tumor.

“Even if the immune system is able to recognize those mutations, a response of sufficient magnitude is essential to eradicate the tumor,” stresses Alex Franzusoff, PhD, CEO of PACT Pharma. “Our surprising finding was that virtually every person with solid cancer has T cells circulating in their blood that already recognize their mutations.” Franzusoff adds that the company has also found that in most instances, neoepitope-specific T cells are very rare.

These observations inspired PACT Pharma to develop an adoptive cell therapy technology that generates large numbers of neoepitope-specific T cells. It compares the sequence of the tumor cells to that of healthy cells, and then predicts which neopeptides will be presented by that individual’s HLAs. Using high-throughput techniques, it generates peptides that mimic candidate neopeptides. It incorporates the peptides into HLA constructs that are used to coat DNA-barcode-labeled nanoparticles, which are incubated with an individual’s T cells. And it captures and sorts the T cells that recognize the HLA constructs.

At this point, PACT Pharma clones the isolated T cells’ neoepitope-specific T-cell receptors (neoTCRs). Then, using nonviral precision genome engineering, the company replaces the endogenous TCRs on fresh T cells with the cloned neoTCRs. In this way, the company generates T cells that can kill the neoantigen-expressing tumors. By applying a broad catalog of HLAs, the company gives itself the ability to produce patient-specific therapeutics for patients of any ethnicity.

PACT Pharma generates large numbers of T cells
PACT Pharma generates large numbers of T cells that can recognize cancer neoepitopes. After identifying the mutation blueprint of each person’s tumor, the company uses its barcoded snare technologies to capture preexisting T cells from the blood that already recognize and target the unique mutations. Then the ideal T-cell receptors for specific mutations are identified. Once the target is authenticated, PACT pharma uses nonviral gene editing to engineer the ideal mutation-targeted T-cell receptors into T cells from the same patient. When reinfused back to the patient, these T cells can eliminate tumor cells that express the unique mutations.

“In solid tumors, it has been difficult to find targets,” says Franzusoff. “Those targets do exist, and we now have developed a high-tech search tool to determine the right target and build a full immune response for each patient that has the potential to eradicate their cancer.”

PACT Pharma’s NeoTCR-P1, an adoptive cell therapy for patients with solid tumors, is being evaluated in a Phase Ia/Ib trial (NCT03970382). It is focusing on six different cancer types with varying degrees of mutational burden.

Cancer vaccines

Gritstone Oncology’s vaccine strategy also focuses on neoantigens. Mass spectrometry was used to generate a large dataset of HLA peptides from human tumor and normal tissue specimens. The company’s EDGE™ platform, a machine learning model for neoantigen prediction, was trained with this dataset along with selected published peptide datasets.

According to Karin Jooss, PhD, executive vice president and CSO of Gritstone Oncology, the model learns key DNA sequence features and other factors like RNA expression that lead to a greater likelihood of peptide presentation by the HLA.

In general, only a very small fraction of tumor-mutated sequences, <1%, are expected to result in the actual presentation of neoantigens on the tumor cell’s surface. Most mutations are patient-specific passenger mutations; thus, most neoantigens will be patient-specific across tumor types. Candidate neoantigens are prioritized for inclusion in each patient’s personalized immunotherapy. This concept is currently being tested in the company’s GRANITE clinical study.

A second clinical program, SLATE, is currently underway. It is evaluating a vaccine that includes neoantigens that are shared between some patients. Patients are being selected based on the presence of the driver mutation and an HLA match.

“Based on human infectious disease vaccine experience, we believe an adenoviral vector is one of the most potent antigen-delivery platforms to prime naïve T cells,” Jooss declares. “Continued strong immune pressure upon the tumor is likely necessary to drive a durable clinical response.”

To sustain high numbers of tumor-specific T cells, the same tumor-specific antigen can be given as a boost immunization in a different vector. This heterologous prime-boost concept has been shown to activate and sustain elevated antigen-specific T-cell responses against infectious disease antigens.

The boost in this vaccine regimen is a self-amplifying mRNA (SAM) formulated in a lipid nanoparticle. A SAM vector comprises RNA that encodes the selected neoantigens plus an RNA polymerase. After intramuscular injection, the RNA starts to replicate, leading to production of large amounts of the delivered target antigens. During the RNA replication, RNA structures that are foreign to a normal cell are generated. The presence of large quantities of antigen in an immune-stimulating environment drives profound antigen-specific T-cell responses.

Gritstone Oncology’s vaccine approach has been tested in nonhuman primates and shown to activate high T-cell titers against the delivered antigens.

Identifying primary and off targets

Eliminating off-target effects can facilitate drug development and produce much safer products. In a 2014 retrospective analysis of its pipeline, AstraZeneca found that the primary driver of failure in preclinical studies was off-target binding.1

“Monoclonal antibodies can be polyspecific 20–25% of the time,” says Benjamin Doranz, PhD, president and CEO of Integral Molecular. Off-target binding and potential adverse side effects are difficult to predict without testing. For close to four decades, the FDA has required tissue cross-reactivity studies for biologics; however, these studies are poorly predictive of in vivo toxicity. By testing antibodies or chimeric antigen receptor (CAR) T cells against an array of native proteins, you know exactly what specificity you are getting.

The company has developed the Membrane Proteome Array (MPA) platform, which encompasses virtually the entire human membrane proteome and expresses all the proteins in their native conformations in live cells. Since many antibodies can be sticky, different cell lines can be used to alleviate background binding. Any binding that is detected occurs between a candidate antibody and a specific membrane protein that the chosen cell overexpresses.

Molecular uses its Membrane Proteome Array
To assess monoclonal antibodies for polyspecificity, Integral Molecular uses its Membrane Proteome Array, which expresses virtually all the proteins from the human membrane proteome in their native conformations in live cells. This technology tests for off-target binding to predict potential adverse side effects. On the left, monoclonal antibody MAb001 exhibits specificity to the target; on the right, monoclonal antibody MAb002 demonstrates an off-target effect.

In 2018, the company published an article that discussed antibodies against the SLC2A4 transporter.2 “Most antibodies were very specific,” Doranz notes. “But one had a very small cross-reactivity with NOTCH1, a completely unrelated protein with only 6% amino acid similarity.”

Integral Molecular’s Shotgun Mutagenesis Epitope Mapping platform was used to identify the exact amino acid binding site on the 12-transmembrane-domain SLC2A4 transporter. Even though SLC2A4 and NOTCH1 had almost no sequence in common, the exact binding motif existed in both targets and in the same type of constrained motif. This technology helps to differentiate mechanisms of action and facilitates intellectual property filings for novel antibodies. Mapping is performed directly in live cells, so there is a 95% success rate even for conformational antibodies and structurally complex targets.

Integral Molecular enjoys the distinction of having received a $5.5 million contract from NIAID to epitope map antibodies against new viral pathogens.

Low false-positive rates

At Retrogenix, a system has been developed that produces membrane proteins within a human cell in situ on a chip. Cells grown on top of DNA-encoding vectors are transfected to overexpress the encoded membrane proteins. The resultant full-length proteins are completely natural.

“Safety screens need high success and low false-positive rates,” insists Jim Freeth, PhD, director and co-founder of Retrogenix. “We use a sophisticated proprietary process to comprehensively identify and continually refine our membrane protein library to include the most important isoforms and exclude proteins which do not have an extracellular domain.”

Retrogenix’s cell microarray technology
Retrogenix’s cell microarray technology may be used to discover the human cell surface and secreted protein targets of antibodies, proteins, viruses, and small molecules. It uses proprietary arrays of expression vectors spotted onto slides. Human cells grown over the top become reverse-transfected, resulting in cell surface expression of each respective protein at distinct slide locations. After the test molecule is applied, specific binding is analyzed and confirmed using an appropriate detection system.

A companion technology focuses on secreted proteins synthetically tethered to the surface of the cells. CAR T cells can also be screened—the cell, the antibody, or the antibody fragment. According to Freeth, an anti-PD1 antibody called SHR-1210 was evaluated in a

Chinese clinical trial and shown to have a peculiar toxicity called capillary hemangioma, which is a benign tumor formed by a collection of excess blood vessels. Off-target interaction screening of an alternative drug developer’s SHR-1210 variants—antibodies that had similar binding sites but different backbones—was undertaken to investigate the toxicity.

The variants reproducibly interacted with PD1 and three off targets. Two were involved in blood vessel formation; one activated the VEGFR2 receptor, causing a functional effect indicating a potential involvement in the toxicity. The alternative developer subsequently reengineered the antibody.

Freeth also relates the example of an effort in which MedImmune and Retrogenix scientists collaborated to identify a regulatory T cell (Treg)-specific surface protein that could function as a drug target.3 The scientists found that a number of antibodies from a phage antibody library specifically bound to the Treg cells, and that the most Treg-selective antibodies bound specifically to tumor necrosis factor receptor 2 (TNFR2). Using negative selection, the scientists established that TNFR2 was expressed by tumor-infiltrating Treg cells. Besides identifying TNFR2 as a primary target, the scientists demonstrated that an anti-TNFR2 antibody caused tumor regression in a syngeneic mouse tumor model.

“We also are investigating a label-free screening approach that will work with small molecules, the screening of which currently requires a radiolabeling step,” Freeth notes. “And we are involved in a pilot study to look for off-target effects with engineered TCRs to complement currently used prediction tools.”

 

References
1. Cook D, Brown D, Alexander R, et al. Lessons learned from the fate of AstraZeneca’s drug pipeline: A five-dimensional framework. Nat. Rev. Drug. Discov. 2014 Jun; 13(6): 419-431. doi: 10.1038/nrd4309. Epub 2014 May 16.
2. Tucker DF, Sullivan JT, Mattia KA, et al. Isolation of state-dependent monoclonal antibodies against the 12-transmembrane-domain glucose transporter 4 using virus-like particles. Proc. Natl. Acad. Sci. USA 2018 May 29; 115(22): E4990-E4999. doi: 10.1073/pnas.1716788115. Epub 2018 May 16.
3. Williams GS, Mistry B, Guillard S, et al. Phenotypic screening reveals TNFR2 as a promising target for cancer immunotherapy. Oncotarget 2016 Oct 18; 7(42): 68278-68291. doi: 10.18632/oncotarget.11943.

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