Developing a Bispecific Antibody as a Novel Drug Delivery System to Target Glioblastoma Stem Cells

Asad Akhund, Karenna Bol, Ryan Jefferis, Kevia Qu, Jean Ye

SPECIFIC AIMS

The long-term goal of this project is to offer an alternative or supplementary drug for the treatment of glioblastoma that will improve patients’ post-surgical prognosis and reduce glioblastoma recurrence. However, the first step to achieve this mission is to create a targeted drug delivery system using a bispecific antibody that will deliver an anticancer drug to glioblastoma stem cells. Herein we have developed a method for creating this antibody, linking the drug, and testing its efficacy to reach target cells in the brain using in vivo models. The drug delivery vehicle will be a bispecific antibody engineered to have two light chains with different affinities for separate antigens. We will target the transferrin receptor with one half of the antibody, which will allow for the vehicle and drug to be transported across the blood brain barrier (BBB). The other antibody target will be nestin, an extracellular matrix protein that has been shown to be present on the surface of cancer stem cells indicating high malignancy. A drug will be linked to the antibody and released when the environment of the surrounding tissue is similar to that of a tumor. This system will be engineered and then tested in vitro and in vivo to determine if it is successful in reaching target cells in the brain.

Specific Aim 1: We will engineer a bispecific antibody-drug conjugate that will target transferrin receptors on brain endothelial cells creating the BBB and nestin in the brain. The transferrin receptor specific-portion is intended to allow for uptake of the antibody-drug conjugate into the brain, and the nestin-specific half will facilitate the targeted attachment of the vehicle to destination cells. Anti-cancer drug molecules will be linked to the antibody in order to allow for their release in target tissues and eventual uptake by the glioblastoma stem cells.

Specific Aim 2: We will perform tests on our antibody-drug conjugate in order to determine the binding affinity of the drug for each of the targets and modify the drug to attain the desired binding affinities if necessary. These will be followed by in vitro tests with mice in order to determine the depth of brain tissue penetration and colocalization with nestin in the brain. These tests will determine if our drug delivery vehicle has the ability to be taken up into the brain and reach target cells.

If in vivo trials show that our drug delivery vehicle is successful in reaching target cells, more research can be conducted in order to determine if the drug would be practical and effective for treatment of glioblastoma patients. Further in vivo testing would be conducted in order to determine if the drug is effective in killing glioblastoma stem cells. Additionally, if the vehicle is proven to be effective, it could be linked with cancer drugs other than the one specified here to improve their efficacy. Eventually we would hope to begin clinical trials. If successful, this drug delivery vehicle would provide novel method to target and treat the cells responsible for the proliferation and recurrence of glioblastoma and thus improve patient prognoses. More broadly, we will have developed a novel drug delivery method capable of shuttling therapeutics to more specific targets. Future work will be able to utilize this technology to not only better the treatment of glioblastoma, but other diseases in the brain and elsewhere as well.

RESEARCH STRATEGY

SignificanceGlioblastoma multiforme (GBM) is a highly malignant form of brain cancer. It is the most common brain

tumor, making up 15% of all primary brain tumors, and it affects approximately 5-8 out of every 100,000 people. The median survival rate is 15 months and it is usually fatal within a year of diagnosis. Glioblastoma is difficult to treat because of its high proliferation rate and extensive vasculature1. Additionally, cell populations within glioblastoma tumors are heterogeneous; the tumor contains a variety of cell types in various stages of differentiation2. This makes the cancer difficult to treat as the cell types react differently to the various drugs and therapies used in conventional treatment regimens. Cancer stem cells are one of the cell types within the glioblastoma tumor.

Glioblastoma is often diagnosed with a brain CT scan following an abnormal neurological exam. The current treatment regimen for GBM begins with microsurgical resection in order to properly diagnose the tumor and relieve pressure on the brain3. Surgery for glioblastoma tumors is challenging because the tumors have tentacle-like protrusions that are difficult to remove entirely1. Most GBM tumors occur in the supratentorial region and span multiple lobes. The location and nature of the tumor determine whether surgery is a viable option and how much of the tumor can be removed. Surgery is followed by external radiation treatment with a minimum dose of 54 Gy in combination with various chemotherapeutic regimens. Chemotherapeutic drugs used in GBM treatment are Temozolomide, Cisplatin/Tamoxifen, ACNU/Alexan, Camubris, and Gliadel. All of these are administered intravenously with the exceptions of Temozolomide, which is taken orally, and Gliadel, which is administered intraoperatively4. The combination of chemotherapy with radiation is more effective than radiation alone5. Even so, in glioblastoma patients, recurrence occurs almost 100% of the time. Glioblastoma multiforme tumors generally recur near the site of the original tumor. 78% of recurrences occur within 2 cm of the boundary of the initial tumor6. A select few patients undergo craniotomy for recurrent tumors; however, the benefits of additional surgery have not been proven to significantly improve prognosis4.

One of the reasons glioblastoma is difficult to treat is the blood brain barrier. In a healthy brain, the blood brain barrier (BBB) inhibits the passage of certain molecules from the bloodstream into the brain tissue. This selectivity works to protect the brain from changes in blood composition and keeps the microenvironment of the brain stable. The endothelial cells in the brain contain tight junctions (TJ) that inhibit the diffusion of small hydrophilic molecules and limit transcytosis. In GBM conditions, some characteristics of the BBB are lost and vessels become more permeable3. Transferrin receptors (TfR) are present on the endothelium of brain capillaries and bind to transferrin (Tf). This process facilitates the transcytosis of iron across the BBB and into the brain7. Transferrin receptors have been well studied for their utility as a shuttle for drugs and large molecules into the brain. TfR mediated transcytosis often involves anti-TfR antibodies8.

Glioblastoma tumors are resistant to chemotherapy and radiation and are highly progressive and proliferative due to the presence of cancer stem cells (CSCs), which are also called tumor-initiating cells1. CSCs are unique in their ability to initiate tumorigenesis, self-renew, and abnormally differentiate. These cells are also able to migrate, allowing for initiation of tumor recurrence despite traditional treatment methods1. Glioblastoma tumors have a microenvironment similar to typical tumors in that it is hypoxic due to separation of cells from main blood vessels. Studies have shown that this acidity contributes to the self-renewal capabilities of CSCs and other tumor cells and promotes the CSC phenotype9.

Various studies have been conducted in order to determine a marker for CSCs. CD133 has been shown to be present on the surface of glioblastoma cancer cells, and it is a commonly used marker in the study of glioblastoma. However CD133+ cells and CD133- cells have both been shown to have stem cell like properties1. Thus, CD133 is not a definitive

Figure 1. (A) Immunofluorescent images depicting presence of Nestin in a primary human GBM sample. (B) IF images showing presence of nestin and lack of CD15/CD133 in a primary human GBM sample14

marker for CSCs in glioblastoma. Nestin, which is an intermediate filament protein, is expressed at the surface of cancer stem cells and has been determined to be a viable marker10. It is typically expressed in proliferating cells during the early stages of development in embryonic and fetal tissue11. In adults, expression of nestin is predominantly limited to neural stem cells found in the subventricular zone of the lateral ventricle and the subgranular zone of the dentate gyrus12. Many markers, such as the transcription factor Sox2 and the post-transcription factor Musashi13, are located intracellularly, making it difficult to utilize them as an antibody receptor. Other studies have identified CD15 and CD133 as viable CSC markers. However, in a study conducted by Jin et al., 27-69% and 8% of glioblastoma samples with CD133-negative or CD15-negative cells, respectively, possessed cancer stem cell traits and gave rise to aggressive glioblastomas, suggesting that CD15 and CD133 may not be robust markers. By contrast, nestin-positive cells were found on CD133-negative and CD15-negative cell samples, as seen in Figure 114, suggesting that it may be a more reliable marker. More so, expression of nestin in CSCs is linked to a higher malignant grade tumors and a lower five-year survival rate9, indicating that it is more capable of identifying cancer stem cells related to more severe cases of glioblastoma.

Our goal is to create a drug-delivery system using an antibody that is able to cross the blood brain barrier and specifically target glioblastoma stem cells. This will allow for the specific and targeted delivery of anticancer drugs to the cells that lead to tumorigenesis in order to stop tumor progression and reduce recurrence. If successful, this method will offer a new option for glioblastoma patients that may result in fewer side effects for the patient and a longer survival rate.

Innovation           Glioblastoma tumors remain one of the most difficult tumor types to treat. Currently GBM treatment methods rely on a combination of surgery, radiation treatment, and chemotherapy. However, several challenges still remain, such as the difficulty in excising the finger-like tendrils of the tumor and the universal likelihood of recurrence15. As mentioned previously, a population of cancer stem cells (CSC) has been discovered, which maintain stem cell properties like rapid proliferation, differentiation, and self renewal abilities16, in addition to chemoresistance and radioresistance. Their connection to the inevitable tumor recurrence is hypothesized to be the result of increased levels of MGMT, a DNA repair enzyme17. Temozolomide, which acts by alkylating DNA, is the primary chemotherapeutic drug of choice. However, a DNA repair mechanism can restore DNA integrity in CSCs, rendering drugs less effective in currently used clinical concentrations18. Targeting CSCs with chemotherapeutic agents may decrease the possibility of glioblastoma recurrence, as well as inducing cell death in the finger-like tendrils.

Previous work has indicated the feasibility of developing a bispecific antibody capable of crossing the blood brain barrier19. However, its use as a drug delivery vehicle has yet been unexplored. Our engineered drug-delivery vehicle will be able to specifically reach these usually inaccessible stem cells and unload a more potent local drug dosage to promote their death. This will be done by creating an antibody drug conjugate utilizing the bispecific antibody. Through this approach we hope to create a specific and effective method for the treatment of brain cancer and prevention of cancer recurrence through the targeted delivery of cancer drugs to brain cancer stem cells. This vehicle may further be altered for targeting a variety of cell types.

Generating a Bispecific AntibodyFirst, we propose a method to generate a bispecific antibody (BsAb) with affinity for both nestin and

human transferrin receptor (TfR). Due to the high costs of commercially available antibodies and issues surrounding undesired light and

heavy chain associations, producing an antibody by proteolitically breaking apart monoclonal antibodies would not be cost effective or result in a high enough yield. We therefore turn to the well-established method of knob in holes to heterodimerize and combine two heavy chains to form a bispecific antibody.

Instead, we will use a promising approach that has been recently developed, in which antibody fragments are produced as “half-antibodies” by engineering cells to conjugate LC-HC fragments before they are exocytosed27. Using this method 28 unique BsAbs were produced in milligram to gram quantities demonstrating its ease of manufacture and scalable abilities. This idea has further been adapted for production

in mammalian Chinese hamster ovary (CHO) cells by Shatz et al. (2013) and shown to have scalable production capabilities equivalent to that of E. coli cells28. These two methods are described in detail below, with CHO cell expression as the desired method for producing our anti-nestin, anti-TfR  BsAb due to its glycosylation capabilities.Speiss et al. (2013) was able to produce half-antibodies in separate cultures, and then combine them using a redox

method. However, this method was taken a step further by co-culturing the bacteria to eliminate purification, pooling, heating and redox steps (Figure 2R). The co-culture method is similar to the standard production of monoclonal antibodies

in CHO cells. During antibody folding, heavy chains rapidly dimerize before association of light and heavy chains, so suppression of self-assembly of heavy chains is done to maintain the proteins as half antibodies. A T366W knob is introduced to associate with a T3663, L368A, or Y407V hole mutation in the CH3 domain of the antibodies. Each half-antibody is produced in the same culture, and optimization of the culture is done to maximize the amount of BsAb formed.

The steps involved in co-culture production of BsAbs are inoculating the co-culture, growing the co-culture without protein expression, inducing half-antibody expression, lysis of the biomass, purification and formulation. To begin the process we must first focus on plasmid construction and expression of antibodies. Antibodies are first cloned into expression vectors by cloning expression a pBR322 vector cut at the EcoRI site29. The plasmid promoter sequence is phoA and transcription terminator sequence is lambda t0. The STII signal sequence with a translation initiation strength of 1 will precede the coding sequence for both light and heavy chains. Once the bacterial cells have been cloned, they must first be separately grown overnight (each culture corresponding to a knob or hole) before they are co-cultured. Growing a culture overnight in Luria Bertani (LB) media diluted into expression media with a suitable W3110 derivative will provide full antibody expression29.

After reaching the stationary phase the two bacterial cell pellets can be combined for a single inoculum. Speiss found that an inoculation ratio of 60:40 (hole to knob) produced an optimal amount of BsAb. However, for our antibody we might find a different ratio to be optimal. Lyse-C will then be used to extract the proteins from the culture. The lysates will be purified over Protein-A affinity resin. Hydrophobic interaction chromatography (HIC) will provide the means to effectively isolate intact BsAbs from excess half-antibodies. Further purification can be performed using CEX chromatography in case there is any endotoxin or other bacterial contaminants. Final formulation will be completed using a histidine-acetate buffer, Tween, and sucrose.

The co-culture yields between 100mg of BsAbs for each liter of cultured cells, and can be further upgraded by optimization of translation rates, chaperones, strains and culture conditions30. Several procedures can be used along the road of production to verify that the correct antibodies are being produced. SDS-PAGE analysis can be used to determine the size (should be 150 kD), electrospray ionization-time of flight (ESI-TOF) mass spectrometry to verify proper formation of the hinge region, SEC to verify stability and dispersion of proteins in a solution, Isoelectric focusing to verify the purity, and mass spectrometry to confirm the right conjugation of heavy and light chains.

The product of this method is an aglycosolated antibody that can be used for experimental purposes for proof of concept studies and non-primate in vivo studies. To show our methods are applicable to humans, a glycosolated antibody will need to be produced. Glyosolation has been shown to have positive effects on therapeutic antibodies, including a longer half-life while in the blood stream30.

Figure 2. Representation of the knob in hole method. Light chain association issues when two different antibody LCs are co-expressed in one cell line as described by Klein et al. (2012) are depicted.

Figure 3. The co-culture procedure outlined by Speiss in which E. Coli is used to produce BsAbs28

Shatz introduces a method of producing glycosolated BsAbs via half-antibody production in CHO cells shown in Figure 3R. This method of production attaches a carbohydrate chain in the asparagine 297 (N297) region of the CH2 domain. The carbohydrate chain is composed of a core complex of N-acetylglucosamine (GlcNac) and mannose with variable additions of galactose, sialic acid, fucose and bisecting GlcNac residues. This combination of carbohydrates attached to this particular area of the Fc is meant to provoke an immune response upon antigen binding as well as full antibody-dependent cell-mediated cytotoxicity (ADCC). For our purposes we will need to alter the production method to achieve glycosolation of a non-immunogenic BsAb that does not induce ADCC.

Selection of the proper variation can be taken from Jefferis et al. (2009) who reviews the proper ratio of galactose residues to oligosaccharides in the presence of fucosebased on desired effector function and potency31. Since fully galactosylated glycoforms may undergo minimal uptake by dendritic cells, it would be preferential to use type G2BF oligosaccharides. Yu has been successful in producing and testing BsAbs using both E. coli and CHO cells to cross the BBB32,33. We hope to adapt these methods to an anti-nestin half-antibody.

Two methods of producing a bispecific antibody capable of crossing the BBB have been outlined thus far. The purpose of describing these two methods is to recognize that production of antibodies is complex and fickle, especially using mammalian cells. Since the results from Speiss et al. (2013) are promising due to the variety of BsAbs they were able to produce, we retain this method as a viable alternative in the event that mammalian cell production methods run awry. In any event, we are confident that one of these methods will yield an effective bispecific antibody ready for drug conjugation and in vivo testing.

Creating a LinkerEarly attempts at creating antibody drug conjugates (ADC) attached the linkers at free cysteines or

lysines, which resulted in a high degree of variability34. There can be up to 40 lysine-drug conjugation sites within an antibody and 0-8 drugs attached per IgG molecule. Therefore, greater than one million different conjugates can be formed. Even when considering antibody species with the same number of drugs per molecule, differences in conjugation sites can alter binding affinities and serum stability34,35. Cysteine conjugation results in less variability than lysine conjugation, but it is still possible to generate more than 100 distinct species36. This variability complicates purification, testing, and FDA approval.

To resolve this issue, we will incorporate 4 unnatural amino acids with unique chemistry into the antibody. Optimal antibody drug conjugates have 4 drug molecules per IgG37,38. Attaching fewer drug molecules reduces efficacy, while attaching more results in reduced half-life, reduced antigen binding, and lower tolerability. Introducing unnatural amino acids into the antibody sequence can be done by incorporating an amber codon (TAG), which does not correspond to a natural amino acid, into the desired position of a PCR primer39. Amplifying a limited section of antibody DNA and subsequently lengthening the cloned sequence to encompass the full cDNA in further rounds of PCR will provide sufficient genetic material for our purposes. We will also need to engineer a tRNA and an amino-acyl tRNA synthetase that are orthogonal, i.e. do not have any cross reactivity, with natural amino acids40,41

Most existing ADCs require the complex to be internalized by the target cell before the linker is cleaved, often relying on lysosomal enzymes41. Since nestin is a structural protein and not a receptor, our antibody will remain at the cell surface and will be attached to the drug with an acid-labile

Figure 4. Shows the process of BsAb production using CHO cells as an expression host. Adapted from Shatz et al. (2013)29

hydrazone linker42. Several drugs, including doxorubicin, have been successfully incorporated into antibody drug conjugates with this linker type37,42,43.

The complex will remain stable in circulation and diffuse through the brain to the GBM tumor after crossing the blood brain barrier. Previous work measuring the diffusion of antibodies through the brain has shown that these large molecules are capable of traveling appreciable distances within a few hours44. The acidic tumor microenvironment will provide the proper conditions for cleavage. The drug will then diffuse into the cells and inactivate them. Wang et al evolved an orthogonal tRNA-synthetase pair to incorporate p-acyl-l-phenylalanine to selectively modify E. coli proteins, and further work applied this technique to producing an acid-labile oxime bond in an antibody-drug conjugate in mammalian cells with excellent yields45,46. Since oxime and hydrazone chemistry is almost identical, we do not anticipate any problems in producing our conjugate. Additionally, hydrazones are even more susceptible to acid-catalyzed hydrolysis than oximes47. This is important because the tumor microenvironment is only slightly acidic, especially compared to the lysosomes in which the oxime-linked antibody drug conjugate was meant to be degraded46,49.

Figure 5. Examples of antibody drug conjugates that created using hydrazone linkers38.

Nestin is found in some neural stem cells, so our antibody won’t just bind to GBM stem cells. However, the drug will only be released among healthy cell populations when they are in danger of becoming cancerous. The acidity of tumors is due to the preference of tumor cells for anaerobic respiration49. Lactic acid from this process diffuses into the surrounding healthy tissue. High levels of acidic stress promote a cancerous phenotype among neural stem cells, so our drug will confine a tumor to its current location in addition to killing existing glioblastoma stem cells.

In Vitro TestingAfter the bispecific antibody has been produced, we will perform a series of in vitro tests to characterize

its physical properties. To quantify the antibody’s affinity for human-TfR and nestin, Surface Plasmon Resonance (SPR) will be used to determine the constant of dissociation for each half of the antibody. This will be done by first capturing biotinylated human transferrin receptor on a CM5 chip coupled with streptavidin. Serially diluted concentrations of the bi-specific antibody will then pulsed across the surface of the chip using a Biacore T100 and the changes in response were used to determine the constant of dissociation (KD)19. This experiment will be repeated but with nestin captured on the chip instead. We seek a higher affinity for nestin (a lower KD) and a lower affinity for transferrin (a higher KD). If these criteria are not met, affinity can be modulated by modifying the light chain of the antibody. This can be achieved via a single amino acid substitution, created by introducing a mutation via PCR into the plasmid used to generate each half of the bispecific antibody. These affinity tests and subsequent modification of the antibody will be done until several variations of the antibody exist for further testing and meet the general criteria for ligand affinity (high against nestin and lower against TfR).

After the linker has been made and the drug molecules have been conjugated onto the antibody, the effectiveness of this process must be characterized. First the average number of drugs attached to the

Figure 5. A rendering of how unnatural amino acids are incorporated into proteins34.

bispecific antibody drug conjugate (BADC) must be determined. This can be done using UV/VIS spectroscopy. For an antibody and drug with different Amax values, the molar ratio of moles of drug per mole of antibody can be calculated. This can be done using measured absorbances of the BADC, extinction coefficients of the antibody and drug, and then simultaneously solving two Beer-Lambert’s Law equation51. The distribution of the number of drugs conjugated to each antibody can be found using electrospray ionization coupled with time-of-flight (TOF) analysis51. Furthermore the amount of unconjugated drug remaining intermixed with the BADCs must be assessed for toxicity concerns when the drug conjugate is injected. This can be done using reverse-phase high performance liquid chromatography (RP-HPLC) where peaks containing the unconjugated drug can be monitored using UV/Vis at the drug’s absorption maxima51. We look for effective drug conjugation –leading to our ideal number of drug molecules attached to each antibody (4) and a small concentration of unconjugated drug.

Furthermore, the ability of the linker to cleave under the right pH environment must be assessed. This will be done by developing solutions of various pHs to mimic that of the tumor microenvironment. A range fop Hs will be made from 6.8 (typical in hypoxic tumors) to 7.4 (physiologic)52. The antibody will then be added to each of these solutions at a set concentration and incubated for 24 hours at 37C. The solution will then be run through a column using RP-HPLC techniques where the remaining antibody will bind to the column and the drug will exit in the moving phase. The concentration of drug remaining in the solution can be measured using UV/Vis to determine the concentration of drug cleaved. We hope to see a high concentration of drug in the acidic solution, indicating that the linker will cleave under the hypoxic conditions of the tumor. However, we do not want to see the linker cleave under normal physiologic pHs, otherwise the drug would be released in circulation before it reached the brain.

The antibody drug conjugate will then be tested in cell culture. First, we will assess the binding and targeting ability of the bispecific antibody. The glioblastoma cell line, U251-SC-Adh cells, will be used as a cell model. These cells have been shown to have a higher rate of proliferation and self-renewal potentiality making them a good cell line for testing50. First, the cells will be transfected with human-TfR-GFP in order to visualize this target receptor. Cells will then be co-incubated with the bispecific antibody and then immunostained using an anti-human secondary with a different color fluorophore. An anti-human secondary can be used for the bispecific primary because the primary is engineered from two anti-human halves. We look for colocalization in the cells between the secondary and the targeted receptor. The experiment will be repeated but with human-nestin-GFP transfected instead for visualization. In this case, we look for stronger colocalization with the nestin because we desire higher nestin affinity. To obtain more statistically relevant data, the cells will also be put through Fluorescently Activated Cell Sorting (FACS). We hope to obtain data similar to those in Figure (1, which shows data taken from a similar study presented by Genentech on a bispecific antibody they engineered to cross the BBB19. We wish to see an even higher number of cells expressing a fluorescent signal for the cells transfected with nestin-GFP since there should be a higher affinity for this ligand.

Figure 6: Displays data from Yu. Et al.19. where HEK293 cells were transfected with human-TfR and sorted through FACS. In the control case (left) no bispecific antibody was added, therefore no signal from the secondary antibody was detected. However, once the bispecific antibody was added, a higher population of cells showed fluorescence from the secondary, indicating that the bispecific antibody was binding to its targeted receptor.

To determine the ability of the linker to cleave in cell culture, we will grow the U251 cells in media at an acidic pH (6.8) to mimic the tumor microenvironment. This can be done by adding 2-(N-morpholino)-ethanesolfonic acid and tris-(hydroxymethyl)-aminomethane to the media to acidify the environment52. These cells as well as those grown at normal pH (7.4) as a control, will be co-incubated with the antibody for 24 hours at 37°C. A cell cyto-toxicity assay will be done (using trypan blue to mark dead cells) on both cultures to determine if the drug is being released and effectively killing cells53. We hope to see a higher cytotoxic effect on the cells grown in an acidic environment.

In vivoIf these in vitro trials are successful, we will move into in vivo animal models. First, we will use mice

models to determine the ability of our bispecific antibody to cross the blood brain barrier, and its ability to reach the targeted cancer stem cells in the glioma on the other side. To do so, we will create our own mouse model using immunocompromised human-TfR knocked in mice that will then be xenografted with the modified U251-SC-Adh cell line transfected with human nestin-GFP, developed prior during in vitro testing. This will be done so the nestin can be visualized within the tumor. As a control, we will also use immunocompromised wild-type mice that will also be xenografted with the modified U251 cell line. All mice will be intravenously injected with our bispecific antibody and analyzed at several time points ranging from an hour after injection to 24 hours after. We use the experiment performed by Yu et.al (2014) to engineer a bispecific antibody to cross the BBB as a benchmark for uptake times and concentrations19. Another group of mice will also be injected with a control anti-nestin antibody to evaluate if the anti-nestin portion of the antibody has any effect in aiding BBB penetration. Similarly another group will be injected with a control IgG with no affinity for nestin or human-TfR. To analyze, the mice brains will be sectioned and immunostained with a secondary antibody against the bispecific primary. In the wild-type mice, we hope to see no fluorescent signal from the secondary antibody inside the brain. This would indicate that bispecific antibody engineered was indeed specific enough for its targeted human TfR and would not bind randomly, even to the analogous mouse TfR, and therefore would not be able to cross the BBB into the brain. Similarly, we hope to see no signal from the mice injected with anti-nestin or control IgG antibody. However, in the knocked-in mice possessing human-TfR, we hope to see a strong signal from the secondary antibody inside the brain, with results similar to those seen in Figure 27a. To further determine the depth of penetration in the brain, each section of the mouse brain will be homogenized and run through Western Blot and ELISA tests to determine the concentration of bispecific antibody. We hope to see a comparable spread of the antibody in various portions of the brain in the knocked-in mice but little to no concentration of control IgG or anti-nestin antibody (Figure 7b).

Figure 7: Images taken and adapted from Yu et al19 A) Displays human-transferrin knocked in mouse (KI/KI) and wildtype mouse (WT) brains immunostained for the bispecific primary antibody 1 hour after intravenous injections. As seen, fluorescence is only detected in the knocked-in mice, indicating antibody specificity for human-TfR. B) Displays concentrations of the bispecific antibody throughout different portions of the brain, 24 hours after intravenous injection in the human-TfR knocked-in mice. As seen, little control IgG (not specific to nestin or human-TfR) or anti-nestin antibody was able to penetrate the brain while the bispecific antibody showed relatively comparable concentration throughout.

To determine the ability of the bispecific antibody to reach its targeted nestin-expressing cells within the tumor, we will immunostain the injected mice with a secondary antibody and look for colocalization of

b)a)

fluorescent signals from the secondary as well as the labeled nestin. We want to see strong colocalization between nestin and the antibody in knocked-in mice, indicating that the antibody was able to reach its target.

We must also test the ability of the drug to cleave under such conditions and must investigate the microenvironment before and after tumor removal. To do so, we will perform surgery on the mice xenografted with glioblastoma (U251 cells). After removal of the primary portion of the tumor, we will assess the pH of the surrounding portions of the brain to determine if an acidic microenvironment still exists. This will be done by using localized proton MR spectroscopy (MRS). Both pre and post-surgery mice will be injected with hyperpolarized 13C bicarbonate and measured within 1-2 minutes54. We hope to see post-surgery mice retaining some acidity in the areas where the tendrils of the tumor remain unremoved, so we can use it to catalyze the cleavage of the hyrdrazone linker on our BADC.

ChallengesWhile nestin is expressed in CSCs, it is also expressed at the surface of neural stem cells, making it

possible that the bispecific antibody will target neural stem cells as opposed to cancer stem cells. We believe that the use of an acid-labile linker will regulate the release of the drug only in the presence of the acidic tumor microenvironment. Thus, if the antibody binds to a neural stem cell, the drug should only be released if the neural stem cell is being actively recruited to cellular layers surrounding the tumor55. If this is not the case and neural stem cells are killed, we believe that the potential benefits in glioblastoma treatment are greater than the cost of losing neural stem cells. Neural stem cells are predominantly involved in two functions: repair in response to central nervous system injury and the production of local circuit neurons in the granule cell layer of the olfactory bulb56 or the dentate gyrus of the hippocampus57.  We believe that given the severe nature of glioblastoma, the benefits of immediately prolonging lifespan outweigh the cost of losing future repair functions, sense of smell, or short term memory.  However, as neural stem cells expressing nestin only encompass around 63% of the neural stem cell population58, not all functionality will be lost.        Ideally, implementation of the bispecific antibody drug delivery vehicle would occur post-surgery to prevent glioblastoma recurrence. Currently, however, no research has been conducted into the acidity of the tumor microenvironment post-surgery. It is possible that without the presence of a large tumor, hypoxia would not be induced and lactic acid would not be produced, leading to a lack of acidity in the tumor microenvironment. This would render the bispecific antibody useless, as the drug would not be able to cleaved from the acid-labile linker.  Additional experiments would need to be conducted in order to obtain more information about post-surgery tumor microenvironment. In the event that the microenvironment and the areas surrounding the tumor tendrils are not acidic, the bispecific antibody may be utilized prior to surgical removal of the tumor and use the existing acidic environment to catalyze the release of the drug to peripheral tendrils of the tumor. This may still be useful in halting the spread of the tumor even after its removal.

Doxorubicin is not commonly used to treat glioblastoma, in part due to difficulties crossing the blood brain barrier and therefore reaching sufficient levels interstitially. More so, clinical trials have yielded poor results with doxorubicin, as high doses are required for systematic administration to reach therapeutic levels. Because doxorubicin is highly neurotoxic, these high doses are largely ineffective in treating glioblastoma. Yet doxorubicin shows significant promise: research has indicated doxorubicin displays robust tumoricidal properties in human glioma cell lines59, and over 20% of rats treated with doxorubicin delivered via nanoparticles exhibited long term remission, as well as overall longer survival rates60. Because there is very little data about the desired doxorubicin dosage, any relevant statistics will need to be obtained from experimentation. It is likely that despite the favorable data, doxorubicin is a poor choice of drug for yielding relevant information on the success of the drug delivery vehicle. Obtaining a high enough dose for therapeutic benefit but low enough to not induce adverse neurotoxic effects may be impossible, or long term resilience may develop. In such a case, other glioblastoma drugs, such as temozolomide or cisplatin, will need to be utilized.

Another challenge may be preventing the mis-binding of our bispecific antibody to other cells expressing transferrin receptors on their surface. In a safety assessment of their generated bispecific antibody created to cross the BBB utilizing anti-TfR affinity, Couch et.al found that the antibody bound to and reduced the reticulocyte counts and acute clinical signs in mice injected with the antibody61. The researchers concluded

the decrease in reticulocyte count was likely due to Fc effector function of the antibody. They found that by

reducing affinity to TfR can mitigate reticulocyte reduction and making the antibody effectorless61. Furthermore, they found using multiple and smaller doses of the antibody may aid reduction in acute clinical signs. Multiple dosing was not found to decrease the effectiveness with which the antibody could cross the BBB. Furthermore, the reduction in reticulocyte count and acute clinical signs was found to be less severe in their studies using non-human primates61. These are important considerations for our project as we move forward in assessing the safety of our vehicle and reducing undesired side effects.

ConclusionOur novel drug delivery vehicle will be capable of transporting a drug across the blood brain barrier and

into the brain, allowing the dosage of a drug to be significantly increased without increasing risk of neurotoxicity. By specifically targeting cancer stem cells post-surgery, the risk of recurrence may be decreased. Ultimately, this vehicle may be used to treat glioblastoma with the hopes of improving patient prognosis. Additionally, this method may be further investigated such that it can be altered and applied to other diseases in the brain.

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