WO2018058133A2

WO2018058133A2 - Compositions and systems for intracranial delivery, and methods of making and using the same

Info

Publication number: WO2018058133A2
Application number: PCT/US2017/053524
Authority: WO
Inventors: Fatih ATIK; Carter SURYADEVARA; Gerald ARCHER; Luis SANCHEZ-PEREZ; John Sampson
Original assignee: Duke University
Priority date: 2016-09-26
Filing date: 2017-09-26
Publication date: 2018-03-29
Also published as: WO2018058133A3

Abstract

Compositions, kits, and systems for intracranial delivery of cells of interest are disclosed, along with methods of making and using the same. Carrier compositions include a glycosaminoglycan having at least one thiol substituent, a gelatin having at least one thiol substituent, water, and a hydrogen-bond acceptor. The hydrogen-bond acceptor is capable of simultaneously making at least two hydrogen bonds with thiol substituents. The glycosaminoglycan and the gelatin are not covalently crosslinked. Delivery compositions include the carrier composition and a plurality of cells of interest suspended in the carrier composition.

Description

COMPOSITIONS AND SYSTEMS FOR INTRACRANIAL DELIVERY, AND METHODS OF MAKING AND USING THE

SAME

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is related to, claims priority to, and incorporates herein by reference for all purposes U.S. Provisional Patent Application 62/399,619, filed September 26, 2016 and entitled "Hydrogels for Intracranial Delivery and Methods of Making and Using Same".

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] N/A

BACKGROUND

[0003] Convection Enhanced Delivery (CED) methods are based on infusion of fluids which contain therapeutic agents to the intracranial area over a certain amount of time. Cell delivery with this method has historically been unsuccessful due to sedimentation of cells inside the containers and tubing systems due to gravity. Saline has been commonly used as a carrier for Convection Enhanced Delivery (CED) and caused this problem.

[0004] Others have attempted cell delivery, and in particular T cell delivery, using CED. For example, Dr. Michael Jensen's lab at the Seattle Children's Research Institute has tried to develop systems for T cell delivery using CED, but those research efforts have thus far proven unsuccessful. Rather than using CED, positive pressure infusion was required, which is sub- optimal. Given the high quality of research that is conducted in Dr. Jensen's lab, the lack of success in achieving T cell delivery using CED is an indication of how difficult this problem is.

[0005] A solution is needed to overcome sedimentation problem by holding cells

homogenously distributed inside the containers and tubing systems. The solution should ideally be biodegradable and would not affect the delivered cell's effectivity when they reach to the target organ. A solution is needed for higher amounts of cells to be delivered over hours and attack target pathology with the same capacity of cells in their optimal environment. BRIEF SUMMARY

[0006] In an aspect, the present disclosure provides a carrier composition. The carrier composition includes a glycosaminoglycan having at least one thiol substituent, a gelatin having at least one thiol substituent, water, and a hydrogen-bond acceptor. The hydrogen-bond acceptor is capable of simultaneously making at least two hydrogen bonds with thiol substituents. The glycosaminoglycan and the gelatin are not covalently crosslinked.

[0007] In another aspect, the present disclosure provides a delivery composition. The delivery composition include the carrier composition described elsewhere herein and a plurality of cells of interest suspended in the carrier composition.

[0008] In yet another aspect, the present disclosure provides a kit. The kit includes an infusion tube and the delivery composition described elsewhere herein within the infusion tube.

[0009] In a further aspect, the present disclosure provides a method. The method includes: a) creating an aqueous suspension including a glycosaminoglycan having at least one thiol substituent and a gelatin having at least one thiol substituent; and b) adding a hydrogen-bond acceptor to the aqueous suspension. The hydrogen-bond acceptor is capable of simultaneously making at least two hydrogen bonds with thiol substituents. The method can optionally further include suspending a plurality of cells of interest in the carrier composition.

[0010] In another aspect, the present disclosure provides a cell delivery system. The cell delivery system includes an infusion tube, a pump operative connected to the infusion tube, and a catheter in fluid communication with the infusion tube. The infusion tube contains within it the delivery composition described elsewhere herein.

[0011] In yet another aspect, the present disclosure provides a method. The method includes infusing a target location within a subject with the delivery composition described herein, thereby delivering a desired amount of cells of interest to the target location.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Fig. 1 is a flowchart showing a method, in accordance with an aspect of the present disclosure. [0013] Fig. 2 is a block diagram of a system, in accordance with an aspect of the present disclosure.

[0014] Fig. 3 is a pair of plots showing cell delivery yield (left) and percent viable cells (right) for various carrier compositions, as described in Example 3.

[0015] Fig. 4 is a plot showing a comparison of total cell infusion comparing hydrogel and saline carrier compositions, as described in Example 3.

[0016] Fig. 5 is a plot showing the results of migration experiments, as described in Example 4.

[0017] Fig. 6 is a plot comparing tumor-specific killing of glioma cells in various

compositions, as described in Example 5.

[0018] Fig. 7 is a plot of target-specific killing of glioma cells delivered from hydrogels, as described in Example 5.

[0019] Fig. 8 is a plot showing temperature dependence of infusion, as described in Example 6.

[0020] Fig. 9 is a plot showing temperature dependence of suspension, as described in Example 6.

DETAILED DESCRIPTION

[0021] Before the present invention is described in further detail, it is to be understood that the invention is not limited to the particular embodiments described. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. The scope of the present invention will be limited only by the claims. As used herein, the singular forms "a", "an", and "the" include plural embodiments unless the context clearly dictates otherwise.

[0022] It should be apparent to those skilled in the art that many additional modifications beside those already described are possible without departing from the inventive concepts. In interpreting this disclosure, all terms should be interpreted in the broadest possible manner consistent with the context. Variations of the term "comprising", "including", or "having" should be interpreted as referring to elements, components, or steps in a non-exclusive manner, so the referenced elements, components, or steps may be combined with other elements, components, or steps that are not expressly referenced. Embodiments referenced as "comprising", "including", or "having" certain elements are also contemplated as "consisting essentially of and "consisting of those elements, unless the context clearly dictates otherwise. It should be appreciated that aspects of the disclosure that are described with respect to a system are applicable to the methods, and vice versa, unless the context explicitly dictates otherwise.

[0023] Numeric ranges disclosed herein are inclusive of their endpoints. For example, a numeric range of between 1 and 10 includes the values 1 and 10. When a series of numeric ranges are disclosed for a given value, the present disclosure expressly contemplates ranges including all combinations of the upper and lower bounds of those ranges. For example, a numeric range of between 1 and 10 or between 2 and 9 is intended to include the numeric ranges of between 1 and 9 and between 2 and 10.

[0024] As used herein, the terms "component," "system," "device" and the like are intended to refer to either hardware, a combination of hardware and software, software, or software in execution. The word "exemplary" is used herein to mean serving as an example, instance, or illustration. Any aspect or design described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other aspects or designs.

[0025] Furthermore, the disclosed subject matter may be implemented as a system, method, apparatus, or article of manufacture using standard programming and/or engineering techniques and/or programming to produce hardware, firmware, software, or any combination thereof to control an electronic based device to implement aspects detailed herein.

[0026] Unless specified or limited otherwise, the terms "connected," and "coupled" and variations thereof are used broadly and encompass both direct and indirect mountings, connections, supports, and couplings. Further, "connected" and "coupled" are not restricted to physical or mechanical connections or couplings. As used herein, unless expressly stated otherwise, "connected" means that one element/feature is directly or indirectly connected to another element/feature, and not necessarily electrically or mechanically. Likewise, unless expressly stated otherwise, "coupled" means that one element/feature is directly or indirectly coupled to another element/feature, and not necessarily electrically or mechanically. [0027] As used herein, the term "processor" may include one or more processors and memories and/or one or more programmable hardware elements. As used herein, the term

"processor" is intended to include any of types of processors, CPUs, microcontrollers, digital signal processors, or other devices capable of executing software instructions.

[0028] As used herein, the term "memory" includes a non-volatile medium, e.g., a magnetic media or hard disk, optical storage, or flash memory; a volatile medium, such as system memory, e.g., random access memory (RAM) such as DRAM, SRAM, EDO RAM, RAMBUS RAM, DR DRAM, etc.; or an installation medium, such as software media, e.g., a CD-ROM, or floppy disks, on which programs may be stored and/or data communications may be buffered. The term "memory" may also include other types of memory or combinations thereof.

[0029] As used herein, "treatment," "therapy" and/or "therapy regimen" refer to the clinical intervention made in response to a disease, disorder or physiological condition manifested by a patient or to which a patient may be susceptible. The aim of treatment includes the alleviation or prevention of symptoms, slowing or stopping the progression or worsening of a disease, disorder, or condition and/or the remission of the disease, disorder or condition.

[0030] The term "effective amount" or "therapeutically effective amount" refers to an amount of a compound, cells, etc. sufficient to effect beneficial or desirable biological and/or clinical results.

[0031] As used herein, the term "subject" and "patient" are used interchangeably herein and refer to both human and nonhuman animals. The term "nonhuman animals" of the disclosure includes all vertebrates, e.g., mammals and non-mammals, such as nonhuman primates, sheep, dog, cat, horse, cow, chickens, amphibians, reptiles, and the like. Preferably, the subject is a human patient in need of intracranial delivery of cells.

[0032] As used herein, the term "cells of interest" refer to any cell(s) that are to be administered to a subject for therapeutic benefit. Cells may include those derived from another subject or the same subject. In some embodiments, the cells have been treated ex vivo (e.g., irradiated, transfected or otherwise modified, and the like). Examples of cells of interest include, but are not limited to, human and mouse T cells and chimeric antigen receptor transduced T cells (CARs). Observations

[0033] Prior to development of the compositions, kits, methods, and systems described below, Applicant attempted to solve the problems discussed in the background in a variety of other ways. It should be apparent that these other approaches are an indication of the difficulty of finding the compositions, kits, methods, and systems described below. It should also be an indication that a person having ordinary skill in the art would not have necessarily thought to attempt the compositions, kits, methods, and systems described below.

[0034] In one effort, Applicant tried methylcellulose compositions as a delivery carrier. While there was modest improvement relative to saline solution, the overall improvement was not sufficient to be practical for broader implementation.

[0035] In a separate effort, Applicant attempted mechanical alterations to the CED system. Specifically, Applicant tried modifying a horizontal CED system to function vertically, hoping that the adjustment would overcome the sedimentation problem described above. However, this also failed, because of electrical charge in the saline causing attraction and attachment between cells. The agglomeration cause clogs within the system.

[0036] In yet another effort, Applicant attempted adding vibrations throughout the system to counteract the sedimentation and clogging effect. Again, this effort was unsuccessful.

[0037] Once Applicant considered the use of hydrogels, other hydrogels were attempted before those discussed below. The results were unsuccessful, with some hydrogels turning solid and thus being non-injectable.

[0038] It should be noted that these efforts were only the efforts of a single research group and others (such as Dr. Jensen discussed above) have attempted solving the problems of existing systems without success.

[0039] As shown below, the compositions, kits, methods, and systems of the present disclosure show exceedingly surprising results when compared with the state of the art saline systems. For example, the compositions, kits, methods, and systems of the present disclosure achieve an increase in cell delivery rate of approximately 40 times. Saline systems achieve roughly a 2% delivery rate, whereas those discussed below achieve 70-80% delivery. This is an astounding increase. Efficacy is also improved with the compositions, kits, methods, and systems of the present disclosure as compared with saline systems, with a result of 20-30 times more effective tumor treatments. These impressive results could not have been predicted and are a sign of the nonobviousness and the presence of inventive step in the compositions, kits, methods, and systems of the present disclosure.

[0040] This disclosure provides the continuous infusion of CAR T cells with high efficiency and no negative effect to therapeutic function. We found the CED process is greatly affected by the sedimentation of the cells in saline, which is compounded by the inner diameter of the syringes and extension tubes routinely used during CED. Also, infusion time and infusion speed is another contributor to sedimentation. We use 20 ml syringes for CED and have found a very high sedimentation rate of cells in saline in contrast to a smaller Hamilton syringe with a 1 mm inner diameter. We tried to correct the sedimentation problem by rotating the instrumentation into a vertical position and thus theoretically preventing sedimentation inside the syringe.

However, our experiments demonstrated that the cells continued to sediment inside the extension tubing and syringe over 5 hours of infusion period, most of the cells were irregularly infused within the first and second hour of CED, and some portions of the infusate contained cell clots which eventually disrupted the infusion and required flushing of the extension tubing.

Conversely, sedimentation of cells in the carrier compositions described below is minimum. Our data indicates that regardless of the syringe and extension tube used in the CED system, the carrier compositions described below provide much higher yield during infusion without damaging migration capacity or cytotoxicity of CAR T cells.

Compositions

[0041] The present disclosure provides a carrier composition. The carrier composition includes a glycosaminoglycan having at least one thiol substituent, a gelatin having at least one thiol substituent, and a hydrogen-bond acceptor. The carrier composition is aqueous and thus includes water. The hydrogen-bond acceptor is capable of simultaneously making at least two hydrogen bonds with thiol substituents. The carrier composition can be a hydrogel.

[0042] The carrier composition has not had a covalent cross-linker added to it. Specifically, the carrier composition has not had a thiol-thiol cross-linker added to it.

[0043] The glycosaminoglycan with at least one thiol substituent can be hyaluronic acid with at least one thiol substituent. The glycosaminoglycan with at least one substituent, including the hyaluronic acid with at least one thiol substituent, can be present in the carrier composition in an amount by weight of between 0.01% to 10%, including but not limited to, between 0.05% and 5%), between 0.1% and 2%, or between 0.5% and 1.5%. Examples of a suitable commercially- available hyaluronic acid with at least one thiol substituent include, but are not limited to, Glycosil®, available from Ascendance Biotechnology, Medford, MA.

[0044] The gelatin with at least one thiol substituent can be present in the carrier composition in an amount by weight of between 0.01% to 10%, including but not limited to, between 0.05% and 5%), between 0.1% and 2%, or between 0.5% and 1.5%. Examples of suitable commercially- available gelatin with at least one thiol substituent include, but are not limited to, Gelin-S®, available from ESI BIO - a Division of BioTime, Alameda, CA.

[0045] It should be appreciated that the disclosure is not attempting to suggest that combining the glycosaminoglycan with at least one thiol substituent and the gelatin with at least one thiol substituent is a new concept, as these ingredients have previously been combined and covalently cross-linked (for example, using Extralink® cross-linker, available commercially from ESI BIO). However, this covalent cross-linking produces a solid hydrogel that is unsuitable for delivery of cells of interest. Applicant surprisingly discovered that the same components can be utilized with a hydrogen bond "cross-link", as described elsewhere herein, to achieve the successful delivery of cells of interest. Nothing regarding the prior usage of these ingredients with the covalent cross-linking suggests the usage described herein.

[0046] The hydrogen-bond acceptor can be oxygen (O2). The hydrogen-bond acceptor can be present in an amount sufficient to create hydrogen bonds with between 10% and 90% of the thiol substituents of the glycosaminoglycan and the gelatin, including but not limited to, between 20% and 80%, between 25% and 75%, or between 40% and 60%.

[0047] The carrier composition can have a total number of thiol substituents between the glycosaminoglycan and the gelatin of between 10,000 and 50,000 thiol substituents per microliter, including but not limited to, between 20,000 and 45,000 thiol substituents per microliter or between 25,000 and 40,000 thiol substituents per microliter.

[0048] The carrier composition can have an average dynamic viscosity of between 1000 cP and 5000 cP, including but not limited to, between 1500 cP and 4000 cP or between 1750 cP and 3000 cP. The carrier composition can have a storage modulus (G¹) of at least 5 Pa, including but not limited to, a storage modulus of between 5 Pa and 25 Pa or between 10 Pa and 20 Pa. The carrier composition can include a loss modulus (G") of at least 0.25 Pa, including but not limited to, a loss modulus of between 0.25 Pa and 0.75 Pa or between 0.3 Pa and 0.5 Pa. The average dynamic viscosity, the storage modulus, and the loss modulus can be measured using known methods and a rheometer. Specifically, the values reported herein were measured using a Kinexus Pro™ Rheometer, available commercially from Malvern Instruments Ltd, Malvern UK. An 8 mm diameter parallel plate attachment was used and measurements were taken at 25 °C. Gels were loaded onto the bottom platen and the top platen was lowered to a gap of 1 mm.

Samples were then trimmed to the proper geometry. The linear viscoelastic region was determined by performing a strain sweep from 0.1 to 10% strain at an oscillation frequency of 1 Hz. The elastic modulus (G'), viscous modulus (G"), and viscosity were calculated by averaging the values across this linear range (0.8-5% strain). Measurements were done in triplicate.

[0049] The carrier composition can be frozen and then thawed and used without loss of performance capabilities. For example, the carrier composition can be stored at a temperature of -80 °C within 24 hours of creation. The carrier composition can be thawed by exposure to a warm water bath (e.g., 37 °C) and gentle mixing.

[0050] The present disclosure provides a delivery composition. The delivery composition includes the carrier composition as described above and a plurality of cells of interest. The plurality of cells of interest are suspended in the carrier composition.

[0051] The cells of interest can be T cells. The T cells can be effector T cells, helper T cells, memory T cells, regulatory T cells, killer T cells, mucosal associated invariant T cells, gamma delta T cells, beta selection T cells, chimeric antigen receptor (CAR) T cells, or combinations thereof. In one specific aspect, the T cells are CAR T cells.

[0052] The delivery composition can contains cells of interest in an amount of between 100,000 cells per ml and 1,000,000,000 cells per ml, including but not limited to, between 1,000,000 cells per ml and 100,000,000 cells per ml or between 5,000,000 cells per ml and 10,000,000 cells per ml. Methods

[0053] Referring to Fig. 1, the present disclosure provides a method 100 of making the carrier composition described above. At process block 102, the method 100 includes creating an aqueous suspension comprising a glycosaminoglycan having at least one thiol substituent and a gelatin having at least one thiol substituent. At process block 104, the method 100 includes adding a hydrogen-bond acceptor to the aqueous suspension. The hydrogen-bond acceptor is capable of simultaneously making at least two hydrogen bonds with thiol substituents. This step converts the aqueous suspension into a carrier composition. At optional process block 106, the method 100 optionally includes suspending a plurality of cells in the carrier composition. This step creates the delivery composition described above.

[0054] The adding a hydrogen-bond acceptor of process block 104 can include exposing the aqueous suspension to oxygen for a predetermine time period at a predetermine temperature. The predetermined time period can be between 12 hours and 24 hours. The predetermined

temperature can be between 25 °C and 30 °C.

[0055] The creating an aqueous suspension of process block 102 can include creating a first aqueous suspension comprising the glycosaminoglycan having at least one thiol substituent, creating a second aqueous suspension comprising the gelatin having at least one thiol substituent, and mixing the first and second aqueous suspension to create the aqueous suspension.

[0056] The aqueous suspension, the first aqueous suspension, and/or the second aqueous suspension can be created using degassed water.

[0057] The present disclosure provides a method of using the delivery composition described above. The method includes infusing a target location within a subject with the delivery composition described above, thereby delivering a desired amount of cells of interest to the target location. The infusing can be via convection enhanced delivery. The target location can be in a subject's cranium and the infusing can be intracranial. The infusing can be at a rate of between 100 and 10 ml/hr.

Kits

[0058] The present disclosure provides a cell delivery kit. The cell delivery kit includes an infusion tube and the delivery composition as described above within the infusion tube. [0059] The infusion tube can be configured to deliver the delivery composition via convection enhanced delivery. The infusion tube can be configured such that the delivery composition exiting the infusion tube is moving horizontally. The infusion tube can be a syringe. The infusion tube can be plastic.

[0060] The infusion tube can have a volume of between 1 ml and 100 ml, including but not limited to, between 5 ml and 75 ml or between 25 ml and 50 ml.

Systems

[0061] Referring to Fig. 2, the present disclosure provides a cell delivery system 200 for delivering the delivery compositions described herein. The cell delivery system 200 includes an infusion tube 202, a pump 204, and a catheter 206. The cell delivery system 200 can optionally include a computer 208, which includes a processor 210 and optional memory 212.

[0062] The infusion tube 202 can have the properties described above with respect to the kits of the present disclosure.

[0063] The pump 204 can be a syringe pump. A non-limiting example of a suitable pump for use with the present disclosure is a MedFusion 3010a syringe pump, available commercially from Smiths Medical, Minneapolis, MN.

[0064] The catheter 206 can be a single piece of can have multiple pieces. The catheter 206 can be rigid, flexible, or can have portions that are rigid and portions that are flexible. The catheter 206 can include a length of tubing (e.g., silicon tubing), a needle, an intracranial catheter, or combinations thereof. For example, the catheter 206 can include a length of tubing with an intracranial catheter or a needle affixed to a distal end. Suitable catheters include, but are not limited to, catheters from the lab of Dr. Michael Vogelbaum at Cleveland Clinic, such as those described in U.S. Patent Nos. 8,808,234 and 8,979,822 and U.S. Patent Application Pub Nos. 2013/0103000, 2016/0015930, and 2016/0136411, the entire contents of which are incorporated herein in their entirety by reference. Other suitable catheters suitable for use in the present disclosure include, but are not limited to, Sophysa ventricular catheters, available commercially from Sophysa USA Inc, Crown Point, IN.

[0065] The catheter 206 can have a length of between 1 cm and 100 cm, including but not limited to, between 5 cm and 75 cm or between 10 cm and 50 cm. [0066] The catheter 206 can take a variety of shapes and sizes suitable for CED, as would be understood by a person having ordinary skill in the fluid dynamics arts. At least a portion of the catheter 206 can have a size of between 10 gauge and 20 gauge. At least a portion of the catheter 206 can have an internal diameter of between 0.1 mm and 4 mm. At least a portion of the catheter 206 can have an external diameter of between 0.5 mm and 5 mm.

[0067] The processor 210 can be operatively coupled to the pump 204 and can be configured to control the pump 204. The memory 212 can have stored thereon instructions that, when executed by the processor, cause the processor to direct the pump to execute an infusion routine.

[0068] The infusion routine can cause the pump to deliver the delivery composition at a constant delivery rate. The infusion routine can cause the pump to deliver the delivery

composition at a variable delivery rate. The infusion routine can have any time-dependent characteristics that might be useful for a given infusion, as would be understood by those having ordinary skill in the art.

EXAMPLES

[0069] Materials and Methods.

[0070] Tumor Lines: The human glioma cell line U87MG, as well as the Epidermal Growth Factor Receptor variant III (EGFRvIII) expressing subline, U87MG.AEGFR, have been previously described by Nishikawa R, Ji XD, Harmon RC, et al. A mutant epidermal growth factor receptor common in human glioma confers enhanced tumorigenicity. Proc Natl Acad Sci U S A. 1994;91(16):7727-31, which is incorporated herein in its entirety by reference. For clarity, any glioma cell line can be used for similar experiments, including mouse and rat models.

[0071] Carrier preparation and Optimization: Injectable, low viscosity hydrogel of the trade name LVHydrogel (i.e., the carrier composition described above) is comprised of two ingredients, thiolized hyaluronic acid and gelatin (denatured collagen) from (Ascendance Biotech^®, MA). Both elements were used in rodent brain without any toxicity and currently being tested on human subjects. These two ingredients were mixed with degassed water and processed as described above. After reaching the proper viscosity level, CAR T cells were resuspended at the indicated concentration in LVHydrogel at room temperature. To verify correct viscosity level, number of thiol groups must be evaluated with a fluorescence thiol assay. According to our optimization studies, the number of thiol groups is ideally 32457.43 ± 1207.35 per microliter.

[0072] Rheology: Rheology was performed as described above.

[0073] Human Pump System: The devices used in CED are a MedFusion 3010a syringe pump (Smiths Medical^®, MN), 20 ml syringes (Becton-Dickinson^®, NJ), and 20 cm long coiled silicon extension tube (SOPHYSA^®, France, model PIT400). The system was placed on a flat surface for the horizontal application. In Vertical Infusion setup, injection pump-syringe and extension tube was placed on a shelf while tip of the syringe looking downward and extension tube is connected to an Eppendorf tube to collect infusate. Infusion rate was adjusted to 500 μΐ/hr. All infusions were performed at room temperature for five hours to replicate the clinical setup.

[0074] Calculating Number of Cells Post infusion: 20 μΐ sample was taken from final infusate and mixed with 180 μΐ trypan blue to make 1/10 dilution. After mixing gently 10 μΐ sample was transferred to hemocytometer and counted under microscope to evaluate number of live CAR T cells. Following formula was used to calculate true number of cells:

Total Number of Viable Cells/ml = (Average cell count from each 16 corner squares) x (Dilution Factor) x 10.000

[0075] Cytotoxicity Assay: 5.0xl0⁶ U87MG and U87MG. AEGFR glioma cells were radioactively labelled with 100 μθ of Chromium51 at 37°C for 90 minutes. Cells were washed three times, co-incubated with saline and LVHydrogel carriers containing EGFRvIII-specific CAR T cells at a 10: 1 ratio (CAR T cells: tumor cells), and incubated at 37°C for a minimum of four hours in a total of 200μΐ8. After incubation, supernatant was harvested and radioactivity was measured to determine the release of chromium 51 and thereby the effective lysis of the glioma cells by the CAR T cells. Percentage of tumor specific cytotoxicity was calculated using the following equation:

Tumor specific cytotoxicity (%) = ((sample lysis - spontaneous)/ (maximum lysis - spontaneous)) x 100

[0076] Migration Assay: The lower chamber of 24-well transwell plates (Corning^®, NY) was loaded with 600 μΐ RPMI (without serum). The human chemokine CXCL10, responsible for the migration of T cells, was added to each chamber at 1 ng/ml, lOng/ml, 100 ng/ml, 500 ng/ml and 1000 ng/ml. The upper chamber of each well was seeded with serum starved 6xl0⁵

EGFRvIII-specific CAR T cells in either saline or LVHydrogel carriers. Transwell plates were then incubated at 37°C for 12 hours. Plates were removed from the incubator and the cells in the lower chamber of the transwell were counted in a hemocytometer using trypan blue staining. Percentage of migration by CAR T cells was calculated by the following formula:

CAR T cell migration (%) = ((Total number of cells that migrated to lower chamber)/(Total number of cells loaded in upper chamber)) x 100

[0077] Statistical Analysis: The difference in CAR T cell yield and viability between saline and LVHydrogel at each hour was assessed using a t-test with Bonferroni correction. A linear regression model including a quadratic term for log-transformed CXCL-10 concentration and corresponding interaction terms was used to assess the difference in CAR T cell migration between saline and LVHydrogel. A 2-way ANOVA with interaction was used to assess tumor- specific cytotoxicity among T cell carriers. Significance was determined at the level of p < 0.05. Values of p > 0.05 were Non-Significant (NS).

[0078] Example 1. Physical Properties.

[0079] While viscosity values for normal saline as well as water exist in the literature, we utilized dynamic mechanical testing to evaluate the rheological properties for the LV

LVHydrogel. In these measurements, we found the average dynamic viscosity of the

LVHydrogel to be 2360 ± 330 centipoise, roughly 2000-fold greater than saline or water alone (Table 1). It should be noted that the estimated viscosity for saline in this reference is for a 1M salt concentration, whereas standard isotonic saline contains 154 mM NaCl. However, saline's viscosity increases with salt concentration, indicating that the LV LVHydrogel viscosity is still well above that of the carrier saline solution. We could identify the storage (G') and loss (G") moduli from these data as 14.85 ± 2.09 and 0.37 ± 0.06 Pa, respectively. While values for water and saline are unavailable, they would be expected to be significantly lower than those established for the LVHydrogel. Furthermore, as the storage modulus is greater than the loss modulus for the LVHydrogel, we can confidently state that we have formed a gel during the synthesis of our hyaluronic acid based LVHydrogel carrier. Table 1

[0080] Comparative Example 2. Saline as Carrier.

[0081] Saline has been the carrier of choice for small chemotherapeutic agent delivery via CED. In light of our interest in delivering therapeutic cells to the brain, and the known advantages of increased infusion dose and distribution that CED offers, we decided to evaluate the efficiency of saline as a carrier for the delivery of CAR T cells. We positioned the infusion pump and extension tube system in a horizontal orientation as it is commonplace in the clinic. To our surprise, a minor fraction of the total CAR T cells resuspended in saline were capable of being delivered. While 3.8xl0⁷ cells/ml were expected to be delivered per hour, less than -l .OxlO⁶ cells/ml were delivered per hour. In order to overcome this problem, we evaluated the impact of rotating the infusion system to a vertical position; however, an erratic rather than continuous delivery was observed, where the number of cells delivered ranged between

-2.5x106 - 6.0xl0⁷ cells/ml in each hour. This vertical position created large clots that intermittently blocked cellular flow and failed to provide uniform delivery. When the efficiency of T cell delivery per hour was calculated, only 2-4% of the T cells were effectively delivered when the pump was positioned horizontally, and a much more variable efficiency, 7-150%), was obtained when the pump was in a vertical position. Altogether, our data demonstrate that saline is not a suitable carrier for CED of CAR T cells, regardless of pump positioning. The failure of saline as a carrier for CED was largely due to the sedimentation of CAR T cells.

[0082] Example 3. LVHydrogel as Carrier.

[0083] In order to solve the sedimentation of CAR T cells in saline and to generate both a high yield and a homogenous distribution of delivered CAR T cells, we developed a non-toxic, biodegradable low viscosity LVHydrogel carrier. To evaluate the delivery capacity of LVHydrogel, T cells were resuspended in LVHydrogel or control saline for 5-hour CED with the pump placed in a horizontal position. The efficiency of cell delivery in saline was dismal with less than -10% delivered in the first hour, and -0.5% delivered at later hours (Fig. 3, left, *p < 0.05). In contrast, LVHydrogel efficiency of delivery was significantly higher. During the first hour the efficiency of CAR T cell delivery in LVHydrogel reached over -90%, while at the later hours the delivery seen was -40% (Fig. 3, left). Of note, viability of the delivered T cells was not compromised during the CED procedure in either saline or LVHydrogel (Fig. 3, right, p > 0.23 NS). These data demonstrate that LVHydrogel as a carrier increases the efficiency of

continuously delivered CAR T cells during the CED procedure, making this an ideal carrier in the context of CED. The performance of the LVHydrogel was also compared with a methyl cellulose composition.

[0084] A 6-hour pump using the conditions described above was conducted with the human pump system described above. One suspension was prepared in LVHydrogel and another was prepared in saline solution. Fig. 4 shows the comparison of the total cells transfused. The LVHydrogel suspension had 74% live cells at the end of infusion. The saline suspension had 2% live cells at the end of infusion.

[0085] Example 4. Migration.

[0086] In order for CAR T cells to effectively mediate tumor killing, these cells need to be able to migrate out of the LVHydrogel matrix.

[0087] To evaluate the impact of LVHydrogel on CAR T cell migration, CAR T cells were resuspended in LVHydrogel or control saline and submitted to in vitro migration. CAR T cells in LVHydrogel displayed an equivalent migration capacity compared to those in saline carrier. Fig. 5 shows the results of these experiments. These experiments demonstrate that CAR T cells can effectively migrate out of the LVHydrogel with similar migration relative to a saline carrier.

[0088] Example 5. Tumor Specific Killing.

[0089] Having demonstrated that CAR T cells have the capacity to migrate out of

LVHydrogel to the same extent as saline, we next investigated if CAR T cells delivered via CED in LVHydrogel or saline can effectively mediate tumor cell killing. After 5-hour CED, CAR T cells from LVHydrogel or saline infusate were incubated with chromium labeled glioma cells bearing the CAR tumor-target in a standard cytotoxicity assay. Cytotoxicity of fresh cells prior to CED was also evaluated as a control. Prior to CED, the cytotoxicity of all CAR T cells was equivalent as expected. However, after CED, CAR T cells from LVHydrogel exhibited significantly superior tumor-specific killing of glioma cells versus saline (Fig. 6, ***p < 0.0001). Furthermore, the cytotoxicity of CAR T cells from LVHydrogel after CED was similar to the fresh non-infused CAR T cells in LVHydrogel, demonstrating that the CED process of delivery did not impair CAR T cell cytotoxicity. In contrast, the cytotoxicity of the infused CAR T cells resuspended in saline was much lower than the fresh CAR T cells in saline. This significant reduction was due to the drastic decrease of CAR T cell delivery observed when saline was used as a carrier. Of note, while the recognition and killing of the tumor by CAR T cells delivered in LVHydrogel still required target-specific engagement of the CAR with the tumor, glioma cells that did not express the CAR target EGFRvIII were not killed either before or after CED in LVHydrogel (Fig. 7, p = 0.7176 NS). Altogether, these experiments demonstrate that CAR T cells delivered in LVHydrogel retain tumor-specific recognition and cytotoxicity to effectively kill the tumor.

[0090] Example 6. Temperature Dependence.

[0091] Four cell compositions prepared as discussed above in LVHydrogel were prepared. One composition was infused for three hours as described above at 25 °C and another at 37 °C. Fig. 8 shows the number of cells (top) and the viability of cells (bottom) infused at these two temperatures. One composition was stored in an Eppendorf tube for three hours at 25 °C and another at 37 °C. Fig. 9 shows the number of cells (top) and the viability of cells (bottom) retained in suspension at these two temperatures. The LVHydrogel compositions have advantageous infusion and stability properties at room temperature.

[0092] Example 7. Intracranial Localization.

[0093] Intracranial CED was conducted to deliver CAR T cells into the brain of rats using the LVHydrogel and saline compositions described above. 2.4xl0⁶ cells in a volume of 24 μΐ were infused. CAR T cells were also delivered to the rats intravenously. The LVHydrogel CED provided significantly higher concentration of cells in the brain of the rates when compared with the saline composition, which in turn provided higher concentration than the intravenous delivery. This observation continued over the course of seven days. At the same time, over the course of the seven days, a significantly higher concentration of the cells were observed in the cervical lymph nodes of the rats when saline CED was utilized for delivery when compared with intravenous delivery, which in turn showed higher concentrations in the cervical lymph nodes when compared with LVHydrogel CED delivery.

[0094] The particular aspects disclosed above are illustrative only, as the technology may be modified and practiced in different but equivalent manners apparent to those skilled in the art having the benefit of the teachings herein. Furthermore, no limitations are intended to the details of construction or design herein shown, other than as described in the claims below. It is therefore evident that the particular aspects disclosed above may be altered or modified and all such variations are considered within the scope and spirit of the technology. Accordingly, the protection sought herein is as set forth in the claims below.

Claims

CLAIMS I/We claim:

1. A carrier composition comprising:

a glycosaminoglycan having at least one thiol substituent;

gelatin having at least one thiol substituent;

water; and

a hydrogen-bond acceptor capable of simultaneously making at least two hydrogen bonds with thiol substituents;

wherein the glycosaminoglycan and the gelatin are not covalently crosslinked.

2. The carrier composition of claim 1, wherein the glycosaminoglycan having at least one thiol substituent is hyaluronic acid having at least one thiol substituent.

3. The carrier composition of claim 1 or 2, wherein the hydrogen-bond acceptor is O2.

4. The carrier composition of any one of the preceding claims, wherein the

glycosaminoglycan having at least one thiol substituent is present in an amount by weight of between 0.01% and 10%.

5. The carrier composition of any one of the preceding claims, wherein the

glycosaminoglycan having at least one thiol substituent is present in an amount by weight of between 0.1% and 2%.

6. The carrier composition of any one of the preceding claims, wherein the

glycosaminoglycan having at least one thiol substituent is present in an amount by weight of between 0.5% and 1.5%.

7. The carrier composition of any one of the preceding claims, wherein the gelatin having at least one thiol substituent is present in an amount by weight of between 0.01% to 10%.

8. The carrier composition of any one of the preceding claims, wherein the gelatin having at least one thiol substituent is present in an amount by weight of between 0.1% and 2%.

9. The carrier composition of any one of the preceding claims, wherein the gelatin having at least one thiol substituent is present in an amount by weight of between 0.5% and 1.5%.

10. The carrier composition of any one of the preceding claims, wherein the

glycosaminoglycan and the gelatin have a combined number of thiol substituents of between 10,000 and 50,000 per microliter.

11. The carrier composition of any one of the preceding claims, wherein the

glycosaminoglycan and the gelatin have a combined number of thiol substituents of between 20,000 and 45,000 per microliter.

12. The carrier composition of any one of the preceding claims, wherein the

glycosaminoglycan and the gelatin have a combined number of thiol substituents of between 25,000 and 40,000 per microliter.

13. The carrier composition of any one of the preceding claims, wherein the hydrogen-bond acceptor is oxygen.

14. The carrier composition of any one of the preceding claims, wherein the hydrogen-bond acceptor is present in an amount sufficient to create hydrogen bonds with between 10% and 90% of the thiol substituents of the glycosaminoglycan and the gelatin.

15. The carrier composition of any one of the preceding claims, wherein the carrier composition has an average dynamic viscosity of between 1000 centipoise and 5000 centipoise.

16. The carrier composition of any one of the preceding claims, wherein the carrier composition has an average dynamic viscosity of between 2000 centipoise and 4000 centipoise.

17. The carrier composition of any one of the preceding claims, wherein the carrier composition has a storage modulus of at least 5 Pascal.

18. The carrier composition of any one of the preceding claims, wherein the carrier composition has a storage modulus of between 5 Pascal and 25 Pascal.

19. The carrier composition of any one of the preceding claims, wherein the carrier composition has a storage modulus of between 10 Pascal and 20 Pascal.

20. The carrier composition of any one of the preceding claims, wherein the carrier composition has a loss modulus of at least 0.25 Pascal.

21. The carrier composition of any one of the preceding claims, wherein the carrier composition has a loss modulus of between 0.25 Pascal and 0.75 Pascal.

22. The carrier composition of any one of the preceding claims, wherein the carrier composition has a loss modulus of between 0.3 Pascal and 0.5 Pascal.

23. A delivery composition comprising the carrier composition of any one of the preceding claims and a plurality of cells of interest suspended in the carrier composition.

24. The delivery composition of claim 23, wherein the plurality of cells of interest is a plurality of T cells.

25. The delivery composition of claim 24, wherein the plurality of T cells is a plurality of at least one of effector T cells, helper T cells, memory T cells, regulatory T cells, killer T cells, mucosal associated invariant T cells, gamma delta T cells, beta selection T cells, and chimeric antigen receptor (CAR) T cells.

26. The delivery composition of claim 25, wherein the plurality of T cells is a plurality of CAR T cells.

27. The delivery composition of any one of claims 23 to the immediately preceding claim, wherein the plurality of cells of interest is present in an amount of between 100,000 cells of interest per millileter and 1,000,000,000 cells of interest per millileter.

28. The delivery composition of any one of claims 23 to the immediately preceding claim, wherein the plurality of cells of interest is present in an amount of between 1,000,000 cells per milliliter and 100,000,000 cells per milliliter.

29. The delivery composition of any one of claims 23 to the immediately preceding claim, wherein the plurality of cells of interest is present in an amount of between 5,000,000 cells per milliliter and 10,000,000 cells per milliliter.

30. A cell delivery kit comprising an infusion tube and the delivery composition of any one of claims 23 to the immediately preceding claim within the infusion tube.

31. The cell delivery kit of claim 30, wherein the infusion tube is configured to deliver the delivery composition via convection enhanced delivery.

32. The cell delivery kit of claim 30 or 31, wherein the infusion tube is a syringe.

33. The cell delivery kit of any one of claims 30 to the immediately preceding claim, wherein the infusion tube is configured such that the delivery composition exiting the infusion tube is moving horizontally.

34. The cell delivery kit of any one of claims 30 to the immediately preceding claim, wherein the infusion tube is plastic.

35. The cell delivery kit of any one of claims 30 to the immediately preceding claim, wherein the infusion tube has a volume of between 1 milliliter and 100 milliliters.

36. A method comprising:

a) creating an aqueous suspension comprising a glycosaminoglycan having at least one thiol substituent and a gelatin having at least one thiol substituent; and

b) adding a hydrogen-bond acceptor to the aqueous suspension, the hydrogen-bond acceptor capable of simultaneously making at least two hydrogen bonds with thiol substituents, thereby converting the aqueous suspension into a carrier composition.

37. The method of claim 36, the method further comprising:

c) suspending a plurality of cells of interest in the carrier composition.

38. The method of claim 36 or 37, wherein the adding a hydrogen-bond acceptor of step b) includes exposing the aqueous suspension to oxygen for a predetermined time period at a predetermined temperature.

39. The method of claim 38, wherein the predetermined time period is between 12 hours and 24 hours.

40. The method of claim 38 or 39, wherein the predetermined temperature is between 25 °C and 30 °C.

41. The method of any one of claims 36 to the immediately preceding claim, wherein the creating an aqueous suspension of step a) includes creating a first aqueous suspension comprising the glycosaminoglycan having at least one thiol substituent, creating a second aqueous suspension comprising the gelatin having at least one thiol substituent, and mixing the first and second aqueous suspension to create the aqueous suspension.

42. The method of any one of claims 36 to the immediately preceding claim, wherein the aqueous suspension is created using degassed water.

43. A cell delivery system comprising:

an infusion tube have contained therein the delivery composition of any one of claims 23 to 27;

a pump operatively connected to the infusion tube; and

a catheter in fluid communication with the infusion tube.

44. The cell delivery system of claim 43, wherein the pump is a syringe pump.

45. The cell delivery system of claim 43 or 44, wherein the infusion tube is a syringe.

46. The cell delivery system of any one of claims 43 to the immediately preceding claim, wherein the pump, the infusion tube, and the catheter are configured for convection enhanced delivery.

47. The cell delivery system of any one of claims 43 to the immediately preceding claim, wherein the infusion tube is configured such that the delivery composition exiting the infusion tube is moving horizontally.

48. The cell delivery system of any one of claims 43 to the immediately preceding claim, wherein the infusion tube has a volume of between 1 millileter and 100 millileters.

49. The cell delivery system of any one of claims 43 to the immediately preceding claim, wherein the catheter has a length of between 1 centimeter and 100 centimeters.

50. The cell delivery system of any one of claims 43 to the immediately preceding claim, wherein the catheter has a length of between 5 centimeters and 75 centimeters.

51. The cell delivery system of any one of claims 43 to the immediately preceding claim, wherein the catheter has a length of between 10 centimeters and 50 centimeters.

52. The cell delivery system of any one of claims 43 to the immediately preceding claim, wherein at least a portion of the catheter has a size of between 10 gauge and 20 gauge.

53. The cell delivery system of any one of claims 43 to the immediately preceding claim, wherein at least a portion of the catheter has an internal diameter of between 0.1 millimeters and

4 millimeters.

54. The cell delivery system of any one of claims 43 to the immediately preceding claim, wherein at least a portion of the catheter has an external diameter of between 0.5 millimeters and

5 millimeters.

55. The cell delivery system of any one of claims 43 to the immediately preceding claim, wherein the catheter includes an intracranial catheter, a needle, a length of tubing, or a combination thereof.

56. The cell delivery system of any one of claims 43 to the immediately preceding claim, wherein the infusion tube is plastic.

57. The cell delivery system of any one of claims 43 to the immediately preceding claim, the cell delivery system further comprising a processor operatively coupled to the pump and configured to control the pump.

58. The cell delivery system of claim 57, the cell delivery system further comprising a memory having stored thereon instructions that, when executed by the processor, cause the processor to direct the pump to execute an infusion routine.

59. The cell delivery system of claim 58, wherein the infusion routine causes the pump to deliver the delivery composition at a constant delivery rate.

60. The cell delivery system of claim 58, wherein the infusion routine causes the pump to deliver the delivery composition at a variable delivery rate.

61. A method comprising:

a) infusing a target location within a subject with the delivery composition of any one of claims 23 to 27, thereby delivering a desired amount of cells of interest to the target location.

62. The method of claim 61, wherein the infusing of step a) is via convection enhanced delivery.

63. The method of claim 61 or 62, wherein the target location is location in the subject's cranium and the infusing is intracranial.

64. The method of any one of claims 61 to the immediately preceding claim, wherein the infusing of step a) is at a rate of between 100 microliters per hour and 10 millileters per hour.