Cell-free implants for in VIVO recruitment, activation, and reprogramming of immune cells

Abstract

Cell-free implants for in vivo recruitment, activation, and reprogramming of immune cells are described. Before implantation, the implants include columnar pores, chemoattractants, immune cell activating factors, and reprogramming factors.

Description

CELL-FREE IMPLANTS FOR IN VIVO RECRUITMENT, ACTIVATION, AND REPROGRAMMING OF IMMUNE CELLSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 548,701 filed February 1 , 2024, which is incorporated herein by reference in its entirety as if fully set forth herein.REFERENCE TO SEQUENCE LISTING

[0002] The Sequence Listing associated with this application is provided in XML format in lieu of a paper copy and is hereby incorporated by reference into the specification. The name of the file containing the Sequence Listing is 3DD4480.xmL The file is 209,961 bytes, was created on January 27, 2025, and is being submitted electronically via Patent Center.FIELD OF THE DISCLOSURE

[0003] The present disclosure provides cell-free implants for in vivo recruitment, activation, and reprogramming of immune cells. Before implantation, the cell-free implants include columnar pores, chemoattractants, immune cell activating factors, and reprogramming factors.BACKGROUND OF THE DISCLOSURE

[0004] Cancer immunotherapy describes a field in which a subject’s immune system is used to treat cancer. One immunotherapy approach involves reprogramming subject-derived immune cells (e.g., T cells) to express chimeric antigen receptors (CAR). CAR are engineered to bind selected target antigens on cancer cells, and when the CAR binds the selected target antigen, it activates the immune cell to kill the cancer cell.

[0005] Therapies that employ such CAR-expressing immune cells have consistently produced positive results in subjects with hematologic malignancies. However, when it comes to solid tumors, the effectiveness of these therapies has been limited.

[0006] Solid tumors include 90% of all cancers, but, thus far, CAR-based immunotherapies have had limited success in eradicating them. Treatment with tumor-infiltrating lymphocytes (TILs) is a promising experimental cell therapy being developed for solid tumors. In this approach, TILs are removed from a subject’s tumor and grown in large numbers in a laboratory. These expanded lymphocytes are then infused back into the subject to help the immune system kill the cancer cells. However, the tissue collection and cell production process is labor-intensive, expensive, and time-consuming (and needs to be repeated for each individual subject), which means thatTIL therapy is available to relatively few subjects and only at select research institutions and companies. Moreover, removal of enough viable tumor tissue from subjects is difficult for smaller lesions, such as pancreatic cancer, or if tumors are unresectable. Some solid tumor types, such as gastrointestinal cancers, are difficult to resect cleanly, without microbial contamination. Even when tumors can be surgically removed, most of the lymphocytes they contain do not recognize relevant tumor antigens. Thus, clinicians must first identify and enrich tumor-specific TIL clones in vitro, a process that takes weeks, and functionally exhausts the T cells. Because of these barriers it is not possible to widely adopt TIL therapy in its current form.

[0007] Another approach to target solid tumors has been the development of implants that house immune cells. For example, the concept of implantable polymer devices as delivery platforms for ex vivo manufactured CAR-T cells for the treatment of solid malignancies is described in Stephan et al., Nat Biotechnol 33, 97-101 (2015); Smith et al., J Clin Invest 127, 2176-2191 (2017); and Coon et al., Nat Biomed Eng 4, 195-206 (2020). Based on these publications, various other implantable and injectable biomaterials that functionally support adoptively transferred immune cells and improve the effectiveness of CAR-T cell therapy have been developed. See, e.g., Kim et al., Biomater Res 27, 60 (2023); Adu-Berchie et al., Nat Commun 14, 3546 (2023); van Schaik et al., Biomed Pharmacother 162, 1 14665 (2023); Bhatta et al., Biomaterials 293, 121972 (2023); Yu etal., Nat Commun 13, 6357 (2022); Grosskopf etal., Sci Adv 8, eabn8264 (2022); Sharon et al., Oncotarget 12, 1201 -1213 (2021 ); Hu et al., Nat Biomed Eng 5, 1038-1047 (2021 ); Weiden et al., Front Immunol 9, 2798 (2018). Even more recently, Agarwalla and colleagues described an approach to substantially reduce processing time of CAR-T cell manufacturing by loading subject-derived T cells and viral particles encoding CAR into an alginate implant. Agarwalla et al., Nat Biotechnol 40, 1250-1258 (2022). While each of these developments represents an important advance, they all suffer the same drawback of TIL based therapies in terms of requiring the removal of a subject’s immune cells that are then expanded ex vivo and re-infused back into the subject. These developments are similarly restricted as labor-intensive, expensive, timeconsuming, and available to relatively few subjects at select research institutions and companies.SUMMARY OF THE DISCLOSURE

[0008] The current disclosure provides a fundamentally different approach to the use of implants to treat solid tumors, in that the implants are cell-free at the time of implantation and until naturally infiltrated by a subject’s immune cells at a tumor implantation site. Because the implants are cell- free before implantation there is no need to reprogram a subject’s immune cells ex vivo. Thus, the disclosed implants dramatically reduce costs and time to treatment by not relying on subject-derived ex vivo cell manufacturing processes and not relying on -80eC cold chain storage. The disclosure represents a distinctly new approach in the use of implants to treat solid tumors. This new approach can be made vastly more available to subjects ,at a wider range of care centers.

[0009] The cell-free implants disclosed herein are designed to recruit, activate, and reprogram a subject’s immune cells once the implant is implanted at a tumor site. The results described here establish that implants can be successfully engineered to produce effective target antigenrecognizing host immune cells in vivo.

[0010] In particular embodiments, before implantation, the disclosed cell-free implants for in vivo recruitment, activation, and reprogramming of immune cells include a scaffold with columnar pores, chemoattractants, immune cell activating factors, and reprogramming factors.BRIEF DESCRIPTION OF THE FIGURES

[0011] Some of the drawings submitted herein may be better understood in color. Applicant considers the color versions of the drawings as part of the original submission and reserves the right to present color images of the drawings in later proceedings.

[0012] FIG. 1. Schematic illustrating how a rationally designed implant recruits, reprograms, expands and releases host tumor-infiltrating lymphocytes (TILs). A porous collagen scaffold was functionalized with an immune cell chemoattractant, a vector encoding a tumor-specific Chimeric Antigen Receptor (CAR), and stimulatory anti-CD3 / CD28 antibodies. Following implantation, these implants effectively recruit host T cells, reprogram them to express tumor recognizing receptors, and rapidly expand and release them into the surrounding tumor tissue.

[0013] FIGs. 2A-2I. Identifying a lead implant formulation. (2A-2C) Optimizing T-cell recruitment. Collagen scaffold implants (either controls or loaded with chemoattractant) were placed at the center of three-dimensional collagen gels containing human T lymphocytes fluorescently tagged with DiD' (1 ,1 '-Dioctadecyl-3, 3,3', 3'-Tetramethylindodicarbocyanine, 4-Chlorobenzenesulfonate Salt). After 72 hours, following implant removal, the implants were transferred into 6-well culture chambers to quantify fluorescence signals. (2A) In vivo imaging system (IVIS) fluorescence imaging of DiD' T-cell signals emanating from isolated implants. (2B) Graph showing mean fluorescence intensities. Horizontal lines indicate mean values, and error bars represent standard deviation of the mean. Pairwise differences in fluorescent signal between the groups were statistically analyzed with the Wilcoxon rank-sum test (two-sided). (2C) Confocal microscopy images of a representative implant functionalized with C-C motif chemokine ligand 21 (CCL21 ) on day 0 (left panel) and after 3 days (right panel). DiD' signal is shown in green. Scale bar: 1 mm. (2D-2F) T-cell reprogramming. (2D) Schematic of the experimental design. Humanantibodies recognizing CD3 and CD28 at a ratio of 1 =1 were incorporated into collagen scaffold implants using lyophilization. Implants were then hydrated with a mixture of lentivirus (encoding Green Fluorescent Protein; GFP) and naive human T cells at the indicated MOIs (ratios of infectious lentiviral particles to cells in culture). In the control group, T cells were transduced with an equal dose of lentiviral vector and stimulatory antibodies in suspension (implant-free). Gene transfer was measured 7 days later by flow cytometry. (2E) Representative flow cytometry plots showing gene transfer. Summary plots of gene transfer efficiencies at various MOIs following transduction in suspension versus implant are shown in (2F). Each bar graph displays the mean ± SD. (2G-2I) T-cell expansion and release. (2G) Representative flow cytometry plots measuring the activation marker CD25 in T cells that were cultured in implants functionalized with anti- CD3 / CD28 antibodies, following a 6-day test period. Total T-cell numbers after 7 days and 14 days are summarized on the right. (2H) In vitro cell-killing assays conducted by adding escalating doses of untransduced or implant-released CAR-T cells into a culture of bioluminescent tumor targets. To generate tumor-specific CAR-T cells, implants were functionalized with lentiviral vector encoding the CAR specific for the tumor antigen ROR1 (Receptor tyrosine kinase-like orphan receptor 1 ). Released T cells were incubated with luciferase-expressing ROR1 + breast tumor target cells and bioluminescence signals were quantitated by MS imaging 24 hours later and summarized as bar graphs in (2I). Pairwise differences in bioluminescence signal between the groups were statistically analyzed with the Wilcoxon rank-sum test (two-sided).

[0014] FIGs. 3A-3C. Implant-embedded lentivirus retains more than 80% activity after lypophilization in the presence of sucrose. (3A-3C) Schematics showing the experimental groups. To investigate conditions enabling lyophilization of lentivirus-functionalized collagen implants while retaining sufficient activity, GFP-encoding virus, was suspended in a phosphate buffered saline (PBS) solution (as shown in 3B) or in a 1 M sucrose solution as a cryoprotectant (see 3C) prior to loading the virus onto collagen implants functionalized with anti-CD3 / CD28 antibodies. Following lyophilization, implants were hydrated with a solution of naive human T cells and cultured for 7 days. For direct comparison, an equal dose of fresh (nonlyophilized) lentivirus was added with T cells to anti-CD3 / CD28 antibody-functionalized implants (illustrated in 3A). Representative flow cytometry plots measuring gene transfer after 7 days are shown on the right. Mean transduction efficiencies (±SD) for each experimental group are shown at the bottom of each fluorescence-activated cell sorting (FACS) plot. N = 9 biologically independent samples / group.

[0015] FIGs. 4A-4E. Implants efficiently transduce host T cells with tumor-specific CAR transgenes. (4A) Schematic representation of the experimental timeline. (4B) Representativesequential bioluminescence imaging of implant-mediated in vivo reprogramming of host T cells into tumor-reactive CAR-T cells. (4C) Overview graph displaying the bioluminescence photon counts following implantation. Each line represents one animal. (4D) Flow cytometry of cells at implantation sites and in spleens of mice 8 days post implantation. The three profiles for each group shown here are representative of two independent experiments including 5 mice per group. Percentages of CAR+ T cells are summarized in (4E). Each symbol represents an individual mouse (n = 10). Horizontal lines indicate the mean.

[0016] FIGs. 5A-5C. Implant-mediated in vivo reprogramming of breast cancer-specific CAR T- cells can improve survival of mice with established disease. (5A) Schematic representation of the experimental timeline. (5B) Growth of subcutaneous MDA-MB-468 ROR1 + breast tumors (tumor volume) of each mouse following implant or mock-treatment. Each symbol represents an individual mouse at a given time point (n = 10). Solid lines intersect the medians of each group at each time point. N.s stands for not significant. (5C) Kaplan-Meier survival curves. Statistical analysis was performed using the log-rank test. N = 11 biologically independent animals.

[0017] FIG. 6. Secondary electron imaging (LEI) scanning electron microscopy (SEM) image of scaffold having columnar pores. The columnar porous architecture permits cells and nutrients to flow completely through the interpenetrating pores and provides an increased surface area for cell attachment, growth, and migration.

[0018] FIG. 7. Sequences supporting the disclosure including lgG4 hinge (SEQ ID NO: 163), lgG4 hinge coding sequence (SEQ ID NOs: 164-166), lgG4-CH2 domain (SEQ ID NOs: 167 and 168), lgG4-CH2 domain coding sequence (SEQ ID NOs: 169 and 170), lgG4 CH3 domain (SEQ ID NO: 171 ), lgG4-CH3 coding sequence (SEQ ID NOs: 172 and 173), CD28 Transmembrane Domain (SEQ ID NOs: 174-176), CD28TM coding sequence (SEQ ID NOs: 177-180), CD3 signaling domain (SEQ ID NOs: 181 -183), CD3zeta signaling domain coding sequence (SEQ ID NOs: 184 and 185), 4-1 BB costimulatory domain (SEQ ID NOs: 186-188), 4-1 BB signaling coding sequence (SEQ ID NOs: 189-191), Thoseaasigna Virus 2A (T2A) Peptide (SEQ ID NOs: 192 and 193), Porcine Teschovirus-1 2a (P2A) Peptide (SEQ ID NO: 194), Equine Rhinitis A Virus (ERAV) 2A (E2A) Peptide (SEQ ID NO: 195), Foot-And-Mouth Disease Virus 2A (F2A) Peptide (SEQ ID NO: 196), T2A coding sequence (SEQ ID NOs: 197 and 198), EGFRt (SEQ ID NO: 199), EGFRt coding sequence (SEQ ID NO: 200), and tCD19 coding sequence (SEQ ID NO: 201 ).DETAILED DESCRIPTION

[0019] Cancer immunotherapy describes a field in which a subject’s immune system is used to treat cancer. One immunotherapy approach involves reprogramming subject-derived immunecells (e.g., T cells) to express chimeric antigen receptors (CAR). CAR are engineered to bind selected target antigens on cancer cells, and when the CAR binds the selected target antigen, it activates the immune cell to kill the cancer cell. Therapies that employ such CAR-expressing immune cells have consistently produced positive results in subjects with hematologic malignancies. However, when it comes to solid tumors, the effectiveness of these therapies has been limited.

[0020] Solid tumors include 90% of all cancers, but, thus far, CAR-based immunotherapies have had limited success in eradicating them. Treatment with tumor-infiltrating lymphocytes (TILs) is another promising experimental cell therapy being developed for solid tumors. In this approach, TILs are removed from a subject’s tumor and grown in large numbers in a laboratory. These expanded lymphocytes are then infused back into the subject to help the immune system kill the cancer cells. However, the tissue collection and cell production process is labor-intensive, expensive, and time-consuming (and needs to be repeated for each individual subject), which means that TIL therapy is available to relatively few subjects and only at select research institutions and companies. Moreover, removal of enough viable tumor tissue from subjects is difficult for smaller lesions, such as pancreatic cancer, or if tumors are unresectable. Some solid tumor types, such as gastrointestinal cancers, are difficult to resect cleanly, without microbial contamination. Even when tumors can be surgically removed, most of the lymphocytes they contain do not recognize relevant tumor antigens. Thus, clinicians must first identify and enrich tumor-specific TIL clones in vitro - a process that takes weeks, and functionally exhausts the T cells. Because of these barriers it is not possible to widely adopt TIL therapy in its current form.

[0021] Another approach to target solid tumors has been the development of implants that house immune cells. For example, the concept of implantable polymer devices as delivery platforms for ex vivo manufactured CAR-T cells for the treatment of solid malignancies is described in Stephan et al., Nat Biotechnol 33, 97-101 (2015); Smith et al., J Clin Invest 127, 2176-2191 (2017); and Coon et al., Nat Biomed Eng 4, 195-206 (2020). Based on these publications, various other implantable and injectable biomaterials that functionally support adoptively transferred immune cells and improve the effectiveness of CAR-T cell therapy have been developed. See, e.g., Kim et al., Biomater Res 27, 60 (2023); Adu-Berchie et al., Nat Commun 14, 3546 (2023); van Schaik et al., Biomed Pharmacother 162, 1 14665 (2023); Bhatta et al., Biomaterials 293, 121972 (2023); Yu etal., Nat Commun 13, 6357 (2022); Grosskopf etal., Sci Adv 8, eabn8264 (2022); Sharon et al., Oncotarget 12, 1201 -1213 (2021 ); Hu et al., Nat Biomed Eng 5, 1038-1047 (2021 ); Weiden et al., Front Immunol 9, 2798 (2018). Even more recently, Agarwalla and colleagues described an approach to substantially reduce processing time of CAR-T cell manufacturing by loadingsubjects-derived T cells and viral particles encoding CAR into an alginate implant. Agarwalla et al., Nat Biotechnol 40, 1250-1258 (2022). While each of these developments represents an important advance, they all suffer the same drawback of TIL based therapies in terms of requiring the removal of a subject’s immune cells that are then expanded ex vivo and re-infused back into the subjects. These developments are similarly restricted as labor-intensive, expensive, timeconsuming, and available to relatively few subjects at select research institutions and companies.

[0022] The current disclosure provides a fundamentally different approach to the use of implants to treat solid tumors, in that the implants are cell-free at the time of implantation and until naturally infiltrated by a subject’s immune cells at a tumor implantation site. Because the implants are cell- free before implantation there is no need to reprogram a subject’s immune cells ex vivo. The disclosed implants dramatically reduce costs and time to treatment by not relying on subject- derived ex vivo cell manufacturing processes and not relying on -80eC cold chain storage. The disclosure represents a distinctly new approach in the use of implants to treat solid tumors. This new approach can be made vastly more available to subjects, at a wider range of care centers.

[0023] The cell-free implants disclosed herein are designed to recruit, activate, and reprogram a subject’s immune cells once the implant is implanted at a tumor site. The results described here establish that implants can be successfully engineered to reprogram antigen-recognizing capabilities into host immune cells in vivo, resulting in anti-cancer activity.

[0024] In particular embodiments, before implantation, the disclosed cell-free implants for in vivo recruitment, activation, and reprogramming of immune cells include a scaffold having columnar pores, chemoattractants, immune cell activating factors (ICAF), and reprogramming factors (RF).

[0025] In particular embodiments, the cell-free implant recruits immune cells in vivo, activates and expands the immune cells and reprograms them to express a therapeutic protein.

[0026] Referring to FIG. 1 , in particular embodiments, the cell-free implant recruits host T cells in vivo, reprograms the T cells to express tumor-specific receptors (e.g., CAR or engineered T cell receptors (eTCR)), expands the T cells, and allows dispersion of the T cells from the implant in proximity to a solid tumor. In these exemplary embodiments, the implant is based on a porous collagen scaffold (inherently supporting rapid T-cell migration (Sadjadi etal., Biophys J 119, 2141 - 2152, 2020), functionalized with a potent chemoattractant (e.g., C-C motif chemokine ligand 21 (CCL21 )) to recruit host T cells, including TILs. Collagen, a biocompatible material with low immunogenicity, is widely used in medical applications such as wound healing, tissue engineering, reconstructive surgeries, bone grafts, and cosmetic surgeries. Thus, protocols for large-scale production of medical-grade collagen implants are in place. To efficiently reprogram and expand recruited T cells within the implant, vectors encoding a tumor-specific receptor (e.g.,CAR (Mitra et al., Front Immunol 14, 1188049 (2023) or eTCR) and stimulatory anti-CD3 / CD28 antibodies are co-encapsulated within the implant. All of these components can be manufactured at large-scale and without the need to prepare a cellular product for each individual subject. Further, the implant can be frozen and stored until use.

[0027] In particular embodiments, collagen sponges composed of Type I bovine collagen can be purchased, for example, from Advanced Biomatrix (Cat#: 5135-EA) for use as an implant scaffold having columnar pores.

[0028] The overall diameter of an implant (e.g., a collagen sponge) can be sized according to a solid tumor treatment site. In particular embodiments, an implant can have an overall diameter of 5 mm-100 mm, 10-95 mm, 15-90 mm, 20-85 mm, 25-80 mm, 30-75 mm, 35-70 mm, 40-65 mm, 45-60 mm, 50-55 mm, 10-30 mm, or 15-25 mm. In particular embodiments, an implant can have an overall diameter of 16 mm, 17 mm, 18 mm, 19 mm, 20 mm, 21 mm, 22 mm, 23 mm, 24 mm, 25 mm, 26 mm, or more than 26 mm. In particular embodiments, an implant can have an overall diameter of 21 mm.

[0029] Implants (e.g., collagen sponges) disclosed herein have a columnar pore structure (see, e.g., FIG. 6). Within a columnar pore structure, in certain embodiments, individual pores are not interconnected and each pore has a beginning and an end on the surface of the implant. In certain embodiments, no more than 20% of individual pores are interconnected and at least 60% of pores have a beginning and an end on the surface of the implant. In certain embodiments, no more than 15% of individual pores are interconnected and at least 70% of pores have a beginning and an end on the surface of the implant. In certain embodiments, no more than 10% of individual pores are interconnected and at least 80% of pores have a beginning and an end on the surface of the implant. In certain embodiments, no more than 5% of individual pores are interconnected and at least 90% of pores have a beginning and an end on the surface of the implant. In certain embodiments, no more than 2% of individual pores are interconnected and at least 95% of pores have a beginning and an end on the surface of the implant. When a pore has a beginning and an end on the surface of an implant, the path from the beginning to the end is straight or substantially straight. A columnar pore with a straight path includes a passage from beginning to end that does not deviate from 180e. In particular embodiments, a columnar pore with a substantially straight path includes a passage from beginning to end that does not deviate by more than 10eor more than 20sfrom 180e. In particular embodiments, a columnar pore with a substantially straight path from beginning to end does not include a passage that requires a 90eor greater turn. These described columnar pore structures should be contrasted with the interconnected pore networks of many previously developed implants.

[0030] In particular embodiments, columnar pores of an implant have an average cross-sectional diameter of 100-400 pm. In particular embodiments, columnar pores of an implant have a cross- sectional diameter of 100-400 pm with an average cross-sectional pore diameter of 200 pm. In particular embodiments, columnar pores of an implant have an average cross-sectional pore diameter of 150 pm, 160 pm, 170 pm, 180 pm, 190 pm, 200 pm, 210 pm, 220 pm, 230 pm, 240 pm, or 250 pm.

[0031] In particular embodiments, chemoattractant and ICAF are added to an implant. Implants having a chemoattractant and an ICAF can be referred to as functionalized implants. RF can then be added into functionalized implants. In particular embodiments, implants are lyophilized and stored. In particular embodiments, functionalized implants are lyophilized, stored, and rehydrated with a media including an RF, and optionally a nutrient. In particular embodiments, functionalized implants that have been lyophilized can be referred to as lyophilized implants.

[0032] In particular embodiments, a method of preparing an implant includes adding a chemoattractant and ICAF to the implant. In particular embodiments, the functionalized implant is lyophilized and stored at, for example, 4eC. Before use (e.g., on the day of use), the implant can be rehydrated in a media including RF, and optionally sucrose.

[0033] In particular embodiments, chemoattractants can include CCL21 (available from, for example, Biolegend, Cat#: B297009), CCL3 (available from, for example, Biolegend, Cat#: B224650), CCL4 (available from, for example, Biolegend Cat#: B324087), and / or CXCL10 (available from, for example, Biolegend Cat#: B317192).

[0034] In particular embodiments, ICAF can include anti-human CD3 and anti-human CD28 antibody (available from, for example, BioXcell, Cat#: BE-0001 -2 and BE0248, respectively).

[0035] In particular embodiments, RF can include a freshly thawed CAR-encoding lentivirus.

[0036] Particular embodiments of implants disclosed herein include, at the time of implantation into a subject, a Type I collagen scaffold having columnar pores, CCL21 , anti-CD3 and anti-CD28 binding domains, and lentiviral RF.

[0037] As primary tumor debulking surgery remains the standard of care for most types of solid tumor cancers and is often the first intervention a subject receives, implants disclosed herein can be surgically implanted where a tumor was just removed or situated at advanced unresectable tumors. This timing and placement ensures that the reprogramming of immune cells attracted to the implant to express a tumor-specific receptor begins immediately, while the subject recovers from surgery.

[0038] In particular embodiments, disclosed implants substantially reduce the required vector dose per subject as they concentrate a small number of viral particles and enhance gene transferinto T cells (FIG. 2D-2F), so the same reprogramming can be achieved with only a fraction of the vector dose.

[0039] Aspects of the disclosure are now described with additional options and details as follows: (i) Implant Materials; (ii) Chemoattractants; (iii) Immune Cell Activating Factors (ICAF); (iv) Reprogramming Factors (RF); (v) Expressed Molecules; (vi) Optional Additional Immune Stimulants; (vii) Implant Preparation; (viii) Methods of Use; (ix) Kits; (x) Exemplary Embodiments; (xi) Experimental Example; and (xii) Closing Paragraphs. These headings are provided for organizational purposes only and do not limit the scope or interpretation of the disclosure.

[0040] (i) Implant Materials. In particular embodiments, an implant disclosed herein includes a scaffold. A “scaffold” refers to a three dimensional structure designed to provide a supportive framework for cell attachment, growth, and / or proliferation. The material that forms the scaffold of implants disclosed herein can be constructed from a variety of materials that allow formation of columnar pores, although collagen is preferred.

[0041] Collagen is the principal extracellular structural protein in a mammal. At least seven types of mammalian collagen have been described. Their common characteristic is a three stranded helix, including 3 polypeptide chains, called alpha-chains. All of the alpha chains have the same configuration, but differ in the composition and sequence of their amino acids, leading to different types of alpha chains. However, all chains have glycine in every third position of the amino acid sequence, allowing the helical conformation to occur.

[0042] Representative collagen materials include placental collagen, recombinant human collagen, tissue engineered human-based collagen, porcine collagen, bovine collagen, autologous collagen, collagen fibers, and human tissue collagen matrix.

[0043] Any type of collagen may be used in the implants disclosed herein provided that it forms columnar pores. In some embodiments, collagen Type I, collagen Type II, collagen Type III, collagen Type IV, collagen Type VI, or a combination thereof, may be used. A collagen may be derived from cell culture, animal tissue, or recombinant means, and may be derived from human, porcine, or bovine sources. Some embodiments include collagen derived from human fibroblast culture. Some embodiments include collagen that has been denatured to gelatin. In particular embodiments, collagen is human or bovine collagen.

[0044] Collagen is available from multiple commercial sources (e.g., Advanced Biomatrix; ZYDERM1®, ZYDERM2®, ZYPLAST®, COSMODERM I®, or COMSMOPLAST® from Allergan; ARTEFILL® from Artes Medical; EVOLENCE® from ColBar Life Science; FG-5017 from Fibrogen; and ISOLAGEN®).

[0045] In some embodiments, a collagen scaffold includes a mixture of collagen extracted fromanimal / human tissues and collagen obtained via commercially available sources at a ratio of 1 :9, 1 :4, 3:7, 2:3, 1 :1 , 3:2, 7:3, 4:1 , or 9:1.

[0046] In some embodiments, the collagen includes a mixture of Type I and Type III collagen. Type I collagen is the most abundant collagen of the human body and is the principal extracellular material present in scar tissue, tendons, skin, artery walls, the endomysium of myofibrils, fibrocartilage, and the organic part of bones and teeth. Type I collagen is composed of three alphal -chains and one alpha2-chain. When clinicians speak of “collagen,” they are usually referring to Type 1.

[0047] Type III collagen is present in rapidly growing tissue, particularly juvenile and healing skin. This is the collagen of granulation tissue and is produced quickly by young fibroblasts before the tougher Type I collagen is synthesized. Type III collagen has inter-chain disulfide bonds, whereas Type I collagen does not. The inter-chain disulfide bonds are one type of crosslinking and can provide additional molecular stability. An increase in crosslinked type collagen may result in a longer persistence of the collagen material when used in a subject as compared to lesser or no crosslinked materials. Crosslinked collagenous tissue, in certain embodiments, as compared to lesser or no crosslinked tissue may have one or more of the following characteristics: increased tensile or structural strength, increased resistance to enzymatic degradation, reduced antigenicity, and reduced immunogenicity.

[0048] In particular embodiments, multiple types of collagen can be used in the collagen solution. For example, the collagen solution can include a first type of collagen and a second type of collagen. In particular embodiments, a collagen solution includes a first type of collagen, a second type of collagen, and a third type of collagen. In particular embodiments, a collagen solution includes a first type of collagen, a second type of collagen, a third type of collagen, and a fourth type of collagen. In some embodiments, collagen having a high ratio of Type III collagen to Type I collagen can be used as they more closely mimic endogenous tissue. In certain embodiments, compositions containing Type III collagen is used in reducing the formation of excess scar tissue in wound healing by signaling to endogenous skin cells that there is sufficient scar tissue or young tissue already formed. In some embodiments, increasing the ratio of Type I to Type III collagen is used to enhance the durability and strength of the implant.

[0049] In particular embodiments, the ratio of a first type of collagen to a second type of collagen is equal to or greater than 1 :1 , 1 :2, 1 :3, 1 :4, 1 :5, 1 :6, 1 :7, 1 :8, 1 :9, 1 :10, 1 :11 , 2:1 , 2:3, 2:5, 2:7, 2:9, 2:11 , 3:1 , 3:2, 3:4, 3:5, 3:7, 3:8, 3:10, 3:11 , 4:1 , 4:3, 4:5, 4:7, 4:9, 4:11 , 5:1 , 5:2, 5:3, 5:4, 5:6, 5:7, 5:8, 5:9, 5:1 1 , 6:1 , 6:5, 6:7, 6:11 , 7:1 , 7:2, 7:3, 7:4, 7:5, 7:6, 7:8, 7:9, 7:10, 7:11 , 8:1 , 8:3, 8:5, 8:7, 8:9, 8:11 , 9:1 , 9:2, 9:4, 9:5, 9:7, 9:8, 9:10, 9:1 1 , 11 :9, 13:7, 17:3, or 19:1. In certainembodiments, the ratio of Type III to Type I collagen is equal to or greater than 3:7, 2:3, 9:11 , 1 :1 , 11 :9, 3:2, 13:7, 7:3, 3:1 , 4:1 , 17:3, 9:1 , or 19:1 . In one embodiment, the ratio of Type III to Type I collagen is 43:57.

[0050] In particular embodiments, the collagen includes a first type of collagen and a second type of collagen. In particular embodiments, the first type of collagen is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% of the weight or volume of the collagen component . In particular embodiments, the second type of collagen is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% of the weight or volume of the collagen component. In certain embodiments, the collagen includes Type I and Type III collagen. In certain embodiments, the Type III collagen is at least 30% of the weight or volume of the collagen component. In certain embodiments, the Type III collagen is 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% of the weight or volume of the collagen component. In certain embodiments, Type III collagen is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of the weight or volume of the collagen component.

[0051] In particular embodiments, the total collagen concentration is at least 5 mg / mL, at least 10 mg / mL, at least 15 mg / mL, at least 20 mg / mL, at least 25 mg / mL, at least 30 mg / mL, at least 35 mg / mL, at least 40 mg / mL, or at least 45 mg / mL in saline solution. In some embodiments, Type I and Type III collagen are present in a 1 :1 ratio. In some embodiments, the Type I and Type III collagen are present in a 1 :1 ratio, with a total collagen concentration of 35 mg / mL in saline solution that is isotonic relative to tissue at the implantation site.

[0052] In some embodiments, the collagen is treated to increase the level of crosslinking present as compared to untreated collagen. In one embodiment, increasing the level of crosslinking is achieved by heat, gamma irradiation, or contact with a synthetic or natural crosslinking agent. Example of crosslinking agents include glutaraldehyde, polyethylene glycol, and formaldehyde. In particular embodiments, the first type and second type of collagen is subjected to further crosslinking prior to use. In certain embodiments, only Type I or Type III collagen is subjected to further crosslinking prior to use.

[0053] In another embodiment, either one or multiple of types of collagen may be crosslinked. In another embodiment, a first type of collagen is crosslinked and a second type of collagen is crosslinked. In another embodiment, a first type of collagen is non-crosslinked and a second type of collagen is non-crosslinked. In another embodiment, a first type of collagen is crosslinked and a second type of collagen is non-crosslinked. In another embodiment, a first type of collagen is non-crosslinked and a second type of collagen is crosslinked.

[0054] In another embodiment, either one or both of Type I or Type III collagen may be crosslinked. In another embodiment, Type I collagen is crosslinked and Type III collagen is crosslinked. In another embodiment, Type I is non-crosslinked and Type III collagen is noncrosslinked. In another embodiment, Type I collagen is crosslinked and Type III collagen is noncrosslinked. In another embodiment, Type I collagen is non-crosslinked and Type III collagen is crosslinked.

[0055] Insoluble collagen, if treated with a proteolytic enzyme, such as pepsin, undergoes fission at the intermolecular crosslinks and becomes soluble in dilute acids. During this treatment with pepsin, the telopeptide groups at both terminals of each collagen molecule are digested, thus leaving collagen with no telopeptide terminal ends. The collagen thus released is called atelocollagen. Since the telopeptide moiety is primarily responsible for antigenicity of collagen, atelocollagen has little antigenicity if any, which makes it very suitable for use as an implant.

[0056] In some embodiments, the collagen scaffold contains entirely atelocollagen. In other embodiments, the collagen scaffold contains a fraction of collagen with telopeptide groups at one or both terminals of the molecule. In some embodiments, the collagen scaffold includes 50% pure human Type I atelocollagen and 50% pure human Type III atelocollagen.

[0057] The stable collagen scaffold maintains at least one of, or all of, the following aspects after effective sterilization and / or prolonged storage: appearance, pH for use in a subject, extrusion force and / or rheological characteristics, concentration, sterility, and osmolarity.

[0058] In particular embodiments, the desired concentration of collagen in the collagen scaffold includes 1 -1000 mg / mL. In particular embodiments, the desired concentration of collagen in the collagen scaffold includes 1 -100 mg / mL. In particular embodiments, the desired concentration of collagen in the collagen scaffold includes 1 -10 mg / mL. In particular embodiments, the desired concentration of collagen in the collagen scaffold includes 1 -5 mg / mL.

[0059] In particular embodiments, the implant material includes at least one biocompatible polymer. Exemplary biocompatible polymers include agar, agarose, alginate, alginate / calcium phosphate cement (CPC), beta-galactosidase (p-GAL), (1 ,2,3,4,6-pentaacetyl a-D-galactose), cellulose, chitin, chitosan (see, for example, Levengood et al., J. Mater. Chem. B, 2014, 2, 3161 - 3184 describing porous chitosan implants), collagen, elastin, gelatin, hyaluronic acid collagen, hydroxyapatite, poly(3-hydroxybutyrate-co-3-hydroxy-hexanoate) (PHBHHx), poly(lactide), poly(caprolactone) (PCL), poly(lactide-co-glycolide) (PLG), polyethylene oxide (PEG), poly( lactic- co-glycolic acid) (PLGA; see, for example, Omar et al., Sci. Transl. Med. 2009 Nov. 25; 1 (8): 8ra19 describing porous PLGA implants), polypropylene oxide (PPO), poly(vinyl alcohol) (PVA), silk, soy protein, and soy protein isolate, alone or in combination with any other polymercomposition, in any concentration and in any ratio. Blending different polymer types in different ratios using various grades can result in characteristics that borrow from each of the contributing polymers. Various terminal group chemistries can also be adopted.

[0060] In particular embodiments, alginate is used as a scaffold material, either separately or in combination with one or more other materials. Alginate is easily processed, water soluble, and non-immunogenic. Alginate is a biodegradable anionic polysaccharide with free hydroxyl groups that offer easy gelling. In alternative embodiments, the polymer may be a polyelectrolyte complex mixture (PEC) formed from a 1 :1 solution of alginate and chitosan.

[0061] In particular embodiments, an implant may be formed from an alginate / calcium carbonate / glucono-delta-lactone mixture, such as 0.5-5% alginate, 0.5-15 g / L calcium carbonate, and 1 -50 g / L gluconon-delta-lactone in a ratio of 2:1 :1 (alginate:CaCO3:GDL). Polymer structures may also include varying amounts of gelatin in combination with varying amounts of alginate. Depending on the materials and material ratios in mixture, the structures may optionally be crosslinked. Collagen / alginate hybrid implants such as those described in Lee at aL, Chem. Mater., 2012, 24(5), 881-891 can also be used.

[0062] In particular embodiments, polymer solutions having varying amounts of polymer dissolved in an acidic solution can be used to form the scaffolds described herein. The concentration of the acid can be adjusted depending on the amount of polymer dissolved. In one aspect, the acidic solution is 1% (v / v) acetic acid. In particular embodiments, the amount of polymer in solution is between 0.5-5% (w / v) and any whole or partial increments therebetween. For example, the amount of polymer in solution (w / v) can be 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5% or 5%. In particular embodiments, the amount of polymer in solution is 2.4% (w / v). In other various embodiments, the polymer is dissolved in at least one of water, acid, acetic acid, camphene, or camphene-naphthalene.

[0063] When gelatin is incorporated, the concentration of the acid can be adjusted depending on the amount of gelatin in combination with polymer (in particular embodiments, alginate) that is dissolved. In one aspect, the acidic solution is 1% (v / v) acetic acid. In particular embodiments, the amount of gelatin in solution is between 1-10% (w / v) and any whole or partial increments therebetween. For example, the amount of alginate in solution (w / v) can be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%. In particular embodiments, the amount of alginate in solution is 5.5% (w / v). In particular embodiments, the polymer solution includes a combination of 2.4% (w / v) alginate solution and a 5.5% (w / v) gelatin solution. In other various embodiments, the gelatin in combination with varying amounts of alginate is dissolved in at least one of water, acid, acetic acid, camphene, or camphene-naphthalene.

[0064] Crosslinking can be performed with, for example, calcium chloride and / or calcium carbonate. Calcium carbonate is a slow crosslinker, with samples taking up to several hours to fully crosslink. To increase the speed of the reaction gluconodeltalactone (GDL) can be added. Calcium chloride is a fast crosslinker and the samples will fully gel in a few minutes. In one method, the addition of CaCh to the polymer solution can occur prior to freezing. Other methods include use of a 5.5% (w / v) solution of calcium carbonate+GDL added to the polymer solution prior to initial freezing.

[0065] In particular embodiments, polymer solutions can be degassed in a speed mixer and poured slowly into casts to prevent bubbles from forming. When pipetting the polymer solutions into small molds, air bubble formation can be avoided by placing a micropipette on the open end of mold grooves and repeatedly flushing the entire canal system until the residual air is flushed out.

[0066] A collagen scaffold can be made by many methods known in the art including gelation. For example, a collagen slurry can be prepared with a desired concentration of collagen. The desired concentration can be reached by lyophilizing the collagen slurry to remove water and resuspending the collagen with a measured amount of water, saline or other diluent to reach the desired concentration. A strong acid or base can be added to the slurry to bring the pH to a desired level to inactivate any enzymes or chemicals used in the processing of the slurry that are desired to be inactivated before implantation. Additional acid or base, or a buffer with a pK between 7 and 8, can be then used to bring the pH of the solution to the desired range for implantation. The osmolarity of the slurry can be adjusted to the desired range using a salt solution, or an acid or base. Once the slurry has the appropriate pH and osmolarity, it can be subjected to heat or cold to cause self-assembly or gelation of the collagen. After gelation, lyophilization of the implant can be used to produce a scaffold, sponge or powder. Alternatively, the solution can be maintained as a gel.

[0067] Freeze casting can be used to form the scaffolds described herein. Various polymer solutions can be freeze casted into various sized casts as would be understood by those skilled in the art. The rate of cooling should be controlled as it affects the size and alignment of pores, as well as the formation of ridges. In particular embodiments, the cooling rate can range between 0.1 -100°C per minute (m) and any whole or partial increments therebetween. In particular embodiments, the cooling rate can range between 1 -10°C / m, and any whole or partial increments therebetween. For example, the cooling rate can be 0.1 , 0.5, 1 , 2, 3, 4, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10°C / m.

[0068] Scaffolds can also be manufactured from various materials using 3D bioprinting (see, e.g.,Singh et al., Polymers 8(1 ): 19, 2016; and An et al., Engineering, 1 (2): 261-268, 2015).

[0069] Implants can be sterile for in vivo use. The solution used to make the implant may be sterilized and / or components of the solution may be isolated under sterile conditions using sterile techniques to produce a sterile composition. The final desired properties of the scaffold may be determinative of how the solution is sterilized because some sterilization techniques may affect properties such as viscosity. If certain components of the solution are not to be sterilized, e.g., a collagen is isolated from natural sources, the remaining components can be combined and sterilized before addition of the collagen, or each component can be sterilized separately. The solution can then be made by mixing each of the sterilized components with the collagen that has been isolated using sterile techniques under sterile conditions. Sterilization may be accomplished, for instance, by autoclaving at temperatures on the order of 115°C to 130°C for 30 minutes to 1 hour. Gamma radiation is another method for sterilizing components. Filtration is also possible, as is sterilization with ethylene oxide.

[0070] While the degree of “solidness” may vary from application to application, generally speaking, scaffolds of the present disclosure can exhibit viscosities in the full range of from liquid to gel-like to solid-like. The viscosity of an implant may be lowered by diluting the solution used to make the implant. The viscosity of a lower viscosity implant may be increased to increase gelation. One method to do this is to lyophilize the solution and rehydrate it in a specific amount of water. Another method to do this is to stop the lyophilization process before it is completed, thus only removing part of the water from the gel and concentrating the implant material. Gelation is the change in viscosity from a fluid-like composition to a solid or gel-like composition. Gelation or viscosity of a solution may also be increased by adding one or more of the following: other extracellular matrix (ECM) molecules, including insoluble collagen, fibrin, fibronectin, and cellulose; non-toxic crosslinking agents, including tissue transglutaminases, lysyl oxidase, fibrin, and fibronectin; and other high viscosity materials with low osmolarity, including alginate and synthetic filler materials.

[0071] (ii) Chemoattractants. In particular embodiments, a chemoattractant is used to attract cells to an implant at a tumor site. In particular embodiments, the attracted cells are immune cells (e.g., T cells) that will be activated and / or reprogrammed.

[0072] In particular embodiments, a cell type in addition to the one that will be activated and reprogrammed can be attracted to an implant. For examples, the second type of attracted cells could include cells that support the activity of the attracted cell type that was activated and reprogrammed. For example, when T cells are activated and reprogrammed, one could also recruit NK cells or invariant NK (iNKT) cells to support tumor-specific T cells. In particularembodiments, more than two cell types can be attracted to an implant.

[0073] In particular embodiments, cells can be attracted to an implant or cells can be attracted to an anatomical site using preconditioning. In particular embodiments, preconditioning includes recruiting immune cells (e.g., T cells) and / or supporting cell types to a tumor site before an implant is administered.

[0074] Particular immune cell chemoattractants are known in the art. By way of example, the following cell / attractant pairs are recognized:

[0075] One of ordinary skill in the art will recognize that different cell types can be attracted / recruited by different chemoattractant treatments. For example, a combination of chemoattractants can be used to recruit T cells and NK cells or T cells and dendritic cells. In particular embodiments, T cells and NK cells can be recruited to the implant by using a chemoattractant including MIP-i p. In particular embodiments, T cells and NK cells can be recruited to the implant by using a chemoattractant including CCL1 , CCL2, CCL3, CCL4, CXCL9, CXCL10, CCL11 , CCL17, CCL21 , CXCR3, CCL22, or IP10; and CCR2, CCR5, chemerin, or CXCR3. In particular embodiments, T cells and NK cells can be recruited to the implant by using a chemoattractant including CCL1 and CCR2, CCL1 and CCR5, CCL1 and chemerin, CCL1 and CXCR3, CCL2 and CCR2, CCL2 and CCR5, CCL2 and chemerin, CCL2 and CXCR3, CCL3 and CCR2, CCL3 and CCR5, CCL3 and chemerin, CCL3 and CXCR3, CCL4 and CCR2, CCL4 and CCR5, CCL4 and chemerin, CCL4 and CXCR3, CXCL9 and CCR2, CXCL9 and CCR5, CXCL9 and chemerin, CCL1 and CXCL8, CCL2 and CX3CL1 , CXCL9 and CXCR3, CXCL10 and CCR2, CXCL10 and CCR5, CXCL10 and chemerin, CXCL10 and CXCR3, CCL1 1 and CCR2, CCL11 and CCR5, CCL11 and chemerin, CCL11 and CXCR3, CL17 and CCR2, CCL17 and CCR5, CCL17 and chemerin, CCL17 and CXCR3, CCL21 and CCR2, CCL21 and CCR5, CCL21 and chemerin, CCL21 and CXCR3, CXCR3 and CCR2, CXCR3 and CCR5, CXCR3 and chemerin, CXCR3 and CXCR3, CCL22 and CCR2, CCL22 and CCR5, CCL22 and chemerin, CCL22 andCXCR3, IP10 and CCR2, IP10 and CCR5, IP10 and chemerin, or IP10 and CXCR3, In particular embodiments, T cells and dendritic cells can be recruited to the implant by using a chemoattractant including CCL1 , CCL2, CCL3, CCL4, CXCL9, CXCL10, CCL11 , CCL17, CCL21 , CXCR3, CCL22, or IP10; and CCL19 or CCL21 . In particular embodiments, T cells and dendritic cells can be recruited to the implant by using a chemoattractant including CCL1 and CCL19, CCL1 and CCL21 , CCL2 and CCL19, CCL2 and CCL21 , CCL3 and CCL19, CCL3 and CCL21 , CCL4 and CCL19, CCL4 and CCL21 , CXCL9 and CCL19, CXCL9 and CCL21 , CXCL10 and CCL19, CXCL10 and CCL21 , CCL1 1 and CCL19, CCL11 and CCL21 , CCL17 and CCL19, CCL17 and CCL21 , CCL21 , CXCR3 and CCL19, CXCR3 and CCL21 , CCL22 and CCL19, CCL22 and CCL21 , IP10 and CCL19, or IP10 and CCL21 .

[0076] In particular embodiments, each implant includes at least 1 ng, 5 ng, 50 ng, 500 ng, 1 pg, 2 pg, 5 ig, 10 pig, 15 pig, 20 pig, 25 pig, 30 pig, 35 pig, 40 pig, or at least 50 pig of chemoattractant. In particular embodiments, a 21 mm implant includes 2 pig of chemoattractant. In particular embodiments, a 21 mm implant includes 20 pg of chemoattractant. In particular embodiments, the chemoattractant includes CCL21 , CCL3, CCL4, or CXCL10. The current disclosure provides that in the context of exemplary implants disclosed herein, CCL21 is especially effective at recruiting T cells (FIGs. 2A-2C).

[0077] In particular embodiments, a combination of chemoattractants can be used with the percentage of one chemoattractant in the implant being 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% when compared to the other chemoattractants.

[0078] (iii) Immune Cell Activating Factors (ICAF). Implants disclosed herein include ICAF. ICAF include any compound that activates an immune cell and can be incorporated in or attached to the implants disclosed herein. Activation of an immune cell refers to the state of an immune cell that has been sufficiently stimulated to induce detectable cellular proliferation, cytokine production, or effector function such as tumor targeting and / or killing. In particular embodiments, an ICAF binds an activating antigen or an activating epitope on an immune cell.

[0079] In particular embodiments, immune cells include T-cells, natural killer (NK) cells, NK-T cells, monocytes / macrophages, and / or dendritic cells. In particular embodiments, an immune cell is a T-cell.

[0080] T-cell activation can be mediated by two distinct signals: those that initiate antigendependent primary activation and provide a T-cell receptor like signal (primary cytoplasmic signaling sequences) and those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal (secondary cytoplasmic signaling sequences). Combinations of ICAFs can activate any combination of T cell activating epitopes that upon binding induce T-cell activation. Examples of such T cell activating epitopes are on T cell markers including CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, 4-1 BB (CD 137), 0X40, lymphocyte function- associated antigen-1 (LFA-1 ), LIGHT, NKG2C, and B7-H3. T cell suppressive receptors that can be blocked include PD-1 , LAG3, TIM-3, BTLA, CTLA-4, and CD200. Antibodies with PD-1 binding domains include Pembrolizumab and Nivolumab while a CTLA-4 blocking antibody includes Ipilimumab.

[0081] CD3 is a primary signal transduction element of T cell receptors and is expressed on all mature T cells. Binding domains for CD3 can be derived from, for example, OKT3, 20G6-F3, 4B4- D7, 4E7-C9, and 18F5-H10.

[0082] OKT3 is described in U.S. Patent No. 5,929,212. In particular embodiments, the OKT3 antibody includes variable light chain and variable heavy chain having a complementarity determining region (CDR) light (L)1 including SASSSVSYMN (SEQ ID NO: 1 ), a CDRL2 including RWIYDTSKLAS (SEQ ID NO: 2), a CDRL3 including QQWSSNPFT (SEQ ID NO: 3), a CDR heavy (H)1 including KASGYTFTRYTMH (SEQ ID NO: 4), a CDRH2 including INPSRGYTNYNQKFKD (SEQ ID NO: 5), and a CDRH3 including YYDDHYCLDY (SEQ ID NO: 6).

[0083] The following sequence is an scFv derived from OKT3 which retains the capacity to bind CD3: QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYN QKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSSGGG GSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYD TSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINR (SEQ ID NO: 7). It may also be used as a CD3 binding domain.

[0084] The 20G6-F3 antibody includes a CDRL1 including QSLVHNNGNTY (SEQ ID NO: 8), a CDRL2 including KVS, a CDRL3 including GQGTQYPFT (SEQ ID NO: 9), a CDRH1 including GFTFTKAW (SEQ ID NO: 10), a CDRH2 including IKDKSNSYAT (SEQ ID NO: 11 ), and a CDRH3 including RGVYYALSPFDY (SEQ ID NO: 12).

[0085] The 4B4-D7 antibody includes a CDRL1 including QSLVHDNGNTY (SEQ ID NO: 13), a CDRL2 including KVS, a CDRL3 including GQGTQYPFT (SEQ ID NO: 9), a CDRH1 including GFTFSNAW (SEQ ID NO: 15), a CDRH2 including IKARSNNYAT (SEQ ID NO: 16), and a CDRH3 including RGTYYASKPFDY (SEQ ID NO: 17).

[0086] The 4E7-C9 antibody includes a CDRL1 including QSLEHNNGNTY (SEQ ID NO: 18), a CDRL2 including KVS, a CDRL3 including GQGTQYPFT (SEQ ID NO: 9), a CDRH1 including GFTFSNAW (SEQ ID NO: 15), a CDRH2 including IKDKSNNYAT (SEQ ID NO: 20), and aCDRH3 including RYVHYGIGYAMDA (SEQ ID NO: 21 ).

[0087] The 18F5-H10 antibody includes a CDRL1 including QSLVHTNGNTY (SEQ ID NO: 22), a CDRL2 including KVS, a CDRL3 including GQGTHYPFT (SEQ ID NO: 23), a CDRH1 including GFTFTNAW (SEQ ID NO: 24), a CDRH2 including KDKSNNYAT (SEQ ID NO: 25), and a CDRH3 including RYVHYRFAYALDA (SEQ ID NO: 26).

[0088] Additional examples of anti-CD3 antibodies, binding domains, and CDRs can be found in WQ2016 / 116626. TR66 may also be used.

[0089] CD28 is a surface glycoprotein present on 80% of peripheral T cells in humans and is present on both resting and activated T cells. CD28 binds to B7-1 (CD80) and B7-2 (CD86) and is the most potent of the known co-stimulatory molecules (June et al., Immunol. Today 15:321 (1994); Linsley et al., Ann. Rev. Immunol. 11 :191 (1993)).

[0090] In particular embodiments, a CD28 binding domain can be derived from CD80, CD86, or the antibodies TGN1412, 9D7, 9.3, KOLT-2, 15E8, 248.23.2, and EX5.3D10.

[0091] In particular embodiments, a binding domain derived from TGN1412 includes a CDRL1 including HASQNIYVWLN (SEQ ID NO: 27), a CDRL2 including KASNLHT (SEQ ID NO: 28), a CDRL3 including QQGQTYPYT (SEQ ID NO: 29), a CDRH1 including SYYIH (SEQ ID NO: 30), a CDRH2 including CIYPGNVNTNYNEKFKD (SEQ ID NO: 31 ), and a CDRH3 including SHYGLDWNFDV (SEQ ID NO: 32).

[0092] In particular embodiments a CD80 / CD86 binding domain is derived from one or more monoclonal antibodies described in U.S. Patent No. 7,531 ,175. In particular embodiments, the CD80 / CD86 binding domain includes a CDRL1 including SVSSSISSSNLH (SEQ ID NO: 33), a CDRL2 including GTSNLAS (SEQ ID NO: 34), a CDRL3 including QQWSSYPLT (SEQ ID NO: 35), a CDRH1 including DYYMH (SEQ ID NO: 36), a CDRH2 including WIDPENGNTLYDPKFQG (SEQ ID NO: 37), and a CDRH3 including EGLFFAY (SEQ ID NO: 38).

[0093] Activated T-cells express 4-1 BB (CD137). 4-1 BB, also called CD137 or TNFSF9 (UniProt ID No. Q0701 1) is a T-cell co-stimulatory receptor.

[0094] 4-1 BB binding domains can be derived from a monoclonal antibody described in U.S. Patent Number 9,382,328B2.

[0095] In particular embodiments, the 4-1 BB binding domain includes a CDRL1 including RASQSVS (SEQ ID NO: 39), a CDRL2 including ASNRAT (SEQ ID NO: 40), a CDRL3 including QRSNWPPALT (SEQ ID NO: 41 ), a CDRH1 including YYWS (SEQ ID NO: 42), a CDRH2 including INH, and a CDRH3 including YGPGNYDWYFDL (SEQ ID NO: 43).

[0096] In particular embodiments, the 4-1 BB binding domain includes a CDRL1 including SGDNIGDQYAH (SEQ ID NO: 44), a CDRL2 including QDKNRPS (SEQ ID NO: 45), a CDRL3including ATYTGFGSLAV (SEQ ID NO: 46), a CDRH1 including GYSFSTYWIS (SEQ ID NO: 47), a CDRH2 including KIYPGDSYTNYSPS (SEQ ID NO: 48), and a CDRH3 including GYGIFDY (SEQ ID NO: 49).

[0097] Cytotoxic T-cells destroy tumor cells. These cells are also known as CD8+ T-cells because they express the CD8 glycoprotein at their surface. These cells recognize their targets by binding to antigen associated with MHC class I, which is present on the surface of nearly every cell of the body.

[0098] In particular embodiments, a CD8 binding domain can be derived from the OKT8 antibody. The OKT8 antibody includes a CDRL1 including RTSRSISQYLA (SEQ ID NO: 50), a CDRL2 including SGSTLQS (SEQ ID NO: 51 ), a CDRL3 including QQHNENPLT (SEQ ID NO: 52), a CDRH1 including GFNIKD (SEQ ID NO: 53), a CDRH2 including RIDPANDNT (SEQ ID NO: 54), and a CDRH3 including GYGYYVFDH (SEQ ID NO: 55).

[0099] In particular embodiments, immune cells can be activated by suppressing the activity of inhibitory epitopes such as PD-1 , LAG3, TIM-3, BTLA, CTLA-4, VISTA and / or CD200. For example, co-delivering anti-PD1 antibody could be valuable in preventing T-cell exhaustion in cells within the implant.

[0100] PD-1 , also called CD279 (UniProt ID No. Q15116) is an inhibitory cell surface receptor involved in regulating the T-cell immune response. In particular embodiments a PD-1 binding domain cam be derived from a monoclonal antibody described in U.S. Patent Publication 2011 / 0271358. In particular embodiments, the PD-1 binding domain includes a CDRL1 including RASQSVSTSGYSYMH (SEQ ID NO: 56), a CDRL2 including FGSNLES (SEQ ID NO: 57), a CDRL3 including QHSWEIPYT (SEQ ID NO: 58), a CDRH1 including SSWIH (SEQ ID NO: 59), a CDRH2 including YIYPSTGFTEYNQKFKD (SEQ ID NO: 60), and a CDRH3 including WRDSSGYHAMDY (SEQ ID NO: 61 ).

[0101] In particular embodiments, a PD-1 binding domain can be derived from a monoclonal antibody described in U.S. Patent Application 20090217401 A1. In particular embodiments, the PD-1 binding domain includes a CDRL1 including RASQSVSSYLA (SEQ ID NO: 62), a CDRL2 including DASNRAT (SEQ ID NO: 63), a CDRL3 including QQSSNWPRT (SEQ ID NO: 64), a CDRH1 including NSGMH (SEQ ID NO: 65), a CDRH2 including VLWYDGSKRYYADSVKG (SEQ ID NO: 66), and a CDRH3 including NDDY (SEQ ID NO: 67).

[0102] LAG3, also called CD223 (UniProt ID No. P18627) binds to HLA class-ll antigens and is involved in activation of lymphocytes. In particular embodiments a LAG3 binding domain can be derived from a monoclonal antibody described in WQ / 2014 / 008218.

[0103] TIM-3, also known as HAVcr-2 or TIMD-3 (UniProt ID No. Q9TDQ0) is a cell surfacereceptor that plays an inhibitory role in innate and adaptive immune responses. In particular embodiments a TIM-3 binding domain can be derived from a monoclonal antibody described in U.S. Patent Publication 2015 / 0218274.

[0104] BTLA, also known as CD272 (UniProt ID No. Q7Z6A9), is an inhibitory receptor that inhibits the immune response of lymphocytes. In particular embodiments a BTLA binding domain (e.g., scFv) can be derived from one or more monoclonal antibodies described in U.S. Patent Publication 2012 / 0288500.

[0105] CTLA-4, also known as CD152 (UniProt ID No. P16410), is an inhibitory receptor that is a major negative regulator of the T-cell response. In particular embodiments a CTLA-4 binding domain can be derived from a monoclonal antibody described in U.S. Patent No. 6,984,720.

[0106] CD200 (also known as ox-2 membrane glycoprotein, UniProt ID No. P41217) is a protein that can deliver inhibitory signals to immune cells. In particular embodiments a CD200 binding domain can be derived from one or more monoclonal antibodies described in U.S. Patent Publication 2013 / 0189258.

[0107] In particular embodiments, natural killer cells (also known as NK cells, K cells, and killer cells) are targeted for localized activation. NK cells can induce apoptosis or cell lysis by releasing granules that disrupt cellular membranes and can secrete cytokines to recruit other immune cells.

[0108] Examples of activating proteins expressed on the surface of NK cells include NKG2D, CD8, CD16, KIR2DL4, KIR2DS1 , KIR2DS2, KIR3DS1 , NKG2C, NKG2E, NKG2D, and several members of the natural cytotoxicity receptor (NCR) family. Examples of NCRs that activate NK cells upon ligand binding include NKp30, NKp44, NKp46, NKp80, and DNAM-1.

[0109] Examples of commercially available antibodies that bind to an NK cell receptor and induce and / or enhance activation of NK cells include: 5C6 and 1 D11 , which bind and activate NKG2D (available from BioLegend® San Diego, CA); mAb 33, which binds and activates KIR2DL4 (available from BioLegend®); P44-8, which binds and activates NKp44 (available from BioLegend®); SK1 , which binds and activates CD8; and 3G8 which binds and activates CD16. Additional NK cell activating antibodies are described in WG / 2005 / 0003172 and US Patent No. 9,415,104.

[0110] Macrophages can also be activated. Macrophages are a type of leukocyte (or white blood cell) that can engulf and digest cells, cellular debris, and / or foreign substances in a process known as phagocytosis. Examples of activating proteins expressed on the surface of macrophages (and their precursors, monocytes) include macrophage markers including CD11 b, CD11c, CD64, CD68, CD119, CD163, CD206, CD209, F4 / 80, IFGR2 Toll-like receptors (TLRs) 1 -9, IL-4Ra, and macrophage receptor with collagenous structure (MARCO). Commercially available antibodiesthat bind to proteins expressed on the surface of macrophages include M1 / 70, which binds and activates CD11 b (available from BioLegend®); KP1 , which binds and activates CD68 (available from ABCAM®, Cambridge, United Kingdom); and ab87099, which binds and activates CD163 (available from ABCAM®).

[0111] Dendritic cells can be activated by conserved pathogen molecules including pathogen- associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). Recognition of PAMPs are mediated by pattern recognition receptor (PRR) such as Toll-like receptors (TLR), C-type lectin receptors (CLR), cytoplasmic NOD-like receptors (NLRs), intracellular retinoic acid-inducible gene-l)-like receptors (RLR), and absent in melanoma 2-like receptors (AIM2).

[0112] The structure of antibodies, CDR sets, and other segments are discussed elsewhere herein.

[0113] (iv) Reprogramming Factors (RF). As used herein, RF “reprogram” a cell to i) express a molecule that it was not expressing before the reprogramming; ii) express more of the molecule than it was before the reprogramming and / or ill) express less of a molecule than it was before the reprogramming.

[0114] Reprogramming of a cell can include, for example, insertion of a coding sequence, alteration of a gene, and / or deletion of a gene. In particular embodiments, immune cells can be reprogrammed by introducing a vector into the immune cells.

[0115] A "vector" is a nucleic acid molecule that is capable of transporting another nucleic acid molecule into a cell. Vectors may be, e.g., viruses, phage, a DNA vector, an RNA vector, a viral vector, a bacterial vector, a plasmid vector, a cosmid vector, and an artificial chromosome vector. An "expression vector" is any type of vector that is capable of directing the expression of a molecule encoded by one or more genes carried by the vector when it is present in the appropriate environment.

[0116] Viral vectors are usually non-replicating or replication-impaired vectors, which means that the viral vector cannot replicate to any significant extent in normal cells (e.g., normal human cells), as measured by conventional means (e.g., via measuring DNA synthesis and / or viral titer). Nonreplicating or replication-impaired vectors may have become so naturally (i.e., they have been isolated as such from nature) or artificially (e.g., by breeding in vitro or by genetic manipulation). There will generally be at least one cell-type in which the replication-impaired viral vector can be grown-for example, modified vaccinia Ankara (MVA) can be grown in CEF cells. Typically, viral vectors are incapable of causing a significant infection in a subject, typically in a mammalian subject.

[0117] Retroviral vectors (see Miller, et al., 1993, Meth. Enzymol. 217:581-599) can be used. In such embodiments, the gene to be expressed is cloned into the retroviral vector for its delivery into cells. In particular embodiments, a retroviral vector includes all of the cis-acting sequences necessary for the packaging and integration of the viral genome, i.e., (a) a long terminal repeat (LTR), or portions thereof, at each end of the vector; (b) primer binding sites for negative and positive strand DNA synthesis; and (c) a packaging signal, necessary for the incorporation of genomic RNA into virions. More detail about retroviral vectors can be found in Boesen, et al., 1994, Biotherapy 6:291 -302; Clowes, et al., 1994, J. Clin. Invest. 93:644-651 ; Kiem, et al., 1994, Blood 83:1467-1473; Salmons and Gunzberg, 1993, Human Gene Therapy 4:129-141 ; and Grossman and Wilson, 1993, Curr. Opin. in Genetics and Devel. 3:110-114. Adenoviruses, adeno-associated viruses (AAV) and alphaviruses can also be used. See Kozarsky and Wilson, 1993, Current Opinion in Genetics and Development 3:499-503, Rosenfeld, et al., 1991 , Science 252:431 -434; Rosenfeld, et al., 1992, Cell 68:143-155; Mastrangeli, et al., 1993, J. Clin. Invest. 91 :225-234; Walsh, et al., 1993, Proc. Soc. Exp. Bioi. Med. 204:289-300; and Lundstrom, 1999, J. Recept. Signal Transduct. Res. 19: 673-686. Other methods of gene delivery include use of mammalian artificial chromosomes (Vos, 1998, Curr. Op. Genet. Dev. 8:351 -359); liposomes (Tarahovsky and Ivanitsky, 1998, Biochemistry (Mose) 63:607-618); ribozymes (Branch and Klotman, 1998, Exp. Nephrol. 6:78-83); and triplex DNA (Chan and Glazer, 1997, J. Mol. Med. 75:267-282).

[0118] "Gammaretroviruses" refers to a genus of the retroviridae family. Exemplary gammaretroviruses include mouse stem cell virus, murine leukemia virus, feline leukemia virus, feline sarcoma virus, and avian reticuloendotheliosis viruses.

[0119] Widely used retroviral vectors include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol. 66:2731 -2739, 1992; Johann et al., J. Virol. 66:1635-1640, 1992; Sommerfelt et al., Virol. 176:58- 59, 1990; Wilson et al., J. Virol. 63:2374-2378, 1989; Miller et al., J. Virol. 65:2220-2224, 1991 ; and PCT / US94 / 05700).

[0120] Particularly suitable are lentiviral vectors. "Lentivirus" refers to a genus of retroviruses that are capable of infecting dividing and non-dividing cells and typically produce high viral titers. Lentiviral vectors have been employed in gene therapy for a number of diseases. For example, hematopoietic gene therapies using lentiviral vectors or gamma retroviral vectors have been used for x-linked adrenoleukodystrophy and beta thalassaemia. See, e.g., Kohn et al., Clin. Immunol. 135:247-54, 2010; Cartier et al., Methods Enzymol. 507:187-198, 2012; and Cavazzana-Calvo etal., Nature 467:318-322, 2010. Several examples of lentiviruses include HIV (human immunodeficiency virus: including HIV type 1 , and HIV type 2); equine infectious anemia virus; feline immunodeficiency virus (FIV); bovine immune deficiency virus (BIV); and simian immunodeficiency virus (SIV).

[0121] In particular embodiments, other retroviral vectors can be used in the practice of the methods of the disclosure. These include, e.g., vectors based on human foamy virus (HFV) or other viruses in the Spumavirus genera.

[0122] Foamy viruses (FVes) are the largest retroviruses known today and are widespread among different mammals, including all non-human primate species, however are absent in humans. This complete apathogenicity qualifies FV vectors as ideal gene transfer vehicles for genetic therapies in humans and clearly distinguishes FV vectors as gene delivery system from HIV-derived and also gammaretrovirus-derived vectors.

[0123] FV vectors are suitable for gene therapy applications because they can (1 ) accommodate large transgenes (> 9kb), (2) transduce slowly dividing cells efficiently, and (3) integrate as a provirus into the genome of target cells, thus enabling stable long-term expression of the transgene(s). FV vectors do need cell division for the pre-integration complex to enter the nucleus, however the complex is stable for at least 30 days and still infective. The intracellular half-life of the FV pre-integration complex is comparable to the one of lentiviruses and significantly higher than for gammaretroviruses, therefore FV are also - similar to LV vectors - able to transduce rarely dividing cells. FV vectors are natural self-inactivating vectors and characterized by the fact that they seem to have hardly any potential to activate neighboring genes. In addition, FV vectors can enter any cells known (although the receptor is not identified yet) and infectious vector particles can be concentrated 100-fold without loss of infectivity due to a stable envelope protein.

[0124] In particular embodiments, the viral vector is a pseudotyped viral vector. Pseudotyped viral vectors (e.g., pseudotyped lentiviral vector) include vector particles bearing enveloped proteins (glycoproteins, GP) derived from other enveloped viruses. Such particles possess the tropism of the virus from which the enveloped proteins is derived. One of the widely used glycoproteins for pseudotyping lentiviral vectors is the vesicular stomatitis virus GP (VSV-G), due to the very broad tropism and stability of the resulting pseudotypes. Pseudotyped viral vectors are well known in the art, and several examples are described, for example, in Cronin et al., Curr. Gene Ther. 5(4):387-398. It includes lentiviral vectors pseudotyped with lyssavirus GPs, lymphocytic choriomeningitis virus (LCMV) GPs, alphavirus GPs (e.g., Ross River virus (RRV), Semliki Forest virus (SFV) and Sindbis virus GPs), Filovirus GPs (e.g., Marburg virus and Ebola Zaire virus GPs), gammaretrovirus GPs(e.g., ecotropic MLV, amphotropic 4070A MLV, 10A1 MLV, xenotropic NZB MLV, mink cell focusforming virus, gibbon ape leukemia (GALV) virus, RD114 GPs) and baculovirus GPs (GP64).

[0125] Additional examples of viral vectors include those derived from adenoviruses (e.g., adenovirus 5 (Ad5), adenovirus 35 (Ad35), adenovirus 11 (Ad11 ), adenovirus 26 (Ad26), adenovirus 48 (Ad48) or adenovirus 50 (Ad50)), adeno-associated virus (AAV; see, e.g., U.S. Pat. No. 5,604,090; Kay et al., Nat. Genet. 24:257 (2000); Nakai et al., Blood 91 :4600 (1998)), alphaviruses, cytomegaloviruses (CMV), flaviviruses, herpes viruses (e.g., herpes simplex), influenza viruses, papilloma viruses (e.g., human and bovine papilloma virus; see, e.g., U.S. Pat. No. 5,719,054), poxviruses, vaccinia viruses, modified vaccinia Ankara (MVA), NYVAC, avipox, fowlpox (e.g., FP9), canarypox (e.g., ALVAC), or strains derived therefrom. See Kozarsky and Wilson, 1993, Current Opinion in Genetics and Development 3:499-503, Rosenfeld, et al., 1991 , Science 252:431-434; Rosenfeld, et al., 1992, Cell 68:143-155; Mastrangeli, et al., 1993, J. Clin. Invest. 91 :225-234; Walsh, et al., 1993, Proc. Soc. Exp. Bioi. Med. 204:289-300; and Lundstrom, 1999, J. Recept. Signal Transduct. Res. 19: 673-686.

[0126] Particular embodiments utilize Herpes simplex virus 1 (HSV-1 ). Appropriate HSV-1 strains include strain JS1 , strain 17+, strain F, strain KOS, and strain Patton.

[0127] The HSV-1 may be modified such that it lacks functional ICP34.5 genes and / or lacks a functional ICP47 gene. ICP34.5 acts as a virulence factor during HSV infection, limits replication in non-dividing cells and renders the virus non-pathogenic. ICP47 down-regulates major histocompatibility complex (MHC) class I expression on the surface of infected host cells and MHC Class I binding to transporter associated with antigen presentation (TAP). Such actions block antigenic peptide transport in the endoplasmic reticulum and loading of MHC class I molecules. Another HSV gene that can be modified is ICP6, the large subunit of ribonucleotide reductase, involved in nucleotide metabolism and viral DNA synthesis in non-dividing cells but not in dividing cells. Thymidine kinase, responsible for phosphorylating acyclovir to acyclovirmonophosphate, virion trans-activator protein vmw65, glycoprotein H, vhs, ICP43, and immediate early genes encoding ICP4, ICP27, ICP22 and / or ICP0, may also be modified.

[0128] Herpes virus strains and how to make such strains are also described in U.S. Pat. Nos. 5,824,318; 6,764,675; 6,770,274; 7,063,835; 7,223,593; 7,749,745; 7,744,899; 8,273,568; 8,420,071 ; and 8,470,577; WIPO Publication Numbers WO199600007; WO199639841 ; WO199907394; W0200054795; W02006002394; and WO201306795; Chinese Patent Numbers CN128303, CN10230334 and CN 10230335; Varghese and Rabkin, (2002) Cancer Gene Therapy 9:967-97, and Cassady and Ness Parker, (2010) The Open Virology Journal 4:103-108.

[0129] There are a large number of suitable viral vectors known in the art including thoseidentified for human gene therapy applications (see Pfeifer and Verma, 2001 , Ann. Rev. Genomics Hum. Genet. 2:177). Methods of using retroviral and lentiviral viral vectors and packaging cells for transducing mammalian host cells with viral particles including transgenes are described in, e.g., US 8,119,772; Walchli, etal., 2011 , PLoS One 6:327930; Zhao, et al., 2005, J. Immunol. 174:4415; Engels, et al., 2003, Hum. Gene Ther. 14:1155; Frecha, et al., 2010, Mol.Ther. 18:1748; and Verhoeyen, et al., 2009, Methods Mol. Biol. 506:97. Retroviral and lentiviral vector constructs and expression systems are also commercially available.

[0130] In particular embodiments, an RF includes components used for targeted genetic engineering (i.e., gene editing agents). The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) / Cas (CRISPR-associated protein) nuclease system is an engineered nuclease system used for genetic engineering that is based on a bacterial system. Information regarding CRISPR-Cas systems and components thereof are described in, for example, US8697359, US8771945, US8795965, US8865406, US8871445, US8889356, US8889418, US8895308, US8906616, US8932814, US8945839, US8993233 and US8999641 and applications related thereto; and WO2014 / 018423, WO2014 / 093595, WO2014 / 093622,WO2014 / 093635, WO2014 / 093655, WO2014 / 093661 , WO2014 / 093694, WO2014 / 093701 , WO2014 / 093709, WO2014 / 093712, WO2014 / 093718, WO2014 / 145599, WO2014 / 204723, WO2014 / 204724, WO2014 / 204725, WO2014 / 204726, WO2014 / 204727, WO2014 / 204728, WO2014 / 204729, WO2015 / 065964, WO2015 / 089351 , WO2015 / 089354, WO2015 / 089364,WO2015 / 089419, WO2015 / 089427, WO2015 / 089462, WO2015 / 089465, WO2015 / 089473 and WO2015 / 089486, WO201620571 1 , WO2017 / 106657, WO2017 / 127807 and applications related thereto.

[0131] Particular embodiments utilize zinc finger nucleases (ZFNs) as gene editing agents. ZFNs are a class of site-specific nucleases engineered to bind and cleave DNA at specific positions. ZFNs are used to introduce double stranded breaks (DSBs) at a specific site in a DNA sequence which enables the ZFNs to target unique sequences within a genome in a variety of different cells. For additional information regarding ZFNs and ZFNs useful within the teachings of the current disclosure, see, e.g., US 6,534,261 ; US 6,607,882; US 6,746,838; US 6,794,136; US 6,824,978; 6,866,997; US 6,933,113; 6,979,539; US 7,013,219; US 7,030,215; US 7,220,719; US 7,241 ,573; US 7,241 ,574; US 7,585,849; US 7,595,376; US 6,903,185; US 6,479,626; US 2003 / 0232410 and US 2009 / 0203140 as well as Gaj et al., Nat Methods, 2012, 9(8):805-7; Ramirez et al., Nucl Acids Res, 2012, 40(12):5560-8; Kim et al., Genome Res, 2012, 22(7): 1327-33; Urnov et al., Nature Reviews Genetics, 2010, 11 :636-646; Miller, et al. Nature biotechnology 25, 778-785 (2007); Bibikova, etal. Science 300, 764 (2003); Bibikova, etal. Genetics 161 , 1169-1175 (2002);Wolfe, et al. Annual review of biophysics and biomolecular structure 29, 183-212 (2000); Kim, et al. Proceedings of the National Academy of Sciences of the United States of America 93, 1 156- 1160 (1996); and Miller, et al. The EMBO journal 4, 1609-1614 (1985).

[0132] Particular embodiments can use transcription activator like effector nucleases (TALENs) as gene editing agents. TALENs refer to fusion proteins including a transcription activator-like effector (TALE) DNA binding protein and a DNA cleavage domain. TALENs are used to edit genes and genomes by inducing double DSBs in the DNA, which induce repair mechanisms in cells. Generally, two TALENs must bind and flank each side of the target DNA site for the DNA cleavage domain to dimerize and induce a DSB. For additional information regarding TALENs, see US 8,440,431 ; US 8,440,432; US 8,450,471 ; US 8,586,363; and US 8,697,853; as well as Joung and Sander, Nat Rev Mol Cell Biol, 2013, 14(l):49-55; Beurdeley et al., Nat Common, 2013, 4: 1762; Scharenberg et al., Curr Gene Ther, 2013, 13(4):291 -303; Gaj et al., Nat Methods, 2012, 9(8):805-7; Miller, et al. Nature biotechnology 29, 143-148 (2011 ); Christian, et al. Genetics 186, 757-761 (2010); Boch, etal. Science 326, 1509-1512 (2009); and Moscou, & Bogdanove, Science 326, 1501 (2009).

[0133] Particular embodiments can utilize MegaTALs as gene editing agents. MegaTALs have a sc rare-cleaving nuclease structure in which a TALE is fused with the DNA cleavage domain of a meganuclease. Meganucleases, also known as homing endonucleases, are single peptide chains that have both DNA recognition and nuclease function in the same domain. In contrast to the TALEN, the megaTAL only requires the delivery of a single peptide chain for functional activity.

[0134] Regardless of what system is used to deliver RF to a cell, many deliver a coding sequence to the cell. In particular embodiments, the coding sequence is a nucleotide or gene.

[0135] The terms “nucleotide,” “nucleobase,” “nucleic acid,” “nucleic acid molecule,” and their equivalents, may refer to an organic molecule that includes a nitrogenous base, a sugar, and a phosphate group. In various cases, a nucleotide is a monomer of DNA or RNA. In other cases, a nucleotide is a polymer of DNA or RNA. A nucleotide, for instance, is a chemical structure.

[0136] The term “gene” refers to a nucleic acid sequence that encodes and results in expression of a molecule, such as a therapeutic molecule (e.g., therapeutic protein). This definition includes various sequence polymorphisms, mutations, and / or variants wherein such alterations do not substantially affect the function of the encoded molecule. The term “gene” may include not only coding sequences but also regulatory regions such as promoters, enhancers, and termination regions. Genes encoding a molecule can be DNA or RNA that directs the expression of the molecule. These nucleic acids may be DNA that is transcribed into RNA or RNA that is translated into protein. The nucleic acids include both the full-length nucleic acid sequences as well as non-full-length sequences derived from the full-length protein. The sequences can also include degenerate codons of the native sequence or sequences that may be introduced to provide codon preference in a specific cell type. Portions of complete genes are referenced throughout the disclosure as is understood by one of ordinary skill in the art.

[0137] "Encoding” refers to the property of specific sequences of nucleotides in a gene, such as a cDNA, or an mRNA, to serve as templates for synthesis of other macromolecules such as a defined sequence of amino acids. Thus, a gene codes for a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. A "gene encoding a molecule" includes all nucleotide sequences that are degenerate versions of each other and that code for the same molecule or molecule of substantially similar form and function.

[0138] Genes encoding therapeutic molecules are provided herein and can also be readily prepared by synthetic or recombinant methods from sequences and other description provided herein. In embodiments, the gene encoding any of these molecules can also have one or more restriction enzyme sites at the 5' and / or 3' ends of the coding sequence in order to provide for easy excision and replacement of the gene encoding the molecule with another gene encoding a different molecule. In embodiments, the gene encoding the molecules can be codon optimized for expression in mammalian cells.

[0139] Nucleic acids sequences encoding more than one portion of an expressed molecules can be operably linked to each other and relevant regulatory sequences. For example, there can be a functional linkage between a regulatory sequence and an exogenous nucleic acid sequence resulting in expression of the latter. For another example, a first nucleic acid sequence can be operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary or helpful, join coding regions, into the same reading frame.

[0140] Operatively linked refers to the linking of DNA sequences (including the order of the sequences, the orientation of the sequences, and the relative spacing of the various sequences) in such a manner that an encoded molecule is expressed. Methods of operatively linking expression control sequences to coding sequences are well known in the art. See, e.g., Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N. Y., 1982; Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N. Y., 1989; and Green and Sambrook, Molecular Cloning: A Laboratory Manual, 4th Edition (2012).

[0141] Expression control sequences are DNA sequences involved in any way in the control of transcription or translation. Suitable expression control sequences and methods of making and using them are well known in the art. Expression control sequences generally include a promoter. The promoter may be inducible or constitutive. It may be naturally-occurring, may be composed of portions of various naturally-occurring promoters, or may be partially or totally synthetic. Guidance for the design of promoters is provided by studies of promoter structure, such as that of Harley and Reynolds, Nucleic Acids Res., 15, 2343-2361 , 1987. Also, the location of the promoter relative to the transcription start may be optimized. See, e.g., Roberts et al., Proc. Natl. Acad. Sci. USA, 76:760-764, 1979.

[0142] The promoter may include, or be modified to include, one or more enhancer elements. In particular embodiments, the promoter will include a plurality of enhancer elements. Promoters including enhancer elements can provide for higher levels of transcription as compared to promoters that do not include them.

[0143] For efficient expression, the coding sequences can be operatively linked to a 3' untranslated sequence. In particular embodiments, the 3' untranslated sequence can include a transcription termination sequence and a polyadenylation sequence. The 3' untranslated region can be obtained, for example, from the flanking regions of genes.

[0144] In particular embodiments, a 5' untranslated leader sequence can also be employed. The 5' untranslated leader sequence is the portion of an mRNA that extends from the 5' CAP site to the translation initiation codon. In particular embodiments, the nucleic acid is stably integrated into the genome of a cell. In particular embodiments, the nucleic acid is stably maintained in a cell as a separate, episomal segment.

[0145] Particular embodiments can deliver nucleotides within a gene editing system. Gene editing systems modify or affect particular sequences of a cell’s endogenous genome. Gene editing systems are useful for targeted genome editing, for example gene disruption, gene editing by homologous recombination, and gene therapy to insert therapeutic genes at the appropriate chromosomal target sites with a human genome.

[0146] Numerous techniques are known in the art for the introduction of foreign genes into cells (see e.g., Loeffler and Behr, Meth. Enzymol, 217, 599-618 (1993); Cohen et al., Meth. Enzymol, 217, 618-644 (1993); Cline, Pharmac. Ther, 29, 69-92 (1985)) and may be used in accordance with the present disclosure, provided that the necessary developmental and physiological functions of the immune cells are not disrupted. In particular embodiments, the technique provides for the stable transfer of the gene to the cell, so that the gene is expressible by the cell and preferably heritable and expressible by its cell progeny. In particular embodiments, the techniqueprovides for transient expression of the gene within a cell.

[0147] Methods commonly known in the art of recombinant DNA technology which can be used to reprogram cells are described in Ausubel et al. (eds.), 1993, Current Protocols in Molecular Biology, John Wiley & Sons, NY; and Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY.

[0148] (v) Expressed Molecules. Expressed molecules can include any molecule that a cell is reprogrammed to express. Expression can include surface expression or excretion from the cell. In particular embodiments, an expressed molecule is a therapeutic protein. In particular embodiments, a therapeutic protein includes a tumor-specific receptor, such as a recombinant receptor. Recombinant receptors include any engineered receptor that functions within a cell, such as CAR, eTCR, or CAR / TCR hybrids. An expressed molecule can include any protein that re-educates or re-targets a recruited immune cell.

[0149] As indicated, CAR include several distinct subcomponents that allow reprogrammed cells (e.g., regulatory T cells) to recognize and kill cells expressing a target antigen (e.g., an antigen preferentially expressed on the surface of cancer cells). The subcomponents include at least an extracellular component and an intracellular component. The extracellular component includes a binding domain that specifically binds a target antigen that is preferentially present on the surface of cells or in the area thereof. When the binding domain binds such antigens, the intracellular component activates the cell to destroy the bound cell. CAR additionally include a transmembrane domain that directly or indirectly links the extracellular component to the intracellular component, and other subcomponents that can increase the CAR’s function. For example, the inclusion of a spacer region and / or one or more linker sequences can allow the CAR to have additional conformational flexibility, often increasing the binding domain’s ability to bind the targeted antigen.

[0150] The subcomponents of a CAR are described in more detail in the following section (v) subheadings.

[0151] (v-a) Binding Domains. Antibodies are one example of binding domains and include whole antibodies or binding fragments of an antibody, e.g., Fv, Fab, Fab', F(ab')2, and single chain (sc) forms and fragments thereof that specifically bind a cellular antigen. Antibodies or antigen binding fragments can include all or a portion of polyclonal antibodies, monoclonal antibodies, human antibodies, humanized antibodies, synthetic antibodies, non-human antibodies, recombinant antibodies, chimeric antibodies, bispecific antibodies, mini bodies, and linear antibodies.

[0152] Antibodies are produced from two genes, a heavy chain gene and a light chain gene. Generally, an antibody includes two identical copies of a heavy chain, and two identical copies of a light chain. Within a variable heavy chain and variable light chain, segments referred to ascomplementary determining regions (CDRs) dictate antigen epitope binding. Each heavy chain has three CDRs (i.e. , CDRH1 , CDRH2, and CDRH3) and each light chain has three CDRs (i.e., CDRL1 , CDRL2, and CDRL3). CDR regions are flanked by framework residues (FR).

[0153] The assignment of amino acids to each domain can be in accordance with Kabat numbering (Kabat etal. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (“Kabat” numbering scheme)); Chothia (Al-Lazikani et al., (1997) JMB 273, 927-948 (“Chothia” numbering scheme)), Martin (Abinandan etal., Mol Immunol. 45:3832-3839 (2008), “Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains”), Gelfand, Contact (MacCallum et al., J. Mol. Biol. 262:732-745 (1996), “Antibody-antigen interactions: Contact analysis and binding site topography,” J. Mol. Biol. 262, 732-745.” (Contact numbering scheme)), IMGT (Lefranc M P et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev Comp Immunol, 2003 January; 27(1 ):55-77 (“IMGT” numbering scheme)), AHo (Honegger A and Pluckthun A, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” J Mol Biol, 2001 Jun. 8; 309(3):657-70, (AHo numbering scheme)), North (North et al., J Mol Biol. 406(2):228-256 (2011 ), “A new clustering of antibody CDR loop conformations”), or other numbering schemes.

[0154] The boundaries of a given CDR or FR may vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignments, while the Chothia scheme is based on structural information. Numbering for both the Kabat and Chothia schemes is based upon the most common antibody region sequence lengths, with insertions accommodated by insertion letters, for example, "30a," and deletions appearing in some antibodies. The two schemes place certain insertions and deletions ("indels") at different positions, resulting in differential numbering. The Contact scheme is based on analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme. In particular embodiments, the antibody CDR sequences disclosed herein are according to Kabat numbering. North numbering uses longer sequences in the structural analysis of the conformations of CDR loops. CDR residues can be identified using software programs such as ABodyBuilder.

[0155] In some instances, scFvs based on the binding domains described herein and for use in a CAR can be prepared according to methods known in the art (see, for example, Bird et al., (1988) Science 242:423-426 and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). ScFv molecules can be produced by linking VH and VL regions of an antibody together using flexible polypeptide linkers. If a short polypeptide linker is employed (e.g., between 5-10 amino acids) intrachain folding is prevented. Interchain folding is also required to bring the two variableregions together to form a functional antigen epitope binding site. For examples of linker orientations and sizes see, e.g., Hollinger et al. 1993 Proc Natl Acad. Sci. U.S.A. 90:6444-6448, US 2005 / 0100543, US 2005 / 0175606, US 2007 / 0014794, and W02006 / 020258 and W02007 / 024715. More particularly, linker sequences that are used to connect the VL and VH of an scFv are generally five to 35 amino acids in length. In particular embodiments, a VL-VH linker includes from five to 35, ten to 30 amino acids or from 15 to 25 amino acids. Variation in the linker length may retain or enhance activity, giving rise to superior efficacy in activity studies. scFv are commonly used as the binding domains of CAR.

[0156] Other binding fragments, such as Fv, Fab, Fab', F(ab')2, can also be used within the CAR disclosed herein. Additional examples of antibody-based binding domain formats for use in a CAR include scFv-based grababodies and soluble VH domain antibodies. These antibodies form binding regions using only heavy chain variable regions. See, for example, Jespers et al., Nat. Biotechnol. 22:1161 , 2004; Cortez-Retamozo et al., Cancer Res. 64:2853, 2004; Baral et al., Nature Med. 12:580, 2006; and Barthelemy et al., J. Biol. Chem. 283:3639, 2008.

[0157] In particular embodiments, the binding domain includes a humanized antibody or an engineered fragment thereof. In particular embodiments, a non-human antibody is humanized, where one or more amino acid residues of the antibody are modified to increase similarity to an antibody naturally produced in a human or fragment thereof. These nonhuman amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. As provided herein, humanized antibodies or antibody fragments include one or more CDRs from nonhuman immunoglobulin molecules and framework regions wherein the amino acid residues including the framework are derived completely or mostly from human germline. A humanized antibody can be produced using a variety of techniques known in the art, including CDR-grafting (see, e.g., European Patent No. EP 239,400; WO 91 / 09967; and US 5,225,539, US 5,530,101 , and US 5,585,089), veneering or resurfacing (see, e.g., EP 592,106 and EP 519,596; Padlan, 1991 , Molecular Immunology, 28(4 / 5):489-498; Studnicka et al., 1994, Protein Engineering, 7(6):805-814; and Roguska et al., 1994, PNAS, 91 :969-973), chain shuffling (see, e.g., US. 5,565,332), and techniques disclosed in, e.g., US 2005 / 0042664, US 2005 / 0048617, US 6,407,213, US 5,766,886, WO 9317105, Tan et al., J. Immunol., 169:1119-25 (2002), Caldas et al., Protein Eng., 13(5):353-60 (2000), Morea et al., Methods, 20(3):267-79 (2000), Baca et al., J. Biol. Chem., 272(16): 10678-84 (1997), Roguska et al., Protein Eng., 9(10):895-904 (1996), Couto et al., Cancer Res., 55 (23 Supp):5973s-5977s (1995), Couto et al., Cancer Res., 55(8):1717-22 (1995), Sandhu J S, Gene, 150(2):409-10 (1994), and Pedersen et al., J. Mol. Biol., 235(3):959-73 (1994). Often, framework residues in the framework regions willbe substituted with the corresponding residue from the CDR donor antibody to alter, for example improve target antigen binding. These framework substitutions are identified by methods well- known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for target antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., US 5,585,089; and Riechmann et al., 1988, Nature, 332:323).

[0158] Functional variants include one or more residue additions or substitutions that do not substantially impact the physiological effects of the protein. Functional fragments include one or more deletions or truncations that do not substantially impact the physiological effects of the protein. A lack of substantial impact can be confirmed by observing experimentally comparable results in an activation study or a binding study. Functional variants and functional fragments of intracellular domains (e.g., intracellular signaling components) transmit activation or inhibition signals comparable to a wild-type reference when in the activated state of the current disclosure. Functional variants and functional fragments of binding domains bind their cognate antigen or ligand at a level comparable to a wild-type reference.

[0159] In particular embodiments, a VL region in a binding domain of the present disclosure is derived from or based on a VL of an antibody disclosed herein and contains one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions (e.g., conservative amino acid substitutions), or a combination of the above-noted changes, when compared with the VL of the antibody disclosed herein. An insertion, deletion or substitution may be anywhere in the VL region, including at the amino- or carboxy-terminus or both ends of this region, provided that each CDR includes zero changes or at most one, two, or three changes and provided a binding domain containing the modified VL region can still specifically bind its target with an affinity similar to the wild type binding domain.

[0160] In particular embodiments, a binding domain VH region of the present disclosure can be derived from or based on a VH of an antibody disclosed herein and can contain one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions (e.g., conservative amino acid substitutions or non-conservative amino acid substitutions), or a combination of the above-noted changes, when compared with the VH of the antibody disclosed herein. An insertion, deletion or substitution may be anywhere in the VH region, including at the amino- or carboxy-terminus or both ends of this region, provided that each CDR includes zero changes or at most one, two, or three changes and provided a binding domain containing the modified VH region can stillspecifically bind its target with an affinity similar to the wild type binding domain.

[0161] In particular embodiments, a binding domain includes or is a sequence that is at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to an amino acid sequence of a light chain variable region (VL) or to a heavy chain variable region (VH), or both, wherein each CDR includes zero changes or at most one, two, or three changes, from an antibody disclosed herein or fragment or derivative thereof that specifically binds to a target antigen.

[0162] In particular embodiments, target antigens are preferentially expressed by solid tumor cancer cells. “Preferentially expressed” means that a target antigen is found at higher levels on solid tumor cancer cells as compared to other cell types. In some instances, a target antigen is only expressed by solid tumor cancer cells. In other instances, the antigen is expressed on the solid tumor cancer cells at least 25%, 35%, 45%, 55%, 65%, 75%, 85%, 95%, 96%, 97%, 98%, 99%, or 100% more than on other cell types.

[0163] The following provides examples of cancer cell antigens associated with different cancer types:

[0164] Exemplary binding domains for target cancer cell antigens include can be generated de novo or derived from known antibodies or binding domains specific for a selected cancer antigen.

[0165] Epithelial cell adhesion molecule (EpCam; also referred to as EGP-40, Trop-1 , 17-1A, KSA, KS1 / 4, AUA1 , GA733-2, and CD326) is overexpressed in certain cancers, including ovarian cancer. It is a 40kd surface glycoprotein having an extracellular domain with two EGF-like repeats. Antibodies targeting EpCam are commercially available (Richter et al., Am. J. Obstet. Gynecol. 2010, 203(6): 582.e1 -582e7). Exemplary antibodies that bind EpCam include MT201 (adecatumumab) and Edrecolomab.

[0166] Tyrosine related protein 1 or gp75 glycoprotein (TYRP1 / gp75) is a melanosomal protein that is involved in malignant melanocyte and melanoma progression (Ghanem et al., Mol. Oncol. 2011 April; 5(2): 150-155). Exemplary antibodies that bind TYRP1 / gp75 include TA99 (Saenger, et al., Cancer Research, 68(23): 9884-9891 , 2008), 20D7 (Patel, et aL, IOS Press, 16(3-4): 127- 1036, 2007), and flanvotumab (IMC-20D7S) (Khalil, et al., Clinical Cancer Research, 22(21 ): 5204-5210, 2016.

[0167] In particular embodiments, a binding domain can be derived from an antibody that binds TYRP1 / gp75, as described in US7951370. In particular embodiments, the antibody that binds TYRP1 / gp75 includes a CDRL1 including RASQSVSSYLA (SEQ ID NO: 62), a CDRL2 including DASNRAT (SEQ ID NO: 63), a CDRL3 including QQRSNWLMYT (SEQ ID NO: 68), a CDRH1 including GYTFTSYAMN (SEQ ID NO: 69), a CDRH2 including WINTNTGNPTYAQGFTG (SEQ ID NO: 70), and a CDRH3 sequence including RYSSSWYLDY (SEQ ID NO: 71 ).

[0168] In particular embodiments, the antibody that binds TYRP1 / gp75 includes a CDRL1 including RASGNIYNYLA (SEQ ID NO: 72), a CDRL2 including DAKTLAD (SEQ ID NO: 73), a CDRL3 including QHFWSLPFT (SEQ ID NO: 74), a CDRH1 including GFNIKDYFLH (SEQ ID NO: 75), a CDRH2 including WINPDNGNTVYDPKFQG (SEQ ID NO: 76), and a CDRH3 including DYTYEKAALDY (SEQ ID NO: 77).

[0169] In particular embodiments, TYRP1 / gp75-binding antibodies include a variable light chain including the sequence:EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSG SGSGTDFTLTISSLEPEDFAVYYCQQRSNWLMYTFGQGTKLEIK (SEQ ID NO: 78) and a variable heavy chain including the sequence: QVQLVQSGSELKKPGASVKISCKASGYTFTSYAMNWVRQAPGQGLECMGWINTNTGNPTYA QGFTGRFVFSMDTSVSTAYLQISSLKAEDTAIYYCAPRYSSSWYLDYWGQGTLVTVSS (SEQ ID NO: 79) or a variable heavy chain including the sequence: QVQLVQSGSELKKPGASVKISCKASGYTFTSYAMNWVRQAPGQGLESMGWINTNTGNPTYA QGFTGRFVFSMDTSVSTAYLQISSLKAEDTAIYYCAPRYSSSWYLDYWGQGTLVTVSS (SEQ ID NO: 80).

[0170] Exemplary binding domains that bind mesothelin include or are derived from anetumab, ravtansine, Amatuximab, and HN1.

[0171] In particular embodiments, the HN1 antibody includes a CDRL1 including RASEGIYHWLA (SEQ ID NO: 81), a CDRL2 including KASSLAS (SEQ ID NO: 82), a CDRL3 including QQYSNYPLT (SEQ ID NO: 83), a CDRH1 including TYYMQ (SEQ ID NO: 84), a CDRH2 including VINPSGVTSYAQKFQG (SEQ ID NO: 85), and a CDRH3 including WALWGDFGMDV (SEQ ID NO: 86).

[0172] US8206710 describes mesothelin-binding antibodies including: a variable light chain including the sequence: MGWSCIILFLVATATGVHSDIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPK RWIYDTSKLASGVPGRFSGSGSGNSYSLTISSVEAEDDATYYCQQWSKHPLTFGSGTKVEIKR TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS TYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 87) and a variable heavy chain including the sequence:MGWSCIILFLVATATGVHSQVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGK SLEWIGLITPYNGASSYNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGF DYWGSGTPVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRD ELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK* (SEQ ID NO: 88); as well as an antibody having a variable light chain including the sequence: MGWSCIILFLVATATGVHSEIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRL LIYDTSKLASGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWSKHPLTFGSGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 89) and a variable heavy chain including the sequence: MGWSCIILFLVATATGVHSQVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQ GLEWMGLITPYNGASSYNQKFRGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGYDGRG FDYWGSGTPVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS RDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 90).

[0173] Additional mesothelin-binding antibodies are described in US891 1732, US7081518, US8357783 and US8425904.

[0174] MUC16 binding domains can be derived from antibodies Oregovomab, ovarex, and abagovomab. US 7723485 describes a MUC16 binding antibody including a variable light chain including sequence:DIQMTQSPSSLSASVGDRVTITGRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFS GSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKS GTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 91 ) and a variable heavy chain including sequence:EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVHQAPGKGLEWVARIYPTNGYTRYADS VKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS VVTVPSSSLGTQTYIGNVNHKPSNTKVDKKVEPKSCDKTHTGPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK (SEQ ID NO: 92).

[0175] WQ2016149368 describes a MUC16 binding antibody including a variable light chain including a CDRL1 including SEDIYSG (SEQ ID NO: 93), a CDRL2 including GAS, a CDRL3 including GYSYSSTL (SEQ ID NO: 94), a CDRH1 including TLGMGVG (SEQ ID NO: 95), a CDRH2 including HIWWDDDKYYNPALKS (SEQ ID NO: 96), and a CDRH3 including IGTAQATDALDY (SEQ ID NO: 97).

[0176] A folate receptor binding antibody includes farletuzumab. In particular embodiments, farletuzumab is described in US9133275. In particular embodiments, farletuzumab includes a variable light chain including a CDRL1 including KASQSVSFAGTSLMH (SEQ ID NO: 98), a CDRL2 including RASNLEA (SEQ ID NO: 99), and a CDRL3 including QQSREYPYT (SEQ ID NO: 100), and a variable heavy chain including a CDRH1 including GYFMN (SEQ ID NO: 101 ), a CDRH2 including RIHPYDGDTFYNQKFQG (SEQ ID NO: 102), and a CDRH3 including YDGSRAMDY (SEQ ID NO: 103). Additional FOLR binding antibodies are described in US10101343B2, US8388972, and US8709432.

[0177] An exemplary EGFR antibody includes cetuximab. In particular embodiments, cetuximab is described in US US7598350. In particular embodiments, cetuximab includes a variable light chain including a CDRL1 including RASQSVSSYLA (SEQ ID NO: 62), a CDRL2 including DASNRAT (SEQ ID NO: 63), a CDRL3 including HQYGSTPLT (SEQ ID NO: 104), a CDRH1 including SGDYYWS (SEQ ID NO: 105), a CDRH2 including YIYYSGSTDYNPSLKS (SEQ ID NO: 106), and a CDRH3 including VSIFGVGTFDY (SEQ ID NO: 107).

[0178] Additional EGFR binding domains are described in US7247301 , US7723484, US7132511 , and US5844093. US7723484 particularly describes an EGFR binding antibody that includes a variable light chain including sequence: EIVLTQSPDFQSVTPKEKVTITCRASYSIGTNIHWYQQKPDQSPKLLIKYASESISGVPSRFSGS GSGTDFTLTINSLEAEDAATYYCQQNNNWPTTFGGGTKVEIK (SEQ ID NO: 108) and a variable heavy chain including sequence: QVTLKESGPVLVKPTETLTLTCTVSGFSLSNWDVHWIRQPPGKALEWLAVIWSGGATDYNTPF NSRLTISKDTSKSQVVLTMTNMDPVDTATYYCARALDYYDYNFAYWGQGTMVTVSS (SEQ ID NO: 109).

[0179] CD19 binding domains are found within antibody FMC63, SJ25C1 and HD37. (SJ25C1 : Bejcek et al. Cancer Res 2005, PMID 7538901 ; HD37: Pezutto et al. JI 1987, PMID 2437199). In particular embodiments, FMC63 CDRs include a CDRLI including RASQDISKYLN (SEQ ID NO: 110), a CDRL2 including SRLHSGV (SEQ ID NO: 11 1), a CDRL3 including GNTLPYTFG (SEQ ID NO: 112), a CDRH1 including DYGVS (SEQ ID NO: 113), a CDRH2 including VTWGSETTYYNSALKS (SEQ ID NO: 114), and a CDRH3 including YAMDYWG (SEQ ID NO: 115).

[0180] A number of antibodies specific for ROR1 are also known to those of skill in the art and can be readily characterized for sequence, antigen binding, and affinity. See, for example, W02008076868, WO / 2008103849, WO201008069, W02010124188, WQ2011079902, WO2011054007, WO2011 159847, WO2012076066, WO2012076727, WO 2012045085, and

[0181] Particular examples of antibodies that bind ROR1 include R1 1 , R12, 2A2, and Y31 .

[0182] The R11 antibody includes a CDRL1 including QASQSIDSNLA (SEQ ID NO: 1 16), a CDRL2 including RASNLAS (SEQ ID NO: 117), a CDRL3 including LGGVGNVSYRTS (SEQ ID NO: 118), a CDRH1 including DYPIS (SEQ ID NO: 1 19), a CDRH2 including FINSGGSTWYASWVKG (SEQ ID NO: 120), and a CDRH3 including GYSTYYCDFNI (SEQ ID NO: 121 ).

[0183] The R12 antibody includes a CDRL1 including TLSSAHKTDTID (SEQ ID NO: 122), a CDRL2 including GSYTKRP (SEQ ID NO: 123), a CDRL3 including GADYIGGYV (SEQ ID NO: 124), a CDRH1 including AYYMS (SEQ ID NO: 125), a CDRH2 including TIYPSSGKTYYATWVNG (SEQ ID NO: 126), and a CDRH3 including DSYADDGALFNI (SEQ ID NO: 127).

[0184] The 2A2 antibody includes a CDRL1 including KASQNVDAAVA (SEQ ID NO: 128), a CDRL2 including SASNRYT (SEQ ID NO: 129), a CDRL3 including QQYDIYPYT (SEQ ID NO: 130), a CDRH1 including DYEMH (SEQ ID NO: 131 ), a CDRH2 including AIDPETGGTAYNQKFKG (SEQ ID NO: 132), and a CDRH3 including YYDYDSFTY (SEQ ID NO: 133).

[0185] The Y31 antibody includes a CDRL1 including QASQSIGSYLA (SEQ ID NO: 134), a CDRL2 including YASNLAS (SEQ ID NO: 135), a CDRL3 including LGSLSNSDNV (SEQ ID NO: 136), a CDRH1 including SHWMS (SEQ ID NO: 137), a CDRH2 including IIAASGSTYYANWAKG (SEQ ID NO: 138), and a CDRH3 including DYGDYRLVTFNI (SEQ ID NO: 139).

[0186] A Her2 binding domain can be derived from the 4D5 antibody. The 4D5 antibody includes a CDRL1 including RASQDVNTAVAW (SEQ ID NO: 140), a CDRL2 including YSASFLES (SEQ ID NO: 141), a CDRL3 including QQHYTTPT (SEQ ID NO: 142), a CDRH1 including SGFNTKDTYIHW (SEQ ID NO: 143), a CDRH2 including RIYPTNGYTRYADSVKGR (SEQ ID NO: 144), and a CDRH3 including WGGDGFYAMDV (SEQ ID NO: 145).

[0187] PD-L1 binding antibodies include the 3G10 antibody and those described in US 2016 / 0222117. In particular embodiments, binding domains derived from the 3G10 antibody include a CDRL1 including RASQSVSSYL (SEQ ID NO: 146), a CDRL2 including DASNRAT (SEQ ID NO: 63), a CDRL3 including QQRSNWPRT (SEQ ID NO: 147), a CDRH1 including DYGFS (SEQ ID NO: 148), a CDRH2 including WITAYNGNTNYAQKLQG (SEQ ID NO: 149), and a CDRH3 including DYFYGMDY (SEQ ID NO: 150).

[0188] PD-L1 binding domains can also include a CDRL1 including RASQDVSTAVA (SEQ ID NO: 151 ), a CDRL2 including SASFLYS (SEQ ID NO: 152), a CDRL3 including QQYLYHPAT(SEQ ID NO: 153), a CDRH1 including SGFTFSDSWIH (SEQ ID NO: 154), a CDRH2 including WISPYGGSTYYADSVKG (SEQ ID NO: 155), and a CDRH3 including RHWPGGFDY (SEQ ID NO: 156) or (ii) a CDRL1 including TGTSSDVGGYNYVS (SEQ ID NO: 157), a CDRL2 including DVSNRPS (SEQ ID NO: 158), a CDRL3 including SSYTSSSTRV (SEQ ID NO: 159), a CDRH1 including SGFTFSSYIMM (SEQ ID NO: 160), a CDRH2 including SIYPSGGITFYADTVKG (SEQ ID NO: 161), and a CDRH3 including IKLGTVTTVDY (SEQ ID NO: 162).

[0189] Additional antibodies with PD-L1 binding domains include Atezolizumab, Avelumab, and Durvalumab.

[0190] (v-b) Spacers. Spacers are used to create appropriate distances and / or flexibility from other recombinant receptor sub-components. As indicated, in particular embodiments, the length of a spacer is customized for binding targeted antigen-expressing cells and mediating destruction. In particular embodiments, a spacer length can be selected based upon the location of a cellular marker antigen epitope, affinity of a binding domain for the antigen epitope, and / or the ability of the recombinant receptor to mediate cell destruction following target antigen binding.

[0191] Spacers typically include those having 10 to 250 amino acids, 10 to 200 amino acids, 10 to 150 amino acids, 10 to 100 amino acids, 10 to 50 amino acids, or 10 to 25 amino acids.

[0192] In particular embodiments, a spacer is 5 amino acids, 8 amino acids, 10 amino acids, 12 amino acids, 14 amino acids, 20 amino acids, 21 amino acids, 26 amino acids, 27 amino acids, 45 amino acids, 50 amino acids, or 75 amino acids. These lengths qualify as short spacers.

[0193] In particular embodiments, a spacer is 76 amino acids, 90 amino acids, 100 amino acids, 110 amino acids, 120 amino acids, 125 amino acids, 128 amino acids, 131 amino acids, 135 amino acids, 140 amino acids, 150 amino acids, 160 amino acids, 170 amino acids, or 179 amino acids. These lengths qualify as intermediate spacers.

[0194] In particular embodiments, a spacer is 180 amino acids, 190 amino acids, 200 amino acids, 210 amino acids, 212 amino acids, 214 amino acids, 216 amino acids, 218 amino acids, 220 amino acids, 228 amino acids, 230 amino acids, 240 amino acids, 250 amino acids, 260 amino acids, or 270 amino acids. These lengths qualify as long spacers.

[0195] Exemplary spacers include all or a portion of an immunoglobulin hinge region. An immunoglobulin hinge region may be a wild-type immunoglobulin hinge region or an altered wildtype immunoglobulin hinge region. In certain embodiments, an immunoglobulin hinge region is a human immunoglobulin hinge region. As used herein, a “wild type immunoglobulin hinge region” refers to a naturally occurring upper and middle hinge amino acid sequences interposed between and connecting the CH1 and CH2 domains (for IgG, IgA, and IgD) or interposed between and connecting the CH1 and CH3 domains (for IgE and IgM) found in the heavy chain of an antibody.

[0196] An immunoglobulin hinge region may be an IgG, IgA, IgD, IgE, or IgM hinge region. An IgG hinge region may be an IgG 1 , lgG2, lgG3, or lgG4 hinge region. Sequences from IgG 1 , lgG2, lgG3, lgG4 or IgD can be used alone or in combination with all or a portion of a CH2 region; all or a portion of a CH3 region; or all or a portion of a CH2 region and all or a portion of a CH3 region.

[0197] In particular embodiments, the spacer is a short spacer including an lgG4 hinge region. In particular embodiments the short spacer is encoded by any of SEQ ID NOs: 164, 165, or 166. In particular embodiments, the lgG4 hinge region includes the sequence as set forth in SEQ ID NO: 163. In particular embodiments, the spacer is an intermediate spacer including an lgG4 hinge region and an lgG4 CH3 region. In particular embodiments, the spacer is a long spacer including an lgG4 hinge region, an lgG4 CH2 region, and an lgG4 CH3 region. In particular embodiments, the lgG4 CH3 region includes the sequence as set forth in SEQ ID NO: 171 or is encoded by SEQ ID NOs: 172 or 173. In particular embodiments, the lgG4 CH2 region includes the sequence as set forth in SEQ ID NOs: 167 or 168 or is encoded by SEQ ID NOs: 169 or 170. In particular embodiments, the long spacer includes a 4 / 2-N / Q mutation in the CH2 domain. Mutations can be used to prevent Fc-gamma receptor binding and activation-induced cell death.

[0198] Other examples of hinge regions that can be used in CAR described herein include the hinge region present in the extracellular regions of type 1 membrane proteins, such as CD8a, CD4, CD28 and CD7, which may be wild-type or variants thereof.

[0199] In particular embodiments, a spacer includes a hinge region that includes a type II C-lectin interdomain (stalk) region or a cluster of differentiation (CD) molecule stalk region. A “stalk region” of a type II C-lectin or CD molecule refers to the portion of the extracellular domain (ECD) of the type II C-lectin or CD molecule that is located between the C-type lectin-like domain (CTLD; e.g., similar to CTLD of natural killer cell receptors) and the hydrophobic portion (transmembrane domain). For example, the ECD of human CD94 (GenBank Accession No. AAC50291.1 ) corresponds to amino acid residues 34-179, but the CTLD corresponds to amino acid residues 61-176, so the stalk region of the human CD94 molecule includes amino acid residues 34-60, which are located between the hydrophobic portion (transmembrane domain) and CTLD (see Boyington et al., Immunity 10:15, 1999; for descriptions of other stalk regions, see also Beavil et al., Proc. Nat'L Acad. Sci. USA 89:153, 1992; and Figdor et aL, Nat. Rev. Immunol. 2:11 , 2002). These type II C-lectin or CD molecules may also have junction amino acids (described below) between the stalk region and the transmembrane region or the CTLD. In another example, the 233 amino acid human NKG2A protein (GenBank Accession No. P26715.1) has a hydrophobic portion (transmembrane domain) ranging from amino acids 71-93 and an ECD ranging from amino acids 94-233. The CTLD includes amino acids 119-231 and the stalk region includesamino acids 99-116, which may be flanked by additional junction amino acids. Other type II C- lectin or CD molecules, as well as their extracellular ligand-binding domains, stalk regions, and CTLDs are known in the art (see, e.g., GenBank Accession Nos. NP 001993.2; AAH07037.1 ; NP 001773.1 ; AAL65234.1 ; CAA04925.1 ; for the sequences of human CD23, CD69, CD72, NKG2A, and NKG2D and their descriptions, respectively).

[0200] (v-c) Transmembrane Domains. As indicated, transmembrane domains within a CAR serve to connect the extracellular component and intracellular component through the cell membrane. The transmembrane domain can anchor the expressed molecule in the modified cell’s membrane.

[0201] The transmembrane domain can be derived either from a natural and / or a synthetic source. When the source is natural, the transmembrane domain can be derived from any membrane-bound or transmembrane protein. Transmembrane domains can include at least the transmembrane region(s) of the a, p or chain of a T-cell receptor, CD28, CD27, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22; CD33, CD37, CD64, CD80, CD86, CD134, CD137 CD154, Toll-like receptor 1 (TLR1 ), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR9. In particular embodiments, a transmembrane domain may include at least the transmembrane region(s) of, e.g., KIRDS2, 0X40, CD2, CD27, LFA-1 (CD 11 a, CD18), ICOS (CD278), 4-1 BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1 ), NKp44, NKp30, NKp46, CD160, CD19, IL2Rp, IL2Ry, IL7R a, ITGA1 , VLA1 , CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDI Id, ITGAE, CD103, ITGAL, CDI la, ITGAM, CDI lb, ITGAX, CDI Ic, ITGB1 , CD29, ITGB2, CD18, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1 , CRT AM, Ly9(CD229), , PSGL1 , CD100 (SEMA4D), SLAMF6 (NTB- A, LylOS), SLAM (SLAMF1 , CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp, NKG2D, or NKG2C. In particular embodiments, a variety of human hinges can be employed as well including the human Ig (immunoglobulin) hinge (e.g., an lgG4 hinge, an IgD hinge), a GS linker (e.g., a GS linker described herein), a KIR2DS2 hinge or a CD8a hinge. In particular embodiments, the CAR includes a CD28 transmembrane domain.

[0202] In particular embodiments, a transmembrane domain has a three-dimensional structure that is thermodynamically stable in a cell membrane, and generally ranges in length from 15 to 30 amino acids. The structure of a transmembrane domain can include an a helix, a p barrel, a p sheet, a p helix, or any combination thereof.

[0203] A transmembrane domain can include one or more additional amino acids adjacent to the transmembrane region, e.g., one or more amino acid within the extracellular region of the CAR (e.g., up to 15 amino acids of the extracellular region) and / or one or more additional amino acidswithin the intracellular region of the CAR (e.g., up to 15 amino acids of the intracellular components). In one aspect, the transmembrane domain is from the same protein that the signaling domain, co-stimulatory domain or the hinge domain is derived from. In another aspect, the transmembrane domain is not derived from the same protein that any other domain of the CAR is derived from. In some instances, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other unintended members of the receptor complex. In particular embodiments, the transmembrane domain is encoded by the nucleic acid sequence encoding the CD28 transmembrane domain (SEQ ID NOs: 177, 178, 179, or 180). In particular embodiments, the transmembrane domain includes the amino acid sequence of the CD28 transmembrane domain (SEQ ID NOs: 174, 175, or 176).

[0204] (v-d) Intracellular Effector Domains. The intracellular effector domains of a CAR are responsible for activation of the cell in which the CAR is expressed. The term “effector domain” is thus meant to include any portion of the intracellular domain sufficient to transduce an activation signal. An effector domain can directly or indirectly promote a biological or physiological response in a cell when receiving the appropriate signal. In certain embodiments, an effector domain is part of a protein or protein complex that receives a signal when bound, or it binds directly to a target molecule, which triggers a signal from the effector domain. An effector domain may directly promote a cellular response when it contains one or more signaling domains or motifs, such as an immunoreceptor tyrosine-based activation motif (ITAM). In other embodiments, an effector domain will indirectly promote a cellular response by associating with one or more other proteins that directly promote a cellular response, such as co-stimulatory domains.

[0205] Effector domains can provide for activation of at least one function of a modified cell upon binding to the cellular marker expressed by a cancer cell. Activation of the modified cell can include one or more of differentiation, proliferation and / or activation or other effector functions. In particular embodiments, an effector domain can include an intracellular signaling component including a T cell receptor and a co-stimulatory domain which can include the cytoplasmic sequence from co-receptor or co-stimulatory molecule.

[0206] An effector domain can include one, two, three or more intracellular signaling components (e.g., receptor signaling domains, cytoplasmic signaling sequences), co-stimulatory domains, or combinations thereof. Exemplary effector domains include signaling and stimulatory domains selected from: CD79B, DAP1 LRP, NKG2D,Wnt, Zap70, or any combination thereof. In particular embodiments, exemplary effector domains include signaling and co-stimulatory domains selected from: CD86, FcyRlla, DAP12, CD30, CD40, PD-1 , lymphocyte function-associated antigen-1 (LFA-1 ), CD2, CD7, LIGHT, NKG2C, B7- H3, a ligand that specifically binds with CD83, CDS, ICAM-1 , GITR, BAFFR, SLAMF7, NKp80CD11 b, ITGAX, CD11c, ITGB1. CD29, ITGB2, CD18, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1 , CRTAM, Ly9 (CD229), PSGL1 , CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, GADS, PAG / Cbp, NKp44, NKp30, NKp46, TLR1 , TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9. In particular embodiments, the effector domain includes a CD3 signaling domain.

[0207] Intracellular signaling component sequences that act in a stimulatory manner may include iTAMs. Examples of iTAMs including primary cytoplasmic signaling sequences include those derived from CD3y, CD35, CD3c, CD3 , CD5, CD22, CD66d, CD79a, CD79b, and common FcRy (FCER1 G), FcyRlla, FcRp (Fee Rib), DAP10, and DAP12. In particular embodiments, variants of CD3 retain at least one, two, three, or all ITAM regions.

[0208] In particular embodiments, an effector domain includes a cytoplasmic portion that associates with a cytoplasmic signaling protein, wherein the cytoplasmic signaling protein is a lymphocyte receptor or signaling domain thereof, a protein including a plurality of ITAMs, a costimulatory domain, or any combination thereof.

[0209] Additional examples of intracellular signaling components include the cytoplasmic sequences of the CD3 chain, and / or co-receptors that act in concert to initiate signal transduction following binding domain engagement.

[0210] A co-stimulatory domain is a domain whose activation can be required for an efficient lymphocyte response to cellular marker binding. Some molecules are interchangeable as intracellular signaling components or co-stimulatory domains. Examples of costimulatory domains include CD27, CD28, 4-1 BB (CD 137), 0X40, CD30, CD40, PD-1 , ICOS, lymphocyte function- associated antigen-1 (LFA-1 ), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83. For example, CD27 co-stimulation has been demonstrated to enhance expansion, effector function, and survival of human CAR T cells in vitro and augments human T cell persistence and anti-cancer activity in vivo (Song et al. Blood. 2012; 119(3):696-706). Further examples of such co-stimulatory domain molecules include CDS, ICAM-1 , GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1 ), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8a, CD8 ,IL2Rp, IL2RY, IL7Ra, ITGA4, VLA1 , CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDIId, ITGAE, CD103, ITGAL, CDIIa, ITGAM, CDI lb, ITGAX, CDIIc, ITGBI, CD29, ITGB2, CD18, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), NKG2D, CEACAM1 , CRTAM, Ly9 (CD229), PSGL1 , CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, LylOS), SLAM (SLAMF1 , CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, and CD19a. In particular embodiments, the costimulatory domain includes a 4-1 BB signaling domain.

[0211] In particular embodiments, the nucleic acid sequences encoding the intracellular signaling components includes CD3 coding sequence (SEQ ID NO: 184 or 185) and a variant of the 4- 1 BB signaling coding sequence (SEQ ID NOs: 189, 190, or 191). In particular embodiments, the amino acid sequence of the intracellular signaling component includes a variant of CD3 (SEQ ID NOs: 181 , 182, or 183) and a portion of the 4-1 BB (SEQ ID NO: 186, 187, or 188) intracellular signaling component.

[0212] In particular embodiments, the intracellular signaling component includes (i) all or a portion of the signaling domain of CD3 , (ii) all or a portion of the signaling domain of 4-1 BB, or (iii) all or a portion of the signaling domain of CD3 and 4-1 BB. In particular embodiments, the intracellular signaling component includes (i) all or a portion of the signaling domain of CD3 , (ii) all or a portion of the signaling domain of 4-1 BB, (iii) all or a portion of the signaling domain of CD28, (iv) or all or a portion of the signaling domain of CD3 , 4-1 BB, and CD28.

[0213] Intracellular components may also include one or more of a protein of a Wnt signaling pathway (e.g., LRP, Ryk, or ROR2), NOTCH signaling pathway (e.g., NOTCH1 , NOTCH2, NOTCH3, or NOTCH4), Hedgehog signaling pathway (e.g., PTCH or SMO), receptor tyrosine kinases (RTKs) (e.g., epidermal growth factor (EGF) receptor family, fibroblast growth factor (FGF) receptor family, hepatocyte growth factor (HGF) receptor family, insulin receptor (IR) family, platelet-derived growth factor (PDGF) receptor family, vascular endothelial growth factor (VEGF) receptor family, tropomycin receptor kinase (Trk) receptor family, ephrin (Eph) receptor family, AXL receptor family, leukocyte tyrosine kinase (LTK) receptor family, tyrosine kinase with immunoglobulin-like and EGF-like domains 1 (TIE) receptor family, receptor tyrosine kinase-like orphan (ROR) receptor family, discoidin domain (DDR) receptor family, rearranged during transfection (RET) receptor family, tyrosine-protein kinase-like (PTK7) receptor family, related to receptor tyrosine kinase (RYK) receptor family, or muscle specific kinase (MuSK) receptor family); G-protein-coupled receptors, GPCRs (Frizzled or Smoothened); serine / threonine kinase receptors (BMPR or TGFR); or cytokine receptors (IL1 R, IL2R, IL7R, or IL15R).

[0214] (v-e) Linkers. As used herein, a linker can include a chemical moiety that serves to connecttwo other subcomponents of the molecule. Some linkers serve no purpose other than to link components while many linkers serve an additional purpose. Linkers can, for example, link VL and VH of antibody derived binding domains of scFvs and serve as junction amino acids between subcomponent portions of a expressed molecule or recombinant receptor (e.g., CAR).

[0215] Linkers can be flexible, rigid, or semi-rigid, depending on the desired function of the linker. Linkers can include junction amino acids. For example, in particular embodiments, linkers provide flexibility and room for conformational movement between different components of an expressed molecule. Commonly used flexible linkers include Gly-Ser linkers. In particular embodiments, the linker sequence includes sets of glycine and serine repeats such as from one to ten repeats of (GlyxSery)n, wherein x and y are independently an integer from 0 to 10 provided that x and y are not both 0 and wherein n is an integer of 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10). Particular examples include (Gly4Ser)n(SEQ ID NO: 202), (Gly3Ser)n(Gly4Ser)n(SEQ ID NO: 203), (Gly3Ser)n(Gly2Ser)n (SEQ ID NO: 204), or (Gly3Ser)n(Gly4Ser)i (SEQ ID NO: 205). In particular embodiments, the linker is (Gly4Ser)4(SEQ ID NO: 206), (Gly4Ser)3(SEQ ID NO: 207), (Gly4Ser)2(SEQ ID NO: 208), (Gly4Ser)i (SEQ ID NO: 209), (Gly3Ser)2(SEQ ID NO: 210), (Gly3Ser)i (SEQ ID NO: 21 1 ), (Gly2Ser)2(SEQ ID NO: 212) or (Gly2Ser)i, GGSGGGSGGSG (SEQ ID NO: 213), GGSGGGSGSG (SEQ ID NO: 214), or GGSGGGSG (SEQ ID NO: 215).

[0216] In particular embodiments, a linker region is (GGGGS)n(SEQ ID NO: 202) wherein n is an integer including, 1 , 2, 3, 4, 5, 6, 7, 8, 9, or more. In particular embodiments, the linker includes the Whitlow linker: GSTSGSGKPGSGEGSTKG (SEQ ID NO: 216; Whitlow et ah, Protein Eng 6(8):989 -95, 1993). In particular embodiments, the spacer is (EAAAK)n(SEQ ID NO: 216) wherein n is an integer including 1 , 2, 3, 4, 5, 6, 7, 8, 9, or more.

[0217] In some situations, flexible linkers may be incapable of maintaining a distance or positioning of an expressed molecule (e.g., CAR) needed for a particular use. In these instances, rigid or semi-rigid linkers may be useful. Examples of rigid or semi-rigid linkers include prolinerich linkers. In particular embodiments, a proline-rich linker is a peptide sequence having more proline residues than would be expected based on chance alone. In particular embodiments, a proline-rich linker is one having at least 30%, at least 35%, at least 36%, at least 39%, at least 40%, at least 48%, at least 50%, or at least 51 % proline residues. Particular examples of prolinerich linkers include fragments of proline-rich salivary proteins (PRPs).

[0218] Linkers can be susceptible to cleavage (cleavable linker), such as, acid-induced cleavage, photo-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage. Alternatively, linkers can be substantially resistant to cleavage (e.g., stable linker or noncleavable linker). In some aspects, the linker is a procharged linker, a hydrophilic linker, ora dicarboxylic acid-based linker.

[0219] Junction amino acids can be a linker which can be used to connect sequences when the distance provided by a spacer is not needed and / or wanted. For example, junction amino acids can be short amino acid sequences that can be used to connect co-stimulatory intracellular signaling components. In particular embodiments, junction amino acids are 9 amino acids or less (e.g., 2, 3, 4, 5, 6, 7, 8, or 9 amino acids). In particular embodiments, a glycine-serine doublet can be used as a suitable junction amino acid linker. In particular embodiments, a single amino acid, e.g., an alanine, a glycine, can be used as a suitable junction amino acid.

[0220] (v-f) Control Features Including Tag Cassettes, Transduction Markers, and / or Suicide Switches. In particular embodiments, genetic constructs including a sequence encoding an expressed molecule (e.g., recombinant receptor) can include one or more tag cassettes and / or transduction markers. Tag cassettes and transduction markers can be used to activate, promote proliferation of, detect, enrich for, isolate, track, deplete and / or eliminate reprogrammed cells in vitro, in vivo and / or ex vivo. "Tag cassette" refers to a unique synthetic peptide sequence affixed to, fused to, or that is part of a genetic construct, to which a cognate binding molecule e.g., ligand, antibody, or other binding partner) is capable of specifically binding where the binding property can be used to activate, promote proliferation of, detect, enrich for, isolate, track, deplete and / or eliminate the tagged protein and / or cells expressing the tagged protein. Transduction markers can serve the same purposes but are derived from naturally occurring molecules and are often expressed using a skipping element that separates the transduction marker from the rest of the expressed molecule coding sequence.

[0221] Tag cassettes that bind cognate binding molecules include, for example, His tag (HHHHHH; SEQ ID NO: 217), Flag tag (DYKDDDDK; SEQ ID NO: 218), Xpress tag (DLYDDDDK; SEQ ID NO: 219), Avi tag (GLNDIFEAQKIEWHE; SEQ ID NO: 220), Calmodulin tag (KRRWKKNFIAVSAANRFKKISSSGAL; SEQ ID NO: 221 ), Polyglutamate tag, HA tag (YPYDVPDYA; SEQ ID NO: 222), Myc tag (EQKLISEEDL; SEQ ID NO: 223), Strep tag (which refers the original STREP® tag (WRHPQFGG; SEQ ID NO: 224), STREP® tag II (WSHPQFEK SEQ ID NO: 225 (IBA Institut fur Bioanalytik, Germany); see, e.g., US 7,981 ,632), Softag 1 (SLAELLNAGLGGS; SEQ ID NO: 226), Softag 3 (TQDPSRVG; SEQ ID NO: 227), and V5 tag (GKPIPNPLLGLDST; SEQ ID NO: 228).

[0222] Conjugate binding molecules that specifically bind tag cassette sequences disclosed herein are commercially available. For example, His tag antibodies are commercially available from suppliers including Life Technologies, Pierce Antibodies, and GenScript. Flag tag antibodies are commercially available from suppliers including Pierce Antibodies, GenScript, and Sigma-Aldrich. Xpress tag antibodies are commercially available from suppliers including Pierce Antibodies, Life Technologies and GenScript. Avi tag antibodies are commercially available from suppliers including Pierce Antibodies, IsBio, and Genecopoeia. Calmodulin tag antibodies are commercially available from suppliers including Santa Cruz Biotechnology, Abeam, and Pierce Antibodies. HA tag antibodies are commercially available from suppliers including Pierce Antibodies, Cell Signal and Abeam. Myc tag antibodies are commercially available from suppliers including Santa Cruz Biotechnology, Abeam, and Cell Signal. Strep tag antibodies are commercially available from suppliers including Abeam, Iba, and Qiagen.

[0223] T ransduction markers may be selected from at least one of a truncated CD19 (tCD19; see Budde et al., Blood 122: 1660, 2013); a truncated human EGFR (tEGFR or EGFRt; see Wang et al., Blood 118: 1255, 201 1 ); an ECD of human CD34; and / or RQR8 which combines target epitopes from CD34 (see Fehse et al, Mol. Therapy 1 ( 5 Pt 1 ); 448-456, 2000) and CD20 antigens (see Philip et al, Blood 124: 1277-1278).

[0224] In particular embodiments, genetic constructs can include a polynucleotide that encodes a self-cleaving polypeptide, wherein the polynucleotide encoding the self-cleaving polypeptide is located between the polynucleotide encoding the expressed molecule and a polynucleotide encoding a transduction marker (e.g., EGFRt). Exemplary self-cleaving polypeptides include 2A peptide from porcine teschovirus-1 (P2A), Thosea asigna virus (T2A), equine rhinitis A virus (E2A), foot-and-mouth disease virus (F2A), or variants thereof. Further exemplary nucleic acid and amino acid sequences of 2A peptides are set forth in, for example, Kim et al. (PLOS One 6:e18556 (2011 ). In particular embodiments, cells are reprogrammed to express a self-cleaving polypeptide. In particular embodiments, the self-cleaving polypeptide includes T2A.

[0225] Control features may be present in multiple copies in a genetic construct or can be expressed as distinct molecules with the use of a skipping element. For example, a genetic construct can have one, two, three, four or five tag cassettes and / or one, two, three, four, or five transduction markers could also be expressed. For example, embodiments can include a genetic construct having two Myc tag cassettes, or a His tag and an HA tag cassette, or a HA tag and a Softag 1 tag cassette, or a Myc tag and a SBP tag cassette. Exemplary transduction markers and cognate pairs are described in US 13 / 463,247.

[0226] One advantage of including at least one control feature in a genetic construct is that cells expressing an expressed molecule administered to a subject can be increased or depleted using the cognate binding molecule to a tag cassette. In certain embodiments, the present disclosure provides a method for depleting a modified cell expressing an expressed molecule (e.g., recombinant receptor) by using an antibody specific for the tag cassette, using a cognate bindingmolecule specific for the control feature, or by using a second modified cell expressing a CAR and having specificity for the control feature. Elimination of modified cells may be accomplished using depletion agents specific for a control feature. For example, if EGFRt is used, then an anti- EGFRt binding domain (e.g., antibody, scFv) fused to or conjugated to a cell-toxic reagent (such as a toxin, radiometal) may be used, or an anti-EGFRt / anti-CD3 bispecific scFv, or an anti-EGFRt CAR T cell may be used.

[0227] In particular embodiments, a polynucleotide encoding an iCaspase9 construct (iCasp9) may be inserted into a genetic construct as a suicide switch.

[0228] In certain embodiments, modified cells expressing an expressed molecule may be detected or tracked in vivo by using antibodies that bind with specificity to a control feature (e.g., anti-Tag antibodies), or by other cognate binding molecules that specifically bind the control feature, which binding partners for the control feature are conjugated to a fluorescent dye, radiotracer, iron-oxide nanoparticle or other imaging agent known in the art for detection by X-ray, CT-scan, MRI-scan, PET-scan, ultrasound, flow-cytometry, near infrared imaging systems, or other imaging modalities (see, e.g., Yu, et al., Theranostics 2:3, 2012).

[0229] Thus, modified cells expressing at least one control feature with an expressed molecule can be, e.g., more readily identified, isolated, sorted, induced to proliferate, tracked, and / or eliminated as compared to a modified cell without a tag cassette.

[0230] Many considerations associated with CAR apply to eTCR as well. eTCR disclosed herein include a binding domain that binds a target antigen (e.g., an scFv) linked to the Caand / or Cp chains of a TCR. A TCR is a heterodimeric fusion protein that typically includes an a and 3 chain. Each chain includes a variable region (Vaand Vp) and a constant region (Caand Cp). In particular embodiments, an eTCR does not include the native TCR variable region but does include the native TCR constant region. In particular embodiments, the eTCR includes an scFv as the variable region of either the a or p chain. In particular embodiments, the eTCR includes an scFv as the variable region of both the a and p chain. In particular embodiments, eTCR include a Caand / or Cp chain sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to an amino acid sequence of a known or identified TCR Caor Cp.

[0231] There are numerous ways to identify and select particular TCR for use as binding domains in eTCR. For example, the sequences of numerous TCR that bind particular antigen fragments are known and publicly available.

[0232] Useful TCR can also be identified by isolating T cells that bind a particular antigen and sequencing the TCR chains. As examples, antigen-specific T cells may be induced by in vitrocultivation of isolated human T cells in the presence of an antigen / MHC complex. TCR genes encoding TCR that bind the antigen / MHC complex can be readily cloned by, for example, the 5' RACE procedure using primers corresponding to the sequences specific to the TCR a-chain gene and the TCR [3-chain gene.

[0233] In particular embodiments, it may be necessary to pair TCR chains following sequencing (i.e., to perform paired chain analysis). Various methods can be utilized to pair isolated a and p chains. In particular embodiments post-sequencing pairing may be unnecessary or relatively simple, for example in embodiments in which the a and p chain pairing information is not lost in the procedure, such as if one were to sequence from single cells. Chain pairing may also be performed using multiwell sequencing. Assays such as PairSEQ® (Adaptive Biotechnologies Corp., Seattle, WA) have also been developed.

[0234] For particular examples of TCR that can be used within the context of the current disclosure, see, for example, WO2018 / 129270; WO2017 / 112944; WO2011 / 039507; US 8,008,438; US2016 / 0083449; US2015 / 0246959; Stromnes, et al. (2015) Cancer cell 28(5): 638- 652; Kobayashi, et al. (2013) Nature Medicine 19: 1542-1546); Varela-Rohena, et al. (2008) Nature Medicine. 14(12): 1390-1395); and Robbins et al. (2008) The Journal of Immunology 180(9): 6116-6131.

[0235] A CAR / TCR hybrid can include any recombinant receptor including features of a CAR and features of a TCR. In particular embodiments, the extracellular components of a TCR are fused with the intracellular components of a CAR, the intracellular components of a TCR are fused with the extracellular components of a CAR, or one or more the extracellular and intracellular components of a TCR are fused with one more more of the extracellular and intracellular components of a CAR. In particular embodiments, a CAR / TCR hybrid includes a TCR-CD3 complex with an immunoglobulin heavy and light chain to form an HLA-independent TCR.

[0236] Other recombinant proteins that re-educate the recruited immune system include Fas- TNFR proteins (e.g., Fas-CD40).

[0237] In particular embodiments, an expressed molecule can include a cytotoxin, an immunomodulatory protein (i.e., a protein that enhances a host immune response to an antigen), a prodrug activator, a tumor suppressor, a prodrug converting enzyme, proteins capable of causing cell to cell fusion, a TAP inhibitor antisense RNA molecule, or a ribozyme. Examples of immunomodulatory proteins include, for example, cytokines. Cytokines include an interleukins, such as IL-1 , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 , IL-12, IL-13, IL-14, IL-15, IL- 16, IL-17, IL-18, IL-20; a, [3 or y-interferons, tumor necrosis factor alpha (TNFa), CD40L, granulocyte macrophage colony stimulating factor (GM-CSF), macrophage colony stimulatingfactor (M-CSF), and granulocyte colony stimulating factor (G-CSF), chemokines (such as neutrophil activating protein (NAP), macrophage chemoattractant and activating factor (MCAF), RANTES, and macrophage inflammatory peptides MIP-1 a and MIP-1 b), complement components and their receptors, immune system accessory molecules (e.g., B7.1 and B7.2), adhesion molecules (e.g., ICAM-1 , 2, and 3), and adhesion receptor molecules. Pro-drug activators include nitroeductase and cytochrome p450. A tumor suppressor includes p53. A prodrug converting enzymes includes cytosine deaminase. Proteins capable of causing cell to cell fusion include gibbon ape leukaemia fusogenic glycoprotein. TAP inhibitors include the bovine herpesvirus (BHV) UL49.5 polypeptide.

[0238] (vi) Optional Additional Immune Stimulants. In particular embodiments, implants disclosed herein can optionally include an immune stimulant in addition to the ICAF. The immune stimulant can be, for example, a cytokine, an antibody, a small molecule, an siRNA, a plasmid DNA, and / or a vaccine adjuvant.

[0239] Exemplary cytokines include any of the cytokines described above including IL-2, IL-7, IL- 12, IL-15, IL-18, IL-21 , TNFa, IFN-a, IFN-p, IFN-y, or GM-CSF. In particular embodiments, the immune stimulant may be a cytokine and or a combination of cytokines, such as IL-2, IL-12 or IL- 15 in combination with IFN-a, IFN-p or IFN-y, or GM-CSF, or any effective combination thereof, or any other effective combination of cytokines. The above-identified cytokines stimulate TH1 responses, but cytokines that stimulate TH2 responses may also be used, such as IL-4, IL-10, IL- 11 , or any effective combination thereof. Also, combinations of cytokines that stimulate T 1 responses along with cytokines that stimulate T 2 responses may be used.

[0240] Exemplary antibodies include anti-PD1 , anti-PDL1 , anti-CTLA-4, anti-TIM3, agonistic anti- CD40, agonistic anti-4-1 BB, and / or bispecific antibodies (e.g., BITE-antibodies: anti-CD3 / anti- tumor antigen). Exemplary small molecule drugs include, TGF-beta inhibitors, SHP-inhibitors, STAT-3 inhibitors, and / or STAT-5 inhibitors. Any siRNA capable of down-regulating immune- suppressive signals or oncogenic pathways (such as kras) can be used whereas any plasmid DNA (such as minicircle DNA) encoding immune-stimulatory proteins can be used. Exemplary vaccine adjuvants, include any kind of Toll-like receptor ligand or combinations thereof (e.g. CpG, Cpg-28 (a TLR9 agonist), Polyriboinosinic polyribocylidylic acid (Poly(l:C)), a-galactoceramide, MPLA, Motolimod (VTX-2337, a novel TLR8 agonist developed by VentiRx), IMO-2055 (EMD1201081 ), TMX-101 (imiquimod), MGN1703 (a TLR9 agonist), G100 (a stabilized emulsion of the TLR4 agonist glucopyranosyl lipid A), Entolimod (a derivative of Salmonella flagellin also known as CBLB502), Hiltonol (a TLR3 agonist), and Imiquimod), and / or inhibitors of heat-shock protein 90 (Hsp90), such as 17-DMAG (17-dimethylaminoethylamino-17-demethoxygeldanamycin).

[0241] Small molecules that increase immunity can include programmed cell death protein 1 (PD- 1 ) and its ligand (PD-L1) inhibitors, RORyt (retinoic acid-related orphan receptor-gamma t) agonists, chemokine targets such as CXCR4 antagonists, CXCL12 antagonists, CXCL10 agonists, TGFp (transforming growth factor P) inhibitors, ALK5 (activin receptor-like kinase 5) inhibitors; SHIP1 (SH2-containing 5’-inositol phosphatase 1) inhibitors, STING (stimulator of interferon genes) agonists, and toll-like receptor (TLR) agonists. Commercially available small molecule PD-1 / PD-L1 inhibitors include CA-170, INCB086550, IMMH-010, MAX-10181 , GS- 4224, ASC61 , and BPI-371153. Commercially available small molecule RORyt agonists include LYC-55716. Commercially available small molecule chemokine receptor antagonists include AZD5069, X4P-001 , Vicriviroc, Maraviroc, and SX-682. Commercially available small molecule TGFp inhibitors include EW7197. Commercially available small molecule STING agonists include ADU-S100, E7766, TAK-676, BMS-986301 , SB-11285, MK-2118, HG-381 , GSK-3745417, and SNX281 . Commercially available small molecule TLR agonists include GSK1795091 , Imiquimod, LHC165, Guretolimod (DSP-0509), JNJ-64794964, MEDI-9197, and Motolimod.

[0242] (vii) Implant Preparation. One key advance provided by the current disclosure is the ability to manufacture an implant that is cell-free and includes RF at the time of implantation. In particular embodiments, a chemoattractant, ICAF, and RF are added to an implant before use. In particular embodiments, a chemoattractant and an ICAF are added to an implant before a RF is added to the implant. In particular embodiments, a chemoattractant and an ICAF are added to an implant and then the implant is stored before a RF is added to the implant. In particular embodiments, a chemoattractant and an ICAF are added to an implant and then the implant is stored at -0eC to 10eC before a RF is added to the implant.

[0243] In particular embodiments, a method of preparing an implant includes i) adding a chemoattractant to the implant and ii) adding an ICAF to the implant. In particular embodiments, the method further includes adding an RF to the implant. In particular embodiments, the method further includes lyophilizing and storing the implant. In particular embodiments, the method further includes rehydrating the implant.

[0244] In particular embodiments, a method of preparing an implant includes incubating the implant in a first media including a chemoattractant and incubating the implant in a second media including an ICAF, thereby preparing a functionalized implant. In particular embodiments, the implant can be incubated in the first media before the implant is incubated in the second media. In particular embodiments, the implant can be incubated in the second media before the implant is incubated in the first media. In particular embodiments, the implant can be incubated in the firstmedia and the second media at the same time. In particular embodiments, the method further includes incubating the functionalized implant in a third media including an RF. In particular embodiments, the method further includes lyophilizing and storing the functionalized implant. In particular embodiments, the method further includes rehydrating the functionalized implant. In particular embodiments, the rehydrating includes incubating the functionalized implant in a third media including an RF.

[0245] In particular embodiments, a method of preparing an implant includes incubating the implant in a first media including a chemoattractant and an ICAF, thereby preparing a functionalized implant. In particular embodiments, the method further includes incubating the functionalized implant in a second media including RF. In particular embodiments, the method further includes lyophilizing and storing the functionalized implant. In particular embodiments, the method further includes rehydrating the functionalized implant. In particular embodiments, the rehydrating includes incubating the functionalized implant in a second media including RF.

[0246] In particular embodiments, a method of preparing an implant includes the following in the following order: i) adding a chemoattractant and ICAF to the implant, and ii) adding RF to the implant. In particular embodiments, a method of preparing an implant includes the following in the following order: i) adding a chemoattractant and an ICAF to the implant, ii) lyophilizing the implant, iii) storing the implant, and iv) rehydrating and adding RF to the implant.

[0247] Chemoattractant and / or ICAF (both of which are described elsewhere herein) can be added to implants by any method known in the art including adsorption, wet chemistry methods, surface treatment with plasma, chemical crosslinking with crosslinking agents, or chemical functionalization with linkers. For example, the chemoattractant and / or ICAF can be mixed within a first media, such as a buffer solution (e.g. a wet chemistry method). Any buffer solution known in the art can be used, particularly buffer solutions containing nutrients. When choosing the first media, ionic concentration, pH, and nutrient composition should be considered. In particular embodiments, the first media includes a sugar.

[0248] Sugars include saccharides, such as monosaccharides, disaccharides, and oligosaccharides. Suitable sugars also include sugar alcohols, such as erythritol, maltitol, mannitol, sorbitol, xylitol, hydrogenated starch hydrolysates, isomalt, and glycosides, such as steviol glycosides, rebaudiosides, and mogrosides. Additional examples include sucrose, maltose, glucose, raffinose, maltodextrin, trehalose, gum arabic, capsul, lactose, levulose, fructose, ribose, dextrose, isomalt, erythritol, sorbitol, mannitol, xylitol, lactitol, maltitol, pentatol, arabinose, pentose, xylose, galactose, leucrose, stevia, stevioside, rebaudioside A, rubusoside, and mogroside V. In particular embodiments, the first media includes sucrose, maltose,maltodextrin, and / or trehalose. In particular embodiments, the sugar includes sucrose.

[0249] In particular embodiments, the first media includes sucrose phosphate-buffered saline (PBS). Sugars, in particular, can be beneficial to deliver nucleic acids to an anatomical site; such benefits include (i) making the carrier more stable and (ii) maintaining the biological activity and potency of a vector.

[0250] In particular embodiments, a nutrient is in the first media at a concentration of 0.01 M to 50 M, 0.1 M to 10M, or 0.1 M to 1 M. In particular embodiments, a nutrient is in the first media at a concentration of 0.01 M, 0.1 M, 0.5 M, or 1 M. In particular embodiments, a nutrient is in the first media at a concentration of 0.5 M.

[0251] In particular embodiments, the chemoattractant and / or ICAF are distributed evenly throughout the implant. In particular embodiments, the chemoattractant and / or ICAF are distributed along a gradient within the implant. For example, the beginning of a columnar pore could include a chemoattractant while the end includes an ICAF (recognizing that the beginning and end in this example are solely defined by presence of chemoattractant or ICAF). Traveling through the pore from beginning to end, the gradient of chemoattractant to ICAF can shift with the amount of chemoattractant reducing and the amount of ICAF increasing. In certain examples, the amount of one component (chemoattractant or ICAF) can remain steady along the length of a columnar pore while the amount of the other component changes along a gradient.

[0252] The chemoattractant and / or ICAF can be added to an implant scaffold through cosubmersion with the scaffold and incubation in the first media.

[0253] In particular embodiments, 1 ng-1 mg, 100 ng-100 pg, 1 pg-50 pg, 2 pg-20pg of a chemoattractant is added to the first media. In particular embodiments, 2 pg of a chemoattractant is added to the first media. In particular embodiments, 20pg of a chemoattractant is added to the first media. In particular embodiments, 2 pg of a CCL21 , CCL3, CCL4, or CXCL10 is added to the first media. In particular embodiments, 20pg of a CCL21 is added to the first media. In particular embodiments, the chemoattractant is added to the first media at a concentration of 1 ng / mL-1 mg / mL, 100 ng / mL-100 pg / mL, 1 pg / mL-100 pg / mL, 5 pg / mL-90 pg / mL, or 8 pg / mL-80 pg / mL.

[0254] In particular embodiments, 1 ng-1 mg, 100 ng-100 pg, 1 pg-50 pg, 2 pg-20pg of an ICAF is added to the first media. In particular embodiments, 2 pg, 4 pg, 10 pg, or 20 pg of an ICAF is added to the first media. In particular embodiments, 2 pg of anti-CD3 and 2 pg of anti-CD28 is added to the first media. In particular embodiments, 10 pg of anti-CD3 and 10 pg of anti-CD28 is added to the first media. In particular embodiments, the ICAF is added to the first media at a concentration of 1 ng / mL-1 mg / mL, 100 ng / mL-100 pg / mL, 1 pg / mL-100 pg / mL, 5 pg / mL-90 pg / mL, or 8 pg / mL-80 pg / mL.

[0255] In particular embodiments, incubating the scaffold within the first media containing the chemoattractant and / or ICAF includes incubating the scaffold for a time period. The time period can be less than 5 minutes, less than 10 minutes, less than 30 minutes, less than 1 hour, less than 3 hours, less than 12 hours, less than 24 hours, or multiple days.

[0256] In particular embodiments, after the addition of the chemoattractant and / or ICAF, functionalized implants are lyophilized and stored for later use.

[0257] Lyophilization, also referred to as freeze drying, is a method of water removal typically used to preserve materials, extend shelf life, or to make a material more convenient to transport. Lyophilization works by freezing the material, then reducing the pressure and adding heat to allow the frozen water in the material to sublimate. Lyophilization can be performed in a freezer, a chilled bath (shell freezer), or on a shelf in a freeze dryer. In particular embodiments, material is lyophilized in a 4.5 liter benchtop freeze dry system (Labconco, Kansas City, Mo.). During the freezing step, the material is cooled to a temperature below its triple point ensuring that sublimation, rather than melting, will occur, thereby preserving its physical form. In particular embodiments, cooling to a temperature below the triple point includes cooling to a temperature of -5°C, -10°C, -15°C, -20°C, -25°C, -30°C, -35°C, -40°C, -45°C, -50°C, -55°C, -60°C, or below before sublimation. The material can be cooled for any period. For example, the material can be cooled for 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 18 hours, 24 hours, 30 hours, 36 hours, 42 hours, 48 hours, 54 hours, 60 hours, 66 hours, 72 hours or more. In some embodiments, the composition can be cooled to a temperature of -20° C for a period of 24 hours.

[0258] After freezing, a material can undergo primary drying, or sublimation, during lyophilization. During sublimation the pressure is lowered and heat is added to the material in order for the water to sublimate. A vacuum can speed sublimation and a cold condenser provides a surface for the water vapor to adhere and solidify. During sublimation, the temperature can be increased to -5°C, -10°C, -15°C, -20°C, -25°C, -30°C, -35°C, -40°C, -45°C, -50°C, or -55°C. In particular embodiments, the pressure is below 610 Pa, below 500 Pa, below 400 Pa, below 300 Pa, below 200 Pa, below 100 Pa, below 50 Pa, below 25 Pa, or below 10 Pa. In particular embodiments, the pressure is applied for 5 seconds to 12 hours, from 5 seconds to 6 hours, from 5 seconds to 2 hours, from 5 seconds to 1 hour, from 5 seconds to 30 minutes, from 5 seconds to 15 minutes, from 5 seconds to 10 minutes, from 5 seconds to 5 minutes, from 5 seconds to 1 minute, from 5 seconds to 45 seconds. During sublimation, at least 99%, 98%, 95%, 90%, 85%, 80%, or 75% of the water within the material can be removed. In particular embodiments, at least 95% of the water within the material is removed. Sublimation can be a slow process and it is important to note that too much heat can alter the structure of the material.

[0259] After sublimation, another drying referred to as adsorption or secondary drying can be performed. During adsorption, ionically-bound water molecules can be removed. Using a temperature higher than that used in sublimation, the bonds between the material and the water molecules are broken. In particular embodiments, the temperature during adsorption is above 0°C, 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, or 50°C. In particular embodiments, lyophilized materials can retain 0-5% residual moisture after adsorption.

[0260] Implants can be stored at a storage temperature for a storage duration. In particular embodiments, the storage temperature is -20°C, -10°C, -4°C, 0°C, 4°C, 10°C, 15°C, 20°C, or 22°C. In particular embodiments, the storage temperature is 4°C. In particular embodiments, the storage duration is overnight, 1 day, 2 days, 5 days, 10 days, a month, two months, 6 months, a year, or longer. In particular embodiments, the storage duration is overnight. In particular embodiments, implants can be stored in a sealed, airtight, or vacuum container. In particular embodiments, implants can be stored in contact with ambient air. In particular embodiments, the implant is not lyophilized or stored, but is instead fully prepared and implanted before storage occurs.

[0261] In particular embodiments, scaffolds and / or implants can be lyophilized at any point during the manufacture of an implant. For example, a collagen scaffold can be lyophilized; a scaffold with chemoattractants can be lyophilized; a scaffold with ICAF can be lyophilized; a scaffold with chemoattractants and ICAF can be lyophilized; or a scaffold with chemoattractants, ICAF, and RF can be lyophilized.

[0262] In particular embodiments, to prepare for use, a stored implant (at any stage of manufacture) can be rehydrated. Implants can be rehydrated within a second media. During rehydration, RF, such as viral vectors can be added to the implant. In particular embodiments, implants are rehydrated in media including RF. Upon completion of rehydration and / or addition of RF, the implant can be kept on ice until it is implanted in a tumor resection bed or in the vicinity of an unresected or unresectable lesion.

[0263] The second media can be any medium safe for the uses described herein. In particular embodiments, the second media includes saline, buffered saline, physiological saline, water, Hanks' solution, Ringer's solution, Dulbecco's Modified Eagle Medium (DMEM), Normosol-R (Abbott Labs), PLASMA-LYTE A® (Baxter Laboratories, Inc., Morton Grove, IL), sucrose PBS, and combinations thereof.

[0264] In particular embodiments, functionalized implants are incubated in a second media for a time period including less than 5 minutes, less than 10 minutes, less than 30 minutes, less than 1 hour, less than 3 hours, less than 12 hours, less than 24 hours, or multiple days. In particularembodiments, 10 iL, 50 iL, 100 pL, 150 pL, 200 pL, 250 pL, 300 pL, 350 pL, 400 pL, 450 pL, 500 pL, 600 pL, 700 pL, 800 pL, 900 pL or more than 1 mL of second media is added to a 21 mm implant. In particular embodiments, 250 pL of second media is added to a 21 mm implant. The amount of RF needed in a second media can be determined by a person skilled in the art and will depend on the RF, incubation time, implant material, second media concentration, and other relevant factors. The amount of second media needed to rehydrate the implant after lyophilization can also be determined by a person skilled in the art.

[0265] In particular embodiments, RF are added to a second media at a titer of at least 1 x 103Tll / mL, at least 1 x 104TU / mL, at least 1 x 105TU / mL at least 1 x 106TU / mL, at least 1 x 107TU / mL, at least 1 x 108TU / mL, at least 1 x 109TU / mL, or at least 1 x 101° TU / mL.

[0266] (viii) Methods of Use. Methods disclosed herein include treating subjects who have a solid tumor cancer. As used herein, “subject” typically refers to a mammal, such as a human, but can also include other animals such as veterinary animals (dogs, cats, reptiles, birds, etc.) livestock (horses, cattle, goats, pigs, chickens, etc.) and research animals (monkeys, rats, mice, fish, etc.). Treating subjects includes delivering therapeutically effective amounts. Therapeutically effective amounts include those that provide effective amounts, prophylactic treatments and / or therapeutic treatments.

[0267] Cancer (medical term: malignant neoplasm) refers to a class of diseases in which a group of cells display uncontrolled growth (division beyond the normal limits), invasion (intrusion on and destruction of adjacent tissues), and sometimes metastasis. "Metastasis" refers to the spread of cancer cells from their original site of proliferation to another part of the body. The formation of metastasis is a very complex process and depends on detachment of malignant cells from the primary tumor, invasion of the extracellular matrix, penetration of the endothelial basement membranes to enter the body cavity and vessels, and then, after being transported by the blood, infiltration of target organs. Finally, the growth of a new tumor, i.e. a secondary tumor or metastatic tumor, at the target site depends on angiogenesis. Tumor metastasis often occurs even after the removal of the primary tumor because tumor cells or components may remain and develop metastatic potential.

[0268] A "tumor" is a swelling or lesion formed by an abnormal growth of cells (called neoplastic cells or tumor cells). A "tumor cell" is an abnormal cell that divides by a rapid, uncontrolled cellular proliferation and continues to divide after the stimuli that initiated the new division cease. Tumors show partial or complete lack of structural organization and functional coordination with the normal tissue, and usually form a distinct mass of tissue, which may be either benign, pre-malignant or malignant.

[0269] As primary tumor debulking surgery remains the standard of care for most types of solid tumor cancers and is often the first intervention a subject receives, implants disclosed herein can be surgically implanted where a tumor was just removed or situated at advanced unresectable tumors. This timing and placement allows the reprogramming of immune cells attracted to the implant (e.g., to express tumor-specific receptors) while the subject recovers from surgery. It also decreases the risk that previous treatments will have depleted immune cells available for recruitment, reprogramming, and activation. In particular embodiments, subject blood will be prescreened ahead of use of an implant disclosed herein to ensure that the subject immune cell levels meet a threshold. In particular embodiments, subject blood will be pre-screened ahead of enrollment into a clinical trial assessing the implants disclosed herein to ensure that subject immune cell levels meet a threshold. Particular tests during pre-screening can include proliferation assays, expansion assays, and cytokine secretion measurements. In particular embodiments, proliferation, expansion, and / or cytokine secretion are measured in response to in vitro activation (e.g., by anti-CD3 / CD28 bead stimulation).

[0270] As will be understood by one of ordinary skill in the art, the implants are implanted in close proximity to a solid tumor, un-resectable tumor cells and / or in tumor resection beds following resection. The implants can be available in a number of different sizes and shapes and can be shape-conformable to fit the particular needs of individual subjects. In particular embodiments, the implants are injected using ultrasound guidance in close proximity to (or in physical contact with) a solid tumor, un-resected or non-resected tumor cells. Depending on the stage, size or severity of a tumor, implants may be provided with different therapeutic strengths. Therapeutic strength can be manipulated by altering the size of the implant, volume of the implant, the number of ICAF within an implant, the amount of RF within an implant, etc. Each of these parameters can be assessed and determined by a treating physician.

[0271] For the purposes of the present disclosure, the term “proximity” refers to a distance within 10 cm, within 9 cm, within 8 cm, within 7 cm, within 6 cm, within 5 cm, within 4 cm, within 3 cm, within 2 cm, within 1 cm, within 0.9 cm, within 0.8 cm, within 0.7 cm, within 0.6 cm, within 0.5 cm, within 0.4 cm, within 0.3 cm, within 0.2 cm, or within 0.1 cm of a solid tumor, an un-resectable tumor, un-resectable tumor cells, and / or a tumor resection bed.

[0272] Use of the implants disclosed herein result in an anti-tumor effect. As used herein, an antitumor effect refers to a biological effect, which can be manifested by a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in life expectancy, or a decrease of various physiological symptoms associated with the cancerous condition. An anti-tumor effect can also be manifested by a decrease in recurrence or an increasein the time before recurrence. Accordingly, the implants disclosed herein can be used to treat a variety of cancers, can prevent, reduce, or significantly delay metastasis, and / or can prevent, reduce, or significantly delay relapse.

[0273] Cancers that can be treated with the anti-tumor effects of the implants and methods disclosed herein include any solid tumor. Examples of solid tumors include adrenal cancer, brain cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, ear, nose and throat (ENT) cancer, endometrial cancer, esophageal cancer, gastrointestinal cancer, gliomas, head and neck cancer, intestinal cancer, kidney cancer, liver cancer, lung cancer, lymph node cancer, melanomas, neuroblastomas, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, seminomas, skin cancer, stomach cancer, teratomas, thyroid cancer, uterine cancer, and metastases thereof. Without limiting the scope of the disclosure, the following cancer types are noted: brain tumor (Glioblastoma), pancreatic adenocarcinoma, ovarian cancer, and thyroid cancer.

[0274] In particular embodiments, the implants described herein substantially reduce the required RF per subject as it concentrates a small number of RF and enhances RF transfer into immune cells (e.g., T cells) (FIGs. 2D-2F), so the same reprogramming can be achieved with only a fraction of the RF dose.

[0275] It is also understood by one of ordinary skill in the art that the implants can be implanted once, at the time of resection or at a first treatment time in a subject with a solid tumor, an un- resectable tumor, un-resectable tumor cells, and / or a tumor resection bed. Additionally, the implants can be implanted a plurality of times to provide ongoing therapy over months or years. Such treatment regimens can be determined by a treating physician.

[0276] (ix) Kits. The current disclosure also includes kits. Kits can include various components to practice methods disclosed herein. For example, depending on the aspect of the methods practiced, kits could include a scaffold (e.g., collagen); chemoattractant (e.g., CCL21 , CCL3, CCL4, and / or CXCL10); ICAF (e.g., anti-CD3 antibodies and / or anti-CD28 antibodies); RF (e.g., vectors (e.g., lentiviral vector, retroviral vector), CRISPR components, ZFNs, TALENs, MegaTALs); nucleic acids encoding a molecule (e.g., DNA or RNA encoding a recombinant receptor, an scFv, a VL, a VH, a transmembrane domain, EGFRt); components to measure the immune cell status of a subject (e.g., an antibody tagged with a fluorescent molecule, PGR amplification sequences); buffers (e.g., saline, physiological saline, buffered saline, phosphate buffered saline (PBS), Ca++ / Mg-i-+ free PBS, water, Hanks' solution, Ringer's solution); nutrients (e.g., sucrose); combination therapy components (e.g., local anesthetics, chemotherapeutic agents); cytokines (e.g., IL-2, IL-7, IL-15, IL-21 ); culture vessels; reference levels; culture plates;a lyophilizer or freeze dry system; a vacuum; etc.

[0277] The Exemplary Embodiments and Experimental Example below are included to demonstrate particular embodiments of the disclosure. Those of ordinary skill in the art should recognize in light of the present disclosure that many changes can be made to the specific embodiments disclosed herein and still obtain a like or similar result without departing from the spirit and scope of the disclosure.

[0278] (x) Exemplary Embodiments.1 . An implant including (i) a scaffold; (ii) a chemoattractant, (iii) an immune cell activating factor, and (iv) a reprogramming factor, wherein the implant does not include cells before implantation into a subject.2. The implant of embodiment 1 , wherein the scaffold includes columnar pores.3. The implant of embodiment 2, wherein the columnar pores average 100pm to 400pm in diameter.4. The implant of embodiments 2 or 3, wherein the columnar pores are straight.5. The implant of any of embodiments 1 -4, wherein the scaffold includes collagen.6. The implant of embodiment 5, wherein the collagen includes collagen type I, collagen type II, collagen type III, collagen type IV, or collagen type VI.7. The implant of embodiments 5 or 6, wherein the collagen includes collagen type I.8. The implant of any of embodiments 5-7, wherein the collagen includes collagen type III.9. The implant of any of embodiments 5-8, wherein the collagen includes collagen type I and collagen type III.10. The implant of any of embodiments 5-9, wherein the collagen includes atelocollagen.11. The implant of any of embodiments 1-10, wherein the chemoattractant includes a CXC chemokine.12. The implant of any of embodiments 1 -11 , wherein the chemoattractant includes CCL1 , CCL2, CCL3, CCL4, CCL5 (RANTES), CCL7, CCL8, CCL11 , CCL13, CCL17, CCL19, CCL21 , CCL22, CCL24, CCL26, CCR2, CCR5, CXCR3, CXCL8, CXCL9, CXCL10, CX3CL1 , IL-8, macrophage inflammatory protein (MIP)-1 a, MIP-1 p, or neutrophil attractant / activation protein-1 (NAP1 ).13. The implant of any of embodiments 1 -12, wherein the chemoattractant includes CCL21 , CCL3, CCL4, or CXCL .14. The implant of any of embodiments 1 -13, wherein the chemoattractant includes CCL21 .15. The implant of any of embodiments 1 -14, wherein the immune cell activating factor includes a binding domain that binds an activating epitope on a T-cell, an NK cell, macrophage, or dendritic cell.16. The implant of embodiment 15, wherein the activating epitope on the T-cell includes IL-15, CD2, CD7, CD3, CD27, CD28, CD30, CD40, CD80, CD83, CD86, 4-1 BB, (CD137), 0X40, CD30, CD40, lymphocyte function-associated antigen-1 (LFA-1 ), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83 ligands or antibodies, CD1d, recombinant CD1d molecules preloaded with a-galactosyl ceramide and / or a recombinant major histocompatibility complex (MHC) molecule loaded with peptide.17. The implant of embodiments 15 or 16, wherein the activating epitope on the NK cell includes IL-15 or CD137.18. The implant of any of embodiments 15-17, wherein the activating epitope on the macrophage includes CD11b, CD11c, CD64, CD68, CD119, CD163, CD206, CD209, F4 / 80, IFGR2 Toll-like receptors (TLRs) 1-9, IL-4Ra, and / or macrophage receptor with collagenous structure (MARCO).19. The implant of any of embodiments 15-18, wherein the activating epitope on the dendritic cell includes a pattern recognition receptor (PRR).20. The implant of any of embodiments 1 -19, wherein the immune cell activating factor includes a binding domain of an anti-CD3 antibody.21. The implant of any of embodiments 1 -20, wherein the immune cell activating factor includes a binding domain of an anti-CD28 antibody.22. The implant of any of embodiments 1 -21 , wherein the immune cell activating factor includes a binding domain of an anti-CD3 antibody and a binding domain of an anti-CD28 antibody.23. The implant of any of embodiments 1 -22, wherein the reprogramming factor includes a viral vector.24. The implant of embodiment 23, wherein the viral vector includes a retroviral vector, an adenoviral vector, or an adeno-associated virus vector.25. The implant of embodiment 24, wherein the retroviral vector includes a lentiviral vector or a foamy virus vector.26. The implant of any of embodiments 23-25, wherein the viral vector includes a pseudotyped viral vector.27. The implant of any of embodiments 1 -26, wherein the reprogramming factor includes a nucleic acid that encodes an expressed molecule.28. The implant of embodiment 27, wherein the expressed molecule includes a chimeric antigen receptor (CAR), an engineered T cell receptor (eTCR), or a CAR / TCR hybrid.29. The implant of embodiment 28, wherein the CAR includes an anti-ROR1 CAR.30. The implant of any of embodiments 1 -29, further including an additional immune stimulant.31 . The implant of embodiment 30, wherein the additional immune stimulant includes a cytokine,an antibody, a small molecule, an siRNA, a plasmid DNA, and / or a vaccine adjuvant.32. The implant of any of embodiments 1 -31 , wherein the implant is 16 mm-26 mm in diameter.33. The implant of any of embodiments 1 -32, wherein the implant is 19 mm-23 mm in diameter.34. The implant of any of embodiments 1 -33, wherein the implant is 21 mm in diameter.35. The implant of any of embodiments 1 -34, including (i) a collagen type I scaffold including columnar straight pores having an average cross-sectional diameter of 100pm to 400pm; (ii) CCL21 , (iii) a binding domain of an anti-CD3 antibody (iv) a binding domain of an anti-CD28 antibody; and (v) a lentiviral vector including a nucleic acid that encodes a CAR, wherein the implant is 16 mm-26 mm in diameter.36. A kit including i) an implant scaffold or scaffold material, ii) a chemoattractant, and iii) an immune cell activating factor.37. The kit of embodiment 36, wherein the implant scaffold includes a collagen sponge.38. The kit of embodiments 36 or 37, wherein the scaffold material includes collagen, agar, agarose, alginate, alginate / calcium phosphate cement (CPC), beta-galactosidase ([3-GAL), (1 ,2,3,4,6-pentaacetyl a-D-galactose), cellulose, chitin, chitosan, elastin, gelatin, hyaluronic acid collagen, hydroxyapatite, poly(3-hydroxybutyrate-co-3-hydroxy-hexanoate) (PHBHHx), poly(lactide), poly(caprolactone) (PCL), poly(lactide-co-glycolide) (PLG), polyethylene oxide (PEO), poly( lactic-co-g lycolic acid) (PLGA), polypropylene oxide (PPO), poly(vinyl alcohol) (PVA), silk, soy protein, or soy protein isolate.39. The kit of any of embodiments 36-38, further including a first media.40. The kit of embodiment 39, wherein the first media includes a nutrient.41 . The kit of embodiment 40, wherein the nutrient includes sugar.42. The kit of embodiment 41 , wherein the sugar includes sucrose, maltose, glucose, raffinose, maltodextrin, trehalose, gum arabic, capsul, lactose, levulose, fructose, ribose, dextrose, isomalt, erythritol, sorbitol, mannitol, xylitol, lactitol, maltitol, pentatol, arabinose, pentose, xylose, galactose, leucrose, stevia, stevioside, rebaudioside A, rubusoside, and mogroside V.43. The kit of any of embodiments 39-42, wherein the first media includes sucrose phosphate- buffered saline (PBS).44. The kit of any of embodiments 39-43, wherein the first media includes a nutrient at a concentration of 0.01 M to 50M.45. The kit of any of embodiments 39-44, wherein the first media includes a nutrient at a concentration of 0.1 M to 1 M.46. The kit of any of embodiments 39-45, wherein the first media includes nutrient at a concentration of 0.5 M.47. The kit of any of embodiments 39-46, wherein the first media includes the chemoattractant.48. The kit of any of embodiments 39-47, wherein the first media includes the immune cell activating factor.49. The kit of any of embodiments 39-48, wherein the first media includes the chemoattractant and the immune cell activating factor.50. The kit of any of embodiments 39-49, wherein the first media includes 1 ng-50 ng chemoattractant.51 . The kit of any of embodiments 39-50, wherein the first media includes 2 pg-20pg chemoattractant.52. The kit of any of embodiments 39-51 , wherein the first media includes 2 pg chemoattractant.53. The kit of any of embodiments 39-52, wherein the first media includes 20 pg chemoattractant.54. The kit of any of embodiments 36-53, wherein the chemoattractant includes a CXC chemokine.55. The kit of any of embodiments 36-54, wherein the chemoattractant includes CCL1 , CCL2, CCL3, CCL4, CCL5 (RANTES), CCL7, CCL8, CCL11 , CCL13, CCL17, CCL19, CCL21 , CCL22, CCL24, CCL26, CCR2, CCR5, CXCR3, CXCL8, CXCL9, CXCL10, CX3CL1 , IL-8, MIP-1a, MIP- 1 P, or neutrophil attractant / activation protein-1 (NAP1 ).56. The kit of any of embodiments 36-55, wherein the chemoattractant includes CCL21 , CCL3, CCL4, or CXCLW.57. The kit of any of embodiments 36-56, wherein the chemoattractant includes CCL21 .58. The kit of any of embodiments 39-57, wherein the first media includes 1 pg-50 pg immune cell activating factor.59. The kit of any of embodiments 39-58, wherein the first media includes 2 pg-20pg immune cell activating factor.60. The kit of any of embodiments 39-59, wherein the first media includes 2 pg, 4 pg, 10 pg, or 20 pg immune cell activating factor.61 . The kit of any of embodiments 39-60, wherein the first media includes 2 pg or 20 pg immune cell activating factor.62. The kit of any of embodiments 36-61 , wherein the immune cell activating factor includes a binding domain that binds an activating epitope on a T-cell, an NK cell, a macrophage, or a dendritic cell.63. The kit of embodiment 62, wherein the activating epitope on the T-cell includes IL-15, CD2, CD7, CD3, CD27, CD28, CD30, CD40, CD80, CD83, CD86, 4-1 BB, (CD137), 0X40, CD30, CD40, lymphocyte function-associated antigen-1 (LFA-1 ), CD2, CD7, LIGHT, NKG2C, B7-H3,and CD83 ligands or antibodies, CD1d, recombinant CD1d molecules preloaded with a-galactosyl ceramide and / or a recombinant major histocompatibility complex (MHC) molecule loaded with peptide.64. The kit of embodiments 62 or 63, wherein the activating epitope on the NK cell includes IL-15 or CD137.65. The kit of any of embodiments 62-64, wherein the activating epitope on the macrophage includes CD11b, CD11c, CD64, CD68, CD119, CD163, CD206, CD209, F4 / 80, IFGR2 Toll-like receptors (TLRs) 1-9, IL-4Ra, and / or macrophage receptor with collagenous structure (MARCO).66. The kit of any of embodiments 62-65, wherein the activating epitope on the dendritic cell includes a pattern recognition receptor (PRR).67. The kit of any of embodiments 36-66, wherein the immune cell activating factor includes a binding domain of an anti-CD3 antibody.68. The kit of any of embodiments 36-67, wherein the immune cell activating factor includes a binding domain of an anti-CD28 antibody.69. The kit of any of embodiments 36-68, wherein the immune cell activating factor includes a binding domain of an anti-CD3 antibody and a binding domain of an anti-CD28 antibody.70. The kit of any of embodiments 36-69, further including a reprogramming factor.71 . The kit of any of embodiments 39-70, further including a second media.72. The kit of embodiment 71 , wherein the second media includes a reprogramming factor.73. The kit of embodiments 71 or 72, wherein the second media includes a titer of at least 1 x 103Tll / mL, at least 1 x 104TU / mL, at least 1 x 105TU / mL at least 1 x 106TU / mL, at least 1 x 107Tll / mL, at least 1 x 108TU / mL, at least 1 x 109TU / mL, or at least 1 x 101° TU / mL of a reprogramming factor.74. The kit of any of embodiments 71-73, wherein the second media includes phosphate buffered saline.75. The kit of any of embodiments 71 -74, wherein the second media includes a nutrient.76. The kit of embodiment 75, wherein the nutrient includes sugar.77. The kit of embodiment 76, wherein the sugar includes sucrose, maltose, glucose, raffinose, maltodextrin, trehalose, gum arable, capsul, lactose, levulose, fructose, ribose, dextrose, isomalt, erythritol, sorbitol, mannitol, xylitol, lactitol, maltitol, pentatol, arabinose, pentose, xylose, galactose, leucrose, stevia, stevioside, rebaudioside A, rubusoside, and mogroside V.78. The kit of any of embodiments 75-77, wherein the second media includes nutrient at a concentration of 0.01 M to 50M.79. The kit of any of embodiments 70-78, wherein the reprogramming factor includes a viralvector.80. The kit of embodiment 79, wherein the viral vector includes a retroviral vector, an adenoviral vector, or an adeno-associated virus vector.81 . The kit of embodiment 80, wherein the retroviral vector includes a lentiviral vector or a foamy virus vector.82. The kit of any of embodiments 79-81 , wherein the viral vector includes a pseudotyped viral vector.83. The kit of any of embodiments 70-82, wherein the reprogramming factor includes a nucleic acid that encodes an expressed molecule.84. The kit of embodiment 83, wherein the expressed molecule includes a chimeric antigen receptor (CAR), an engineered T cell receptor (eTCR), or a CAR / TCR hybrid.85. The kit of any of embodiments 36-84, further including an additional immune stimulant.86. The kit of embodiment 85, wherein the additional immune stimulant includes a cytokine, an antibody, a small molecule, an siRNA, a plasmid DNA, and / or a vaccine adjuvant.87. The kit of any of embodiments 36-86, wherein the implant scaffold is 16 mm-26 mm in diameter.88. The kit of any of embodiments 36-87, wherein the implant scaffold is 19 mm-23 mm in diameter.89. The kit of any of embodiments 36-88, wherein the implant scaffold is 21 mm in diameter.90. The kit of any of embodiments 36-89, wherein the kit includes a collagen type I scaffold including columnar straight pores having an average cross-sectional diameter of 100pm to 400pm; (ii) CCL21 , (iii) a binding domain of an anti-CD3 antibody (iv) a binding domain of an anti- CD28 antibody; and (v) a lentiviral vector including a nucleic acid that encodes a CAR, wherein the implant scaffold is 16 mm-26 mm in diameter.91 . A method of preparing a functionalized implant including: obtaining an implant scaffold, wherein the implant scaffold includes columnar pores; adding a chemoattractant to the implant scaffold; and adding an immune cell activating factor to the implant scaffold, thereby preparing the functionalized scaffold.92. The method of embodiment 91 , wherein the obtaining includes selecting an implant scaffold.93. The method of embodiments 91 or 92, wherein the obtaining includes manufacturing an implant scaffold.94. The method of any of embodiments 91-93, wherein the adding a chemoattractant and the adding an immune cell activating factor are performed simultaneously.95. The method of any of embodiments 91-94, wherein the adding a chemoattractant and the adding an immune cell activating factor are performed sequentially.96. The method of any of embodiments 91-95, wherein the adding the chemoattract is by adsorption, wet chemistry, surface treatment with plasma, chemical crosslinking, or chemical functionalization with linkers.97. The method of any of embodiments 91-96, wherein the adding the immune cell activating factor is by adsorption, wet chemistry, surface treatment with plasma, chemical crosslinking, or chemical functionalization with linkers.98. The method of any of embodiments 91 -97, wherein the adding the chemoattractant and immune cell activating factor is by adsorption, wet chemistry, surface treatment with plasma, chemical crosslinking, or chemical functionalization with linkers.99. The method of any of embodiments 91-98, wherein the adding the chemoattract is by submerging the implant scaffold in a first media including the chemoattractant and / or the immune cell activating factor.100. The method of embodiment 99, wherein the first media includes a nutrient.101. The method of embodiment 100, wherein the nutrient includes sugar.102. The method of embodiment 101 , wherein the sugar includes sucrose, maltose, glucose, raffinose, maltodextrin, trehalose, gum arabic, capsul, lactose, levulose, fructose, ribose, dextrose, isomalt, erythritol, sorbitol, mannitol, xylitol, lactitol, maltitol, pentatol, arabinose, pentose, xylose, galactose, leucrose, stevia, stevioside, rebaudioside A, rubusoside, and mogroside V.103. The method of any of embodiments 99-102, wherein the first media includes sucrose phosphate-buffered saline (PBS).104. The method of any of embodiments 99-103, wherein the first media includes nutrient at a concentration of 0.01 M to 50M.105. The method of any of embodiments 99-104, wherein the first media includes nutrient at a concentration of 0.1 M to 1 M.106. The method of any of embodiments 99-105, wherein the first media includes nutrient at a concentration of 0.5 M.107. The method of any of embodiments 99-106, wherein the first media includes 1 pg-50 ng chemoattractant.108. The method of any of embodiments 99-107, wherein the first media includes 2 pg-20qg chemoattractant.109. The method of any of embodiments 99-108, wherein the first media includes 2 qgchemoattractant.110. The method of any of embodiments 99-109, wherein the first media includes 20 pg chemoattractant.111. The method of any of embodiments 91 -110, wherein the chemoattractant includes a CXC chemokine.112. The method of any of embodiments 91 -1 11 , wherein the chemoattractant includes CCL1 , CCL2, CCL3, CCL4, CCL5 (RANTES), CCL7, CCL8, CCL11 , CCL13, CCL17, CCL19, CCL21 , CCL22, CCL24, CCL26, CCR2, CCR5, CXCR3, CXCL8, CXCL9, CXCL10, CX3CL1 , IL-8, MIP- 1 a, MIP-1 p, or neutrophil attractant / activation protein-1 (NAP1 ).113. The method of any of embodiments 91-112, wherein the chemoattractant includes CCL21 , CCL3, CCL4, or CXCL .114. The method of any of embodiments 91-113, wherein the chemoattractant includes CCL21 .115. The method of any of embodiments 99-114, wherein the first media includes 1 pg-50 pg immune cell activating factor.116. The method of any of embodiments 99-115, wherein the first media includes 2 pg-20pg immune cell activating factor.117. The method of any of embodiments 99-1 16, wherein the first media includes 2 pg, 4 pg, 10 pg, or 20 pg immune cell activating factor.118. The method of any of embodiments 99-117, wherein the first media includes 2 pg or 20 pg immune cell activating factor.119. The method of any of embodiments 99-118, wherein the first media includes 20 pg chemoattractant and 1 pg-50 pg immune cell activating factor.120. The method of any of embodiments 99-119, wherein the first media includes 2 pg-20 pg chemoattractant and 1 pg-50 pg immune cell activating factor.121. The method of any of embodiments 91 -120, wherein the immune cell activating factor includes a binding domain that binds an activating epitope on a T-cell, an NK cell, a macrophage, or a dendritic cell.122. The method of embodiment 121 , wherein the activating epitope on the T-cell includes IL- 15, CD2, CD7, CD3, CD27, CD28, CD30, CD40, CD80, CD83, CD86, 4-1 BB, (CD137), 0X40, CD30, CD40, lymphocyte function-associated antigen- 1 (LFA-1 ), CD2, CD7, LIGHT, NKG2C, B7- H3, and CD83 ligands or antibodies, CD1d, recombinant CD1d molecules preloaded with a- galactosyl ceramide and / or a recombinant major histocompatibility complex (MHC) molecule loaded with peptide.123. The method of embodiments 121 or 122, wherein the activating epitope on the NK cellincludes IL-15 or CD137.124. The method of any of embodiments 121 -123, wherein the activating epitope on the macrophage includes CD11b, CD11 c, CD64, CD68, CD119, CD163, CD206, CD209, F4 / 80, IFGR2 Toll-like receptors (TLRs) 1 -9, IL-4Ra, and / or macrophage receptor with collagenous structure (MARCO).125. The method of any of embodiments 121 -124, wherein the activating epitope on the dendritic cell includes pattern recognition receptor (PRR).126. The method of any of embodiments 91 -125, wherein the immune cell activating factor includes a binding domain of an anti-CD3 antibody.127. The method of any of embodiments 91 -126, wherein the immune cell activating factor includes a binding domain of an anti-CD28 antibody.128. The method of any of embodiments 91 -127, wherein the immune cell activating factor includes a binding domain of an anti-CD3 antibody and a binding domain of an anti-CD28 antibody.129. The method of any of embodiments 91 -128, further including lyophilizing the functionalized scaffold.130. The method of embodiment 129, wherein the lyophilizing includes freezing the implant, sublimating the implant, and adsorbing the implant.131. The method of embodiment 130, wherein the freezing includes lowering the temperature to a temperature below the triple point for a freezing period.132. The method of embodiment 131 , wherein the temperature below the triple point includes -5°C, -10°C, -15°C, -20°C, -25°C, -30°C, -35°C, -40° C, -45°C, -50°C, -55°C, or -60°C.133. The method of embodiments 131 or 132, wherein the temperature below the triple point -20°C.134. The method of any of embodiments 131 -133, wherein the freezing period is 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 18 hours, 24 hours, 30 hours, 36 hours, 42 hours, 48 hours, 54 hours, 60 hours, 66 hours, or 72 hours.135. The method of any of embodiments 131-134, wherein the freezing period is 24 hours.136. The method of any of embodiments 130-135, wherein the sublimating includes lowering the pressure to a sublimation pressure and heating the implant to a sublimation temperature.137. The method of embodiment 136, wherein the sublimation pressure includes a pressure around the implant of below 610 Pa, below 500 Pa, below 400 Pa, below 300 Pa, below 200 Pa, below 100 Pa, below 50 Pa, below 25 Pa, or below 10 Pa.138. The method of embodiments 136 or 137, wherein the sublimation temperature includes atemperature of -5°C, -10°C, -15°C, -20°C, -25°C, -30°C, -35°C, -40°C, -45°C, -50°C, or -55°C.139. The method of any of embodiments 130-138, wherein the sublimating includes dehydrating the implant.140. The method of any of embodiments 130-139, wherein the adsorbing includes heating the implant to an adsorption temperature.141. The method of embodiment 140, wherein the adsorption temperature is higher than the sublimation temperature.142. The method of embodiments 140 or 141 , wherein the adsorption temperature includes a temperature above 0°C, 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, or 50°C.143. The method of any of embodiments 129-142, further including storing the lyophilized implant at a storage temperature and for a storage duration.144. The method of the embodiment 143, wherein the storage temperature is a temperature of -22°C to 22°C.145. The method of the embodiments 143 or 144, wherein the storage temperature is a temperature of -10°C to 10°C.146. The method of the any of embodiments 143-145, wherein the storage temperature is a temperature of 0°C to 10°C.147. The method of the any of embodiments 143-146, wherein the storage temperature is a temperature of 2°C to 8°C.148. The method of the any of embodiments 143-147, wherein the storage temperature is a temperature of 4°C.149. The method of any of embodiments 143-148, wherein the storage temperature is not - 80°C.150. The method of any of embodiments 143-149, wherein the storage duration includes overnight, 1 day, 2 days, 5 days, 10 days, one month, two months, 6 months, or a year.151 . The method of any of embodiments 129-150, further including rehydrating the lyophilized implant by submerging the lyophilized implant in a second media.152. The method of embodiment 151 , wherein the second media includes a reprogramming factor.153. The method of embodiments 151 or 152, wherein the second media includes phosphate buffered saline.154. The method of any of embodiments 151 -153, wherein the second media includes a nutrient.155. The method of embodiment 154, wherein the nutrient includes sugar.156. The method of embodiment 155, wherein the sugar includes sucrose, maltose, glucose, raffinose, maltodextrin, trehalose, gum arabic, capsul, lactose, levulose, fructose, ribose, dextrose, isomalt, erythritol, sorbitol, mannitol, xylitol, lactitol, maltitol, pentatol, arabinose, pentose, xylose, galactose, leucrose, stevia, stevioside, rebaudioside A, rubusoside, and mogroside V.157. The method of any of embodiments 151-156, wherein the second media includes nutrient at a concentration of 0.01 M to 50M.158. The method of any of embodiments 152-157, wherein the reprogramming factor includes a viral vector.159. The method of embodiment 158, wherein the viral vector includes a retroviral vector, an adenoviral vector, or an adeno-associated virus vector.160. The method of embodiment 159, wherein the retroviral vector includes a lentiviral vector or a foamy virus vector.161 . The method of embodiments 159 or 160, wherein the viral vector includes a pseudotyped viral vector.162. The method of any of embodiments 152-161 , wherein the reprogramming factor includes a nucleic acid that encodes an expressed molecule.163. The method of embodiment 162, wherein the expressed molecule includes a chimeric antigen receptor (CAR), an engineered T cell receptor (eTCR), or a CAR / TCR hybrid.164. The method of any of embodiments 151 -163, wherein the submerging is for a time period that is less than 5 minutes, less than 10 minutes, less than 30 minutes, less than 1 hour, less than 3 hours, less than 12 hours, or less than 24 hours.165. The method of any of embodiments 151 -164, wherein the second media includes reprogramming factors at a titer of at least 1 x 103TU / mL, at least 1 x 104TU / mL, at least 1 x 105TU / mL at least 1 x 106TU / mL, at least 1 x 107TU / mL, at least 1 x 108TU / mL, at least 1 x 109TU / mL, or at least 1 x 1010 TU / mL.166. A method of treating a solid tumor in a subject in need thereof including: implanting the implant of any of embodiments 1 -35 into the subject within a proximity to the solid tumor or tumor resection bed, thereby treating the solid tumor in the subject.167. The method of embodiment 166, wherein the implanting is within a tumor resection bed.168. The method of embodiments 166 or 167, wherein the solid tumor includes an adrenal cancer, a brain cancer, a breast cancer, a cervical cancer, a colon cancer, a colorectal cancer, an ear, nose and throat (ENT) cancer, an endometrial cancer, an esophageal cancer, agastrointestinal cancer, a glioma, a head and neck cancer, an intestinal cancer, a kidney cancer, a liver cancer, a lung cancer, a lymph node cancer, a melanoma, a neuroblastoma, an ovarian cancer, a pancreatic cancer, a prostate cancer, a rectal cancer, a seminoma, a skin cancer, a stomach cancer, a teratoma, a thyroid cancer, or a uterine cancer, or a metastasis thereof.169. The method of any of embodiments 166-168, wherein the subject is a mammal.170. The method of embodiment 169, wherein the mammal is a human.171 . The method of any of embodiments 166-170, wherein the implanting occurs during a tumor de-bulking surgery.172. The method of embodiment 171 , wherein the tumor de-bulking surgery is a primary tumor de-bulking surgery.173. The method of any of embodiments 166-172, wherein the subject has been pre-screened for an immune cell status before the treating.

[0279] (xi) Experimental Example. Materials and methods. Cell lines: MDA-MB-468 breast cancer cells were obtained from ATCC (Cat# HTB-132) and cultured in Dulbecco’s modified Eagle’s medium with GlutaMAX (DMEM, GIBCO). DMEM was reconstituted with 10% fetal bovine serum (FBS) and 1% Penicillin / Streptomycin (P / S). To generate ROR1 + tumor targets, MDA-MB- 468 cells were stably transduced with human ROR-encoding lentiviral vector (purchased from G&P Biosciences: SKU#: LTV0043). Successfully transduced ROR1 + cells were isolated using fluorescence-activated cell sorting. For in vitro tumor killing assays, a luciferase-expressing version of the ROR1 + MDA-BM-468 breast tumor cell line was generated by lentiviral transduction with firefly luciferase (F-luc). All cell lines tested negative for mycoplasma using a DNA-based PCR test (DDC Medical).

[0280] Mice and in vivo tumor model: Female NSG-MHC l / ll DKO mice (NOD.Cg-PrkdcscidH2- K1b4m'BpeH2-Ab197'e lM™ H2-D1Mm1BpeH2rglm1Wjl / SzJ) were purchased from Jackson Laboratory (Strain #:025216). To model triple-negative human breast cancer, 10 million ROR1 + MDA-MB- 468 breast cancer cells were resuspended in 200 pL Cultrex® ECM (R&D Systems) and injected into the right inferior mammary fat pad of NSG-MHC l / ll DKO mice.

[0281] Tumor monitoring: To monitor tumor progression of subcutaneous ROR1 + MDA-MB-468 breast tumors, a digital caliper was used to measure width and length of tumors and the volume was then calculated using the formula TT / 6 X W X W.

[0282] Lentiviral vectors: GFP-encoding lentiviral particles, used for in vitro gene transfer experiments in FIGs. 2A-2I and 3A-3C, were purchased from Fenics Bio (Cat#: LV-1016-100). The lentiviral plasmid encoding the ROR1 -specific CAR was provided by Dr. Stanley Riddell (Fred Hutchinson Cancer Center). More specifically, the CAR was constructed using VL and VHsegments of 2A2 and R12 as described in Hudecek, eta!., Clin Cancer Res 19, 3153-3164 (2013). The scFV was linked by a spacer domain (lgG4 hinge domain, SEQ ID NO: 163) to the transmembrane domain of human CD28 and to a signaling module including the 42 AA cytoplasmic domain of human 4-1 BB, which was linked to the 1 12 AA cytoplasmic domain of isoform 3 of human CD3<. The construct encoded a T2A ribosomal skip element and a truncated EGFR (tEGFR) sequence downstream of the CAR, which served as a transduction tracking marker for CAR-modified T-cells. Replication incompetent, HIV-based, VSV-G pseudotyped lentiviral particles were made at a titer of 1 x 108TU / mL.

[0283] Preparation of functionalized collagen scaffolds: Collagen sponges composed of Type I bovine collagen were purchased from Advanced Biomatrix (Cat#: 5135-EA). The overall diameter of the collagen sponges was 21 mm, with an average pore diameter of 200 pm. A chemoattractant and antibody mix was prepared in 0.5M sucrose PBS. For in vitro studies, 2 pg of anti-human CD3 and 2 pg of anti-human CD28 antibody (BioXcell, Cat#: BE-0001 -2 and BE0248, respectively) and 2 pg of CCL21 (Biolegend, Cat#: B297009), or CCL3 (Biolegend, Cat#: B224650), or CCL4 (Biolegend Cat#: B324087), or CXCL10 (Biolegend Cat#: B317192) were added onto each implant. For in vivo studies, 10 pg of anti-human CD3 and 10 pg of anti-human CD28 antibody and 20 pg of CCL21 were added onto each implant. The chemoattractant + antibody mix was distributed equally on collagen sponges, lyophilized for 30 minutes, and stored at 4°C overnight. The following day (day of implantation), implants were rehydrated with 250 pL of freshly thawed CAR-encoding lentiviral suspension (1 x 108TU / mL) in PBS and kept on ice until use.

[0284] Confocal imaging: To visualize T cells within implants (FIG. 2C), images were collected using an Andor Dragonfly 200 High Speed Confocal microscope (Oxford Instruments). DiD' was excited with the 637 nm laser and collected with a 698 / 77 nm Cy5 band pass emission filter. Images were acquired with an Andor Zyla 4.2 Plus sCMOS camera controlled with Fusion software (version 2.4.0.13). The resulting images were viewed and analyzed using Imaged (Version 1.53t).

[0285] In vitro tumor killing assays: Lentiviral particles encoding anti-ROR1 CAR and 2 pg of antihuman CD3 and anti-human CD28 antibody in 1 M sucrose PBS were added to 21 mm collagen sponges and lyophilized for 30 minutes and stored at 4°C. The collagen sponges were rehydrated with 1 x106T cells resuspended in complete T-cell medium. After 72 hours, effector T cells were removed from the implant and cultured in complete T-cell medium with 10% Cellkines® (ZeptoMetrix, Cat#: 0801017) for two additional days. The total number of live CAR+ T cells was quantified using the trypan blue exclusion method combined with flow cytometry staining for thetEGFR (which serves as the transduction marker for anti-ROR1 CAR expression). Luciferaseexpressing ROR1 + MDA-MB-468 breast tumor cells were seeded at a density of 6x104cells per well in a 96-well plate. ROR1 -CAR-transduced T cells and untransduced control T cells were cocultured with tumor cells at 0:1 , 0.2:1 and 3:1 Effector: Target (E:T) ratios. After 24 hours, residual tumors were visualized using bioluminescence I VIS imaging.

[0286] In vivo bioluminescence imaging: D-Luciferin (Xenogen) in PBS (15 mg / mL) was used as a substrate for firefly luciferase imaging. Bioluminescence images were collected with a Xenogen I VIS Spectrum Imaging System (Xenogen). Mice were anesthetized with 2% isoflurane (Forane, Baxter Healthcare) before and during imaging. For imaging of in vivo CAR-T cell reprogramming (FIGs. 4B, 4C), each mouse was injected intraperitoneally (i.p.) with 300 pg of D-Luciferin, and images were collected 10 minutes later. Acquisition times ranged from 10 seconds to 5 minutes. Data were analyzed using IV IS Living Image 4.8.0 software.

[0287] Flow cytometry: Data were acquired using a BD LSRFortessa or FacsCanto II cell analyzer running FACSDIVA software, and analyzed with FlowJo v10.8. Antibodies used in flow cytometry were purchased from eBioscience.

[0288] Statistics: Unless otherwise stated, graphs show mean ± standard error of the mean. Statistical analysis was done with Prism software (Graphpad). Results with a P-value <0.05 were considered significant.

[0289] Study approval: The care and use of mice in this study were approved by the Institutional Animal Care & Use Committee (IACUC) at the Fred Hutchinson Cancer Center and complied with all relevant ethical regulations for animal testing and research (Assurance #A3226-01 , IACUC Protocol Number 50782).

[0290] Results. Rationally designed biomaterial implants recruit, reprogram, expand and release human T cells. To identify a potent chemokine that recruits host T cells into an implant, a T- lymphocyte chemotaxis screen in porous collagen implants was performed. Collagen was chosen because it best supports the attachment, migration and proliferation of T cells (Sadjadi et al., Biophys J 1 19, 2141-2152 (2020)). All the implants had large pores ranging from 100 pm to 400 pm across, with the average diameter being 200 pm, which permits cells and nutrients to easily flow through the device. Collagen discs were functionalized with 2 pg of the lymphocyte chemoattractant C-C Motif Chemokine Ligand (CCL)-21 , CCL3, CCL4, or C-X-C Motif Chemokine Ligand 10 (CXCL10), before placing them into collagen gel loaded with fluorescently tagged human T cells. After 48 hours, implants were isolated and fluorescent signals were recorded using IVIS imaging. CCL21 triggered a mean 5.84-fold boost in T-cell recruitment, compared to PBS control (FIGs. 2A, 2B), whereas CCL3, CCL4 or CXCL10 promoted lesser recruitment activity(2.24-fold, 1.52-fold, and 1.83-fold increase vs. PBS; FIGs. 2A, 2B). Fluorescent microscopy confirmed that T lymphocytes efficiently penetrate and migrate throughout the implants (FIG. 2C).

[0291] To assess their ability to reprogram infiltrating T cells, implants were functionalized with agonistic anti-CD3 and anti-CD28 antibodies and GFP-encoding lentivirus (FIG. 2D). GFP- expressing T-cell populations were compared to those produced in an implant-free suspension culture. Microporous collagen implants facilitated the interaction of lentivirus and T cells and enabled efficient gene transfer, even at the relatively low titer of 0.1 or 1 multiplicity of infection (MOI; FIGs. 2E, 2F). In contrast, incubating T cells with equal amounts of virus in suspension resulted in poor gene transfer (12.1 -fold lower at MOI of 1 ; FIG. 2F). Notably, lentivirus / antibody- loaded implant that was freeze-dried prior to use, using sucrose as a lyoprotectant, maintained 84% of its lentiviral activity (FIG. 3). This is an important finding as lyophilization would allow convenient storage of the final product at 2-8eC, eliminating the need for disruptive -80eC cold chains that require special equipment and logistics.

[0292] Following reprogramming, implants also must expand and release therapeutic T lymphocytes, so cell expansion and anti-tumor effects in vitro was evaluated next. A 4-fold and 66-fold expansion of embedded T cells following a 7-day and 14-day culture period, respectively was observed (FIG. 2G). To determine whether T cells that migrate out of implants are functional, their ability to lyse tumor cells was measured. Human MDA-MB-468 breast cancer cells expressing firefly luciferase and the receptor tyrosine kinase-like orphan receptor (ROR1 ) tumor antigen were co-cultured with implant-released T cells that had been reprogrammed with a chimeric antigen receptor (CAR) that targets cells expressing ROR1. Using bioluminescence as a readout, implants efficiently generated fully functional CAR-T cells that efficiently destroyed their targets (FIGs. 2H, 2I). Altogether, the in vitro findings establish that collagen implant augmented with chemoattractant, stimulatory cues and viral vector can provide an appropriate interface to rapidly create human CAR-T cells.

[0293] Implants efficiently reprogram host T cells with tumor-recognizing capabilities. One goal was to edit lymphocyte targeting in vivo in order to bring about cancer regression. Accordingly, how rapidly and efficiently functionalized implants can recruit host T cells and reprogram them with tumor-specific CAR genes in vivo was next examined (FIG. 4A). As a clinically meaningful in vivo test system, a triple-negative breast cancer was modeled by injecting MDA-MB-468 tumor cells that express the tumor antigen ROR1 into the right inferior mammary fat pad of immunodeficient NSG-MHC l / ll DKO mice (also known as NSG-( *D&)riLi|i( / A)nuH, NSG- (KbDb)nui!(IAr,u)). After 22 days, breast tumors were fully established and mice were injected with 20x106CD3+ human T cells. Three days later, mice were randomized and surgically implantedwith collagen implants formulated with CCL21 , anti-CD3 / CD28 antibodies, and lentivirus coexpressing anti-ROR1 CAR and firefly luciferase. A second group was treated with implants lacking the T-cell reprogramming viral vectors. Implant-mediated in vivo reprogramming of CAR- T cells was then serially quantified by using bioluminescence imaging. No bioluminescence above background levels could be detected in mice treated with vector-free implants (FIG. 4B, left panel). However, mice receiving implants functionalized with lentivirus showed strong luciferase activity in the tumor area, which was first detectable in all mice in this group at day 2 and peaked at day 8 following implantation of the devices (FIG. 4B, right panel, FIG. 4C). To verify that the recorded luciferase signals accurately reflect in vivo reprogramming of CAR-T cells in the host, the experiments were repeated and CAR+ T cells were quantified at the implantation site and the spleen of mice by flow cytometry 8 days after surgery. Treatment with implants carrying ROR1 - CAR lentivirus achieved a mean 39.2± 19.5% CAR gene transfer into host T cells at tumor sites (FIGs. 4D, 4E). As expected, in vivo T-cell reprogramming was mostly confined to the peritumoral tissue where the devices were implanted. CAR+ T cells that had egressed from implants were found at some other host tissues, such as the spleen, albeit at small percentages (mean 3.9± 1.1 % CAR+ population among CD3+ cells; FIGs. 4D, 4E).

[0294] Implant-mediated in vivo T-cell reprogramming brings about tumor regression. To measure the antitumor benefits provided by CAR-programming biopolymer implants, mice (humanized with T cells) bearing orthotopic ROR1 + MDA-MB-468 breast tumors were treated with implants formulated with CCL21 , anti-CD3 / CD28 antibodies, and ROR1-CAR lentivirus. A separate group of mice received no implant. Control mice were not injected with human T cells to exclude the possibility that any observed anti-tumor effects are mediated by xenogeneic graft versus host disease. Tumor growth was serially quantified using a digital caliper and differences in mouse survival were monitored (FIG. 5A). Humanizing mice with T cells provided no anti-tumor effects over controls, as in both these groups the breast tumors gradually progressed and the mice showed the same survivorship curves (median survival: 39 versus 38 days, respectively; FIGs. 5B, 5C). By contrast, all animals treated with CAR-programming implants were protected against tumor progression and even displayed a mean 19.1% shrinkage in tumor volume over time (FIG. 5B).

[0295] In summary, a cell-free polymer implant designed to recruit, reprogram and expand host T cells at tumor lesions in vivo is described. The implant can be fabricated on a large scale and is stable to lyophilization. Using a mouse breast cancer model, the implants quickly and efficiently acquire cancer-specific host lymphocytes at the tumor site in quantities sufficient to bring about long-term tumor regression. Given that surgical care is the mainstay of cancer treatment for manysubjects, this technology can be easily implemented in a clinical setting as an add-on to surgery for solid tumors. Furthermore, the approach can be broadened to recruit and reprogram other therapeutically desirable host cells, such as macrophages, natural killer cells or dendritic cells, potentially boosting the antitumor effectiveness of the implant even more.

[0296] (xii) Closing Paragraphs. The nucleic acid and amino acid sequences provided herein are shown using letter abbreviations for nucleotide bases and amino acid residues, as defined in 37 C.F.R. §1 .831 -1 .835 and set forth in WIPO Standard ST.26 (implemented on July 1 , 2022). Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included in embodiments where it would be appropriate.

[0297] To the extent not explicitly provided herein, coding sequences for proteins disclosed herein and protein sequences for coding sequences disclosed herein can be readily derived from one of ordinary skill in the art.

[0298] Variants of the sequences disclosed and referenced herein are also included. Functional variants include one or more residue additions or substitutions that do not substantially impact the physiological effects of the protein. Functional fragments include one or more deletions or truncations that do not substantially impact the physiological effects of the protein. A lack of substantial impact can be confirmed by observing experimentally comparable results in an activation study or a binding study. Functional variants and functional fragments of intracellular signaling components transmit activation or inhibition signals comparable to a wild-type reference when in the activated state of the current disclosure. Functional variants and functional fragments of binding domains bind their cognate antigen or ligand at a level comparable to a wild-type reference.

[0299] Guidance in determining which amino acid residues can be substituted, inserted, or deleted without abolishing biological activity can be found using computer programs well known in the art, such as DNASTAR™ (Madison, Wisconsin) software. Preferably, amino acid changes in the protein variants disclosed herein are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. A conservative amino acid change involves substitution of one of a family of amino acids which are related in their side chains.

[0300] In a peptide or protein, suitable conservative substitutions of amino acids are known to those of skill in this art and generally can be made without altering a biological activity of a resulting molecule. Those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub. Co., p. 224). Naturally occurring amino acids are generally divided intoconservative substitution families as follows: Group 1 : Alanine (Ala), Glycine (Gly), Serine (Ser), and Threonine (Thr); Group 2: (acidic): Aspartic acid (Asp), and Glutamic acid (Glu); Group 3: (acidic; also classified as polar, negatively charged residues and their amides): Asparagine (Asn), Glutamine (Gin), Asp, and Glu; Group 4: Gin and Asn; Group 5: (basic; also classified as polar, positively charged residues): Arginine (Arg), Lysine (Lys), and Histidine (His); Group 6 (large aliphatic, nonpolar residues): Isoleucine (lie), Leucine (Leu), Methionine (Met), Valine (Vai) and Cysteine (Cys); Group 7 (uncharged polar): Tyrosine (Tyr), Gly, Asn, Gin, Cys, Ser, and Thr; Group 8 (large aromatic residues): Phenylalanine (Phe), Tryptophan (Trp), and Tyr; Group 9 (nonpolar): Proline (Pro), Ala, Vai, Leu, lie, Phe, Met, and Trp; Group 11 (aliphatic): Gly, Ala, Vai, Leu, and lie; Group 10 (small aliphatic, nonpolar or slightly polar residues): Ala, Ser, Thr, Pro, and Gly; and Group 12 (sulfur-containing): Met and Cys. Additional information can be found in Creighton (1984) Proteins, W.H. Freeman and Company.

[0301] In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982, J. Mol. Biol. 157(1 ), 105-32). Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982). These values are: He (+4.5); Vai (+4.2); Leu (+3.8); Phe (+2.8); Cys (+2.5); Met (+1 .9); Ala (+1 .8); Gly (-0.4); Thr (-0.7); Ser (-0.8); Trp (-0.9); Tyr (-1.3); Pro (-1.6); His (-3.2); Glutamate (-3.5); Gin (-3.5); aspartate (-3.5); Asn (-3.5); Lys (-3.9); and Arg (-4.5).

[0302] It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e., still obtain a biological functionally equivalent protein. In making such changes, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred. It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity.

[0303] As detailed in U.S. Pat. No. 4,554,101 , the following hydrophilicity values have been assigned to amino acid residues: Arg (+3.0); Lys (+3.0); aspartate (+3.0±1); glutamate (+3.0±1 ); Ser (+0.3); Asn (+0.2); Gin (+0.2); Gly (0); Thr (-0.4); Pro (-0.5±1); Ala (-0.5); His (-0.5); Cys (-1.0); Met (-1.3); Vai (-1.5); Leu (-1.8); lie (-1.8); Tyr (-2.3); Phe (-2.5); Trp (-3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values are within ±2 ispreferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

[0304] As outlined above, amino acid substitutions may be based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.

[0305] As indicated elsewhere, variants of genes can include codon optimized variants, sequence polymorphisms, splice variants, and / or mutations that do not affect the function of an encoded product to a statistically significant degree.

[0306] Variants of the protein, nucleic acid, and gene sequences disclosed herein also include sequences with at least 70% sequence identity, 80% sequence identity, 85% sequence, 90% sequence identity, 95% sequence identity, 96% sequence identity, 97% sequence identity, 98% sequence identity, or 99% sequence identity to the protein, nucleic acid, or gene sequences disclosed herein.

[0307] “% sequence identity” refers to a relationship between two or more sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between protein, nucleic acid, or gene sequences as determined by the match between strings of such sequences. "Identity" (often referred to as "similarity") can be readily calculated by known methods, including (but not limited to) those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, NY (1994); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (Von Heijne, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Oxford University Press, NY (1992). Preferred methods to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR, Inc., Madison, Wisconsin). Multiple alignment of the sequences can also be performed using the Clustal method of alignment (Higgins and Sharp CABIOS, 5, 151 -153 (1989) with default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Relevant programs also include the GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wisconsin); BLASTP, BLASTN, BLASTX (Altschul, et al., J. Mol. Biol. 215:403-410 (1990); DNASTAR (DNASTAR, Inc., Madison, Wisconsin); and the PASTA program incorporating the Smith-Waterman algorithm (Pearson, Comput. Methods Genome Res., [Proc.Int. Symp.] (1994), Meeting Date 1992, 11 1 -20. Editor(s): Suhai, Sandor. Publisher: Plenum, New York, N.Y. Within the context of this disclosure, it will be understood that where sequence analysis software is used for analysis, the results of the analysis are based on the "default values" of the program referenced. As used herein "default values" will mean any set of values or parameters, which originally load with the software when first initialized.

[0308] Variants also include nucleic acid molecules that hybridize under stringent hybridization conditions to a sequence disclosed herein and provide the same function as the reference sequence. Exemplary stringent hybridization conditions include an overnight incubation at 42 °C in a solution including 50% formamide, 5XSSC (750 mM NaCI, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5XDenhardt's solution, 10% dextran sulfate, and 20 ng / ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1 XSSC at 50 °C. Changes in the stringency of hybridization and signal detection are primarily accomplished through the manipulation of formamide concentration (lower percentages of formamide result in lowered stringency); salt conditions, or temperature. For example, moderately high stringency conditions include an overnight incubation at 37°C in a solution including 6XSSPE (20XSSPE=3M NaCI; 0.2M NaH2PO4; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 pg / ml salmon sperm blocking DNA; followed by washes at 50 °C with 1XSSPE, 0.1% SDS. In addition, to achieve even lower stringency, washes performed following stringent hybridization can be done at higher salt concentrations (e.g., 5XSSC). Variations in the above conditions may be accomplished through the inclusion and / or substitution of alternate blocking reagents used to suppress background in hybridization experiments. Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. The inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility.

[0309] "Binds" refers to an association of a binding domain (of, for example, a CAR binding domain or an antibody binding domain) to its cognate binding molecule with an affinity or Ka( / .e., an equilibrium association constant of a particular binding interaction with units of 1 / M) equal to or greater than 105M’1, while not significantly associating with any other molecules or components in a relevant environment sample. Binding domains may be classified as "high affinity" or "low affinity". In particular embodiments, "high affinity" binding domains refer to those binding domains with a Kaof at least 107M'1, at least 108M'1, at least 109M'1, at least 1010M-1, at least 1011M'1, at least 1012M’1, or at least 1013M-1. In particular embodiments, "low affinity" binding domains refer to those binding domains with a Kaof up to 107M-1, up to 106M-1, up to 105M-1. Alternatively, affinity may be defined as an equilibrium dissociation constant (Kd) of a particular bindinginteraction with units of M (e.g., 10‘5M to 10'13M). In certain embodiments, a binding domain may have "enhanced affinity," which refers to a selected or engineered binding domains with stronger binding to a cognate binding molecule than a wild type (or parent) binding domain. For example, enhanced affinity may be due to a Ka(equilibrium association constant) for the cognate binding molecule that is higher than the reference binding domain or due to a Kd (dissociation constant) for the cognate binding molecule that is less than that of the reference binding domain, or due to an off-rate (KOft) for the cognate binding molecule that is less than that of the reference binding domain. A variety of assays are known for detecting binding domains that specifically bind a particular cognate binding molecule as well as determining binding affinities, such as Western blot, ELISA, and BIACORE® analysis (see also, e.g., Scatchard, etal., 1949, Ann. N. Y. Acad. Sci. 51-.Q60 and U.S. Patent Nos. 5,283,173, 5,468,614, or the equivalent).

[0310] Unless otherwise indicated, the practice of the present disclosure can employ conventional techniques of immunology, molecular biology, microbiology, cell biology and recombinant DNA. These methods are described in the following publications. See, e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual, 4th Edition (2012); F. M. Ausubel, et al. eds., Current Protocols in Molecular Biology, (2003); the series Methods In Enzymology (Academic Press, Inc.); Behlke, et aL, Polymerase Chain Reaction: Theory and Technology (2019); Greenfield, ed. Antibodies, A Laboratory Manual, Second Edition (2014); and Capes- Davis and R. I. Freshney, eds. Freshney's Culture of Animal Cells 8th Edition (2021).

[0311] As will be understood by one of ordinary skill in the art, each embodiment disclosed herein can comprise, consist essentially of or consist of its particular stated element, step, ingredient or component. Thus, the terms “include” or “including” should be interpreted to recite: “comprise, consist of, or consist essentially of.” The transition term “comprise” or “comprises” means has, but is not limited to, and allows for the inclusion of unspecified elements, steps, ingredients, or components, even in major amounts. The transitional phrase “consisting of” excludes any element, step, ingredient or component not specified. The transition phrase “consisting essentially of” limits the scope of the embodiment to the specified elements, steps, ingredients or components and to those that do not materially affect the embodiment. A material effect would cause a statistically significant reduction in the anti-tumor effects, as described herein.

[0312] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to beobtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. When further clarity is required, the term “about” has the meaning reasonably ascribed to it by a person skilled in the art when used in conjunction with a stated numerical value or range, i.e. denoting somewhat more or somewhat less than the stated value or range, to within a range of ±20% of the stated value; ±19% of the stated value; ±18% of the stated value; ±17% of the stated value; ±16% of the stated value; ±15% of the stated value; ±14% of the stated value; ±13% of the stated value; ±12% of the stated value; ±11 % of the stated value; ±10% of the stated value; ±9% of the stated value; ±8% of the stated value; ±7% of the stated value; ±6% of the stated value; ±5% of the stated value; ±4% of the stated value; ±3% of the stated value; ±2% of the stated value; or ±1% of the stated value.

[0313] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0314] The terms “a,” “an,” “the” and similar referents used in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0315] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, thespecification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0316] Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0317] Furthermore, numerous references have been made to patents, printed publications, journal articles and other written text throughout this specification (referenced materials herein). Each of the referenced materials are individually incorporated herein by reference in their entirety for their referenced teaching.

[0318] In closing, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention may be utilized in accordance with the teachings herein. Accordingly, the present invention is not limited to that precisely as shown and described.

[0319] The particulars shown herein are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of various embodiments of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for the fundamental understanding of the invention, the description taken with the drawings and / or examples making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.

[0320] Definitions and explanations used in the present disclosure are meant and intended to be controlling in any future construction unless clearly and unambiguously modified in the examples or when application of the meaning renders any construction meaningless or essentially meaningless. In cases where the construction of the term would render it meaningless or essentially meaningless, the definition should be taken from Webster's Dictionary, 3rd Edition or a dictionary known to those of ordinary skill in the art, such as the Oxford Dictionary ofBiochemistry and Molecular Biology (Eds. Attwood T et al., Oxford University Press, Oxford, 2006).

Claims

CLAIMSWhat is claimed is:

1. An implant comprising (i) a scaffold comprising type I collagen and straight columnar pores; (ii) a chemoattractant comprising CCL21 , CCL3, CCL4, or CXCL10; (iii) an immune cell activating factor comprising an anti-CD3 antibody; and (iv) a reprogramming factor comprising a nucleic acid that encodes a chimeric antigen receptor (CAR), wherein the implant does not comprise cells before implantation into a subject.

2. An implant comprising (i) a scaffold; (ii) a chemoattractant, (iii) an immune cell activating factor, and (iv) a reprogramming factor, wherein the implant does not comprise cells before implantation into a subject.

3. The implant of claim 2, wherein the scaffold comprises columnar pores.

4. The implant of claim 3, wherein the columnar pores average 10Opm to 400pm in diameter.

5. The implant of claim 3, wherein the columnar pores are straight.

6. The implant of claim 2, wherein the scaffold comprises collagen.

7. The implant of claim 6, wherein the collagen comprises collagen type I, collagen type II, collagen type III, collagen type IV, or collagen type VI.

8. The implant of claim 6, wherein the collagen comprises collagen type I.

9. The implant of claim 6, wherein the collagen comprises collagen type III.

10. The implant of claim 6, wherein the collagen comprises collagen type I and collagen type III.11 . The implant of claim 6, wherein the collagen comprises atelocollagen.

12. The implant of claim 2, wherein the chemoattractant comprises a CXC chemokine.

13. The implant of claim 2, wherein the chemoattractant comprises CCL1 , CCL2, CCL3, CCL4, CCL5 (RANTES), CCL7, CCL8, CCL11 , CCL13, CCL17, CCL19, CCL21 , CCL22, CCL24, CCL26, CCR2, CCR5, CXCR3, CXCL8, CXCL9, CXCL10, CX3CL1 , IL-8, macrophage inflammatory protein (MIP)-1 a, MIP-1 p, or neutrophil attractant / activation protein-1 (NAP1 ).

14. The implant of claim 2, wherein the chemoattractant comprises CCL21 , CCL3, CCL4, or CXCL10.

15. The implant of claim 2, wherein the chemoattractant comprises CCL21.

16. The implant of claim 2, wherein the immune cell activating factor comprises a binding domain that binds an activating epitope on a T-cell, an NK cell, macrophage, or dendritic cell.

17. The implant of claim 16, wherein the activating epitope on the T-cell comprises IL-15, CD2, CD7, CD3, CD27, CD28, CD30, CD40, CD80, CD83, CD86, 4-1 BB, (CD137), 0X40, CD30, CD40, lymphocyte function-associated antigen-1 (LFA-1 ), CD2, CD7, LIGHT, NKG2C, B7-H3,and CD83 ligands or antibodies, CD1d, recombinant CD1d molecules preloaded with a- galactosyl ceramide and / or a recombinant major histocompatibility complex (MHC) molecule loaded with peptide.

18. The implant of claim 16, wherein the activating epitope on the NK cell comprises IL-15 or CD137.

19. The implant of claim 16, wherein the activating epitope on the macrophage comprises CD11 b, CD1 1c, CD64, CD68, CD119, CD163, CD206, CD209, F4 / 80, IFGR2 Toll-like receptors (TLRs) 1 -9, IL-4Ro, and / or macrophage receptor with collagenous structure (MARCO).

20. The implant of claim 16, wherein the activating epitope on the dendritic cell comprises a pattern recognition receptor (PRR).

21. The implant of claim 2, wherein the immune cell activating factor comprises a binding domain of an anti-CD3 antibody.

22. The implant of claim 2, wherein the immune cell activating factor comprises a binding domain of an anti-CD28 antibody.

23. The implant of claim 2, wherein the immune cell activating factor comprises a binding domain of an anti-CD3 antibody and a binding domain of an anti-CD28 antibody.

24. The implant of claim 2, wherein the reprogramming factor comprises a viral vector.

25. The implant of claim 24, wherein the viral vector comprises a retroviral vector, an adenoviral vector, or an adeno-associated virus vector.

26. The implant of claim 25, wherein the retroviral vector comprises a lentiviral vector or a foamy virus vector.

27. The implant of claim 24, wherein the viral vector comprises a pseudotyped viral vector.

28. The implant of claim 2, wherein the reprogramming factor comprises a nucleic acid that encodes an expressed molecule.

29. The implant of claim 28, wherein the expressed molecule comprises a chimeric antigen receptor (CAR), an engineered T cell receptor (eTCR), or a CAR / TCR hybrid.

30. The implant of claim 29, wherein the CAR comprises an anti-ROR1 CAR.31 . The implant of claim 2, further comprising an additional immune stimulant.

32. The implant of claim 31 , wherein the additional immune stimulant comprises a cytokine, an antibody, a small molecule, an siRNA, a plasmid DNA, and / or a vaccine adjuvant.

33. The implant of claim 2, wherein the implant is 16 mm-26 mm in diameter.

34. The implant of claim 2, wherein the implant is 19 mm-23 mm in diameter.

35. The implant of claim 2, wherein the implant is 21 mm in diameter.

36. The implant of claim 2, comprising (i) a collagen type I scaffold comprising columnarstraight pores having an average cross-sectional diameter of 100pm to 400pm; (ii) CCL21 , (iii) a binding domain of an anti-CD3 antibody (iv) a binding domain of an anti-CD28 antibody; and (v) a lentiviral vector comprising a nucleic acid that encodes a CAR, wherein the implant is 16 mm-26 mm in diameter.

37. A kit comprising i) an implant scaffold or scaffold material, ii) a chemoattractant, and iii) an immune cell activating factor.

38. The kit of claim 37, wherein the implant scaffold comprises a collagen sponge.

39. The kit of claim 37, wherein the scaffold material comprises collagen, agar, agarose, alginate, alginate / calcium phosphate cement (CPC), beta-galactosidase ( -GAL), (1 ,2, 3,4,6- pentaacetyl a-D-galactose), cellulose, chitin, chitosan, elastin, gelatin, hyaluronic acid collagen, hydroxyapatite, poly(3-hydroxybutyrate-co-3-hydroxy-hexanoate) (PHBHHx), poly(lactide), poly(caprolactone) (PCL), poly(lactide-co-glycolide) (PLG), polyethylene oxide (PEG), poly(lactic-co-glycolic acid) (PLGA), polypropylene oxide (PPG), poly(vinyl alcohol) (PVA), silk, soy protein, or soy protein isolate.

40. The kit of claim 37, further comprising a first media.41 . The kit of claim 40, wherein the first media comprises a nutrient.

42. The kit of claim 41 , wherein the nutrient comprises sugar.

43. The kit of claim 42, wherein the sugar comprises sucrose, maltose, glucose, raffinose, maltodextrin, trehalose, gum arabic, capsul, lactose, levulose, fructose, ribose, dextrose, isomalt, erythritol, sorbitol, mannitol, xylitol, lactitol, maltitol, pentatol, arabinose, pentose, xylose, galactose, leucrose, stevia, stevioside, rebaudioside A, rubusoside, and mogroside V.

44. The kit of claim 40, wherein the first media comprises sucrose phosphate-buffered saline (PBS).

45. The kit of claim 40, wherein the first media comprises a nutrient at a concentration of 0.01 M to 50M.

46. The kit of claim 40, wherein the first media comprises a nutrient at a concentration of 0.1 M to 1 M.

47. The kit of claim 40, wherein the first media comprises nutrient at a concentration of 0.5 M.

48. The kit of claim 40, wherein the first media comprises the chemoattractant.

49. The kit of claim 40, wherein the first media comprises the immune cell activating factor.

50. The kit of claim 40, wherein the first media comprises the chemoattractant and the immune cell activating factor.51 . The kit of claim 40, wherein the first media comprises 1 gg-50 pg chemoattractant.

52. The kit of claim 40, wherein the first media comprises 2 gg-20|jg chemoattractant.

53. The kit of claim 40, wherein the first media comprises 2 pg chemoattractant.

54. The kit of claim 40, wherein the first media comprises 20 pg chemoattractant.

55. The kit of claim 37, wherein the chemoattractant comprises a CXC chemokine.

56. The kit of claim 37, wherein the chemoattractant comprises CCL1 , CCL2, CCL3, CCL4, CCL5 (RANTES), CCL7, CCL8, CCL11 , CCL13, CCL17, CCL19, CCL21 , CCL22, CCL24, CCL26, CCR2, CCR5, CXCR3, CXCL8, CXCL9, CXCL10, CX3CL1 , IL-8, MIP-1 a, MIP-1 , or neutrophil attractant / activation protein-1 (NAP1 ).

57. The kit of claim 37, wherein the chemoattractant comprises CCL21 , CCL3, CCL4, or CXCL10.

58. The kit of claim 37, wherein the chemoattractant comprises CCL21 .

59. The kit of claim 40, wherein the first media comprises 1 pg-50 pg immune cell activating factor.

60. The kit of claim 40, wherein the first media comprises 2 pg-20pg immune cell activating factor.61 . The kit of claim 40, wherein the first media comprises 2 pg, 4 pg, 10 pg, or 20 pg immune cell activating factor.

62. The kit of claim 40, wherein the first media comprises 2 pg or 20 pg immune cell activating factor.

63. The kit of claim 37, wherein the immune cell activating factor comprises a binding domain that binds an activating epitope on a T-cell, an NK cell, a macrophage, or a dendritic cell.

64. The kit of claim 63, wherein the activating epitope on the T-cell comprises IL-15, CD2, CD7, CD3, CD27, CD28, CD30, CD40, CD80, CD83, CD86, 4-1 BB, (CD137), 0X40, CD30, CD40, lymphocyte function-associated antigen-1 (LFA-1 ), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83 ligands or antibodies, CD1d, recombinant CD1d molecules preloaded with a- galactosyl ceramide and / or a recombinant major histocompatibility complex (MHC) molecule loaded with peptide.

65. The kit of claim 63, wherein the activating epitope on the NK cell comprises IL-15 or CD137.

66. The kit of claim 63, wherein the activating epitope on the macrophage comprises CD1 1b, CD11c, CD64, CD68, CD119, CD163, CD206, CD209, F4 / 80, IFGR2 Toll-like receptors (TLRs) 1 -9, IL-4Ra, and / or macrophage receptor with collagenous structure (MARCO).

67. The kit of claim 63, wherein the activating epitope on the dendritic cell comprises a pattern recognition receptor (PRR).

68. The kit of claim 37, wherein the immune cell activating factor comprises a binding domainof an anti-CD3 antibody.

69. The kit of claim 37, wherein the immune cell activating factor comprises a binding domain of an anti-CD28 antibody.

70. The kit of claim 37, wherein the immune cell activating factor comprises a binding domain of an anti-CD3 antibody and a binding domain of an anti-CD28 antibody.71 . The kit of claim 37, further comprising a reprogramming factor.

72. The kit of claim 40, further comprising a second media.

73. The kit of claim 72, wherein the second media comprises a reprogramming factor.

74. The kit of claim 72, wherein the second media comprises a titer of at least 1 x 103TU / mL, at least 1 x 104TU / mL, at least 1 x 105TU / mL at least 1 x 10sTU / mL, at least 1 x 107TU / mL, at least 1 x 108TU / mL, at least 1 x 109TU / mL, or at least 1 x 101° TU / mL of a reprogramming factor.

75. The kit of claim 72, wherein the second media comprises phosphate buffered saline.

76. The kit of claim 72, wherein the second media comprises a nutrient.

77. The kit of claim 76, wherein the nutrient comprises sugar.

78. The kit of claim 77, wherein the sugar comprises sucrose, maltose, glucose, raffinose, maltodextrin, trehalose, gum arabic, capsul, lactose, levulose, fructose, ribose, dextrose, isomalt, erythritol, sorbitol, mannitol, xylitol, lactitol, maltitol, pentatol, arabinose, pentose, xylose, galactose, leucrose, stevia, stevioside, rebaudioside A, rubusoside, and mogroside V.

79. The kit of claim 76, wherein the second media comprises nutrient at a concentration of 0.01 M to 50M.

80. The kit of claim 71 , wherein the reprogramming factor comprises a viral vector.81 . The kit of claim 80, wherein the viral vector comprises a retroviral vector, an adenoviral vector, or an adeno-associated virus vector.

82. The kit of claim 81 , wherein the retroviral vector comprises a lentiviral vector or a foamy virus vector.

83. The kit of claim 80, wherein the viral vector comprises a pseudotyped viral vector.

84. The kit of claim 71 , wherein the reprogramming factor comprises a nucleic acid that encodes an expressed molecule.

85. The kit of claim 84, wherein the expressed molecule comprises a chimeric antigen receptor (CAR), an engineered T cell receptor (eTCR), or a CAR / TCR hybrid.

86. The kit of claim 37, further comprising an additional immune stimulant.

87. The kit of claim 86, wherein the additional immune stimulant comprises a cytokine, an antibody, a small molecule, an siRNA, a plasmid DNA, and / or a vaccine adjuvant.

88. The kit of claim 37, wherein the implant scaffold is 16 mm-26 mm in diameter.

89. The kit of claim 37, wherein the implant scaffold is 19 mm-23 mm in diameter.

90. The kit of claim 37, wherein the implant scaffold is 21 mm in diameter.

91. The kit of claim 37, wherein the kit comprises a collagen type I scaffold comprising columnar straight pores having an average cross-sectional diameter of 100pm to 400pm; (ii) CCL21 , (iii) a binding domain of an anti-CD3 antibody (iv) a binding domain of an anti-CD28 antibody; and (v) a lentiviral vector comprising a nucleic acid that encodes a CAR, wherein the implant scaffold is 16 mm-26 mm in diameter.

92. A method of preparing a functionalized implant comprising: obtaining an implant scaffold, wherein the implant scaffold comprises columnar pores; adding a chemoattractant to the implant scaffold; and adding an immune cell activating factor to the implant scaffold, thereby preparing the functionalized scaffold.

93. The method of claim 92, wherein the obtaining comprises selecting an implant scaffold.

94. The method of claim 92, wherein the obtaining comprises manufacturing an implant scaffold.

95. The method of claim 92, wherein the adding a chemoattractant and the adding an immune cell activating factor are performed simultaneously.

96. The method of claim 92, wherein the adding a chemoattractant and the adding an immune cell activating factor are performed sequentially.

97. The method of claim 92, wherein the adding the chemoattract is by adsorption, wet chemistry, surface treatment with plasma, chemical crosslinking, or chemical functionalization with linkers.

98. The method of claim 92, wherein the adding the immune cell activating factor is by adsorption, wet chemistry, surface treatment with plasma, chemical crosslinking, or chemical functionalization with linkers.

99. The method of claim 92, wherein the adding the chemoattractant and immune cell activating factor is by adsorption, wet chemistry, surface treatment with plasma, chemical crosslinking, or chemical functionalization with linkers.

100. The method of claim 92, wherein the adding the chemoattract is by submerging the implant scaffold in a first media comprising the chemoattractant and / or the immune cell activating factor.

101. The method of claim 100, wherein the first media comprises a nutrient.

102. The method of claim 101 , wherein the nutrient comprises sugar.

103. The method of claim 102, wherein the sugar comprises sucrose, maltose, glucose,raffinose, maltodextrin, trehalose, gum arabic, capsul, lactose, levulose, fructose, ribose, dextrose, isomalt, erythritol, sorbitol, mannitol, xylitol, lactitol, maltitol, pentatol, arabinose, pentose, xylose, galactose, leucrose, stevia, stevioside, rebaudioside A, rubusoside, and mogroside V.

104. The method of claim 100, wherein the first media comprises sucrose phosphate-buffered saline (PBS).

105. The method of claim 100, wherein the first media comprises nutrient at a concentration of 0.01 M to 50M.

106. The method of claim 100, wherein the first media comprises nutrient at a concentration of 0.1 M to 1 M.

107. The method of claim 100, wherein the first media comprises nutrient at a concentration of 0.5 M.

108. The method of claim 100, wherein the first media comprises 1 pg-50 pg chemoattractant.

109. The method of claim 100, wherein the first media comprises 2 pg-20pg chemoattractant.

110. The method of claim 100, wherein the first media comprises 2 pg chemoattractant.

111. The method of claim 100, wherein the first media comprises 20 pg chemoattractant.

112. The method of claim 92, wherein the chemoattractant comprises a CXC chemokine.

113. The method of claim 92, wherein the chemoattractant comprises CCL1 , CCL2, CCL3, CCL4, CCL5 (RANTES), CCL7, CCL8, CCL11 , CCL13, CCL17, CCL19, CCL21 , CCL22, CCL24, CCL26, CCR2, CCR5, CXCR3, CXCL8, CXCL9, CXCL10, CX3CL1 , IL-8, MIP-1a, MIP- 1 P, or neutrophil attractant / activation protein-1 (NAP1 ).

114. The method of claim 92, wherein the chemoattractant comprises CCL21 , CCL3, CCL4, or CXCL10.

115. The method of claim 92, wherein the chemoattractant comprises CCL21 .

116. The method of claim 100, wherein the first media comprises 1 pg-50 pg immune cell activating factor.

117. The method of claim 100, wherein the first media comprises 2 pg-20pg immune cell activating factor.

118. The method of claim 100, wherein the first media comprises 2 pg, 4 pg, 10 pg, or 20 pg immune cell activating factor.

119. The method of claim 100, wherein the first media comprises 2 pg or 20 pg immune cell activating factor.

120. The method of claim 100, wherein the first media comprises 20 pg chemoattractant and 1 pg-50 pg immune cell activating factor.

121. The method of claim 100, wherein the first media comprises 2 pg-20 pig chemoattractant and 1 pg-50 pg immune cell activating factor.

122. The method of claim 92, wherein the immune cell activating factor comprises a binding domain that binds an activating epitope on a T-cell, an NK cell, a macrophage, or a dendritic cell.

123. The method of claim 122, wherein the activating epitope on the T-cell comprises IL-15, CD2, CD7, CD3, CD27, CD28, CD30, CD40, CD80, CD83, CD86, 4-1 BB, (CD137), 0X40, CD30, CD40, lymphocyte function-associated antigen-1 (LFA-1 ), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83 ligands or antibodies, CD1d, recombinant CD1 d molecules preloaded with a- galactosyl ceramide and / or a recombinant major histocompatibility complex (MHO) molecule loaded with peptide.

124. The method of claim 122, wherein the activating epitope on the NK cell comprises IL-15 or CD137.

125. The method of claim 122, wherein the activating epitope on the macrophage comprises CD11 b, CD1 1 c, CD64, CD68, CD119, CD163, CD206, CD209, F4 / 80, IFGR2 Toll-like receptors (TLRs) 1 -9, IL-4Ro, and / or macrophage receptor with collagenous structure (MARCO).

126. The method of claim 122, wherein the activating epitope on the dendritic cell comprises pattern recognition receptor (PRR).

127. The method of claim 92, wherein the immune cell activating factor comprises a binding domain of an anti-CD3 antibody.

128. The method of claim 92, wherein the immune cell activating factor comprises a binding domain of an anti-CD28 antibody.

129. The method of claim 92, wherein the immune cell activating factor comprises a binding domain of an anti-CD3 antibody and a binding domain of an anti-CD28 antibody.

130. The method of claim 92, further comprising lyophilizing the functionalized scaffold.

131. The method of claim 130, wherein the lyophilizing comprises freezing the implant, sublimating the implant, and adsorbing the implant.

132. The method of claim 131 , wherein the freezing comprises lowering the temperature to a temperature below the triple point for a freezing period.

133. The method of claim 132, wherein the temperature below the triple point comprises -5°C, -10°C, -15°C, -20°C, -25°C, -30°C, -35°C, -40° C, -45°C, -50°C, -55°C, or -60°C.

134. The method of claim 132, wherein the temperature below the triple point -20°C.

135. The method of claim 132, wherein the freezing period is 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 18 hours, 24 hours, 30 hours, 36 hours, 42 hours, 48 hours, 54 hours, 60 hours, 66hours, or 72 hours.

136. The method of claim 132, wherein the freezing period is 24 hours.

137. The method of claim 131 , wherein the sublimating comprises lowering the pressure to a sublimation pressure and heating the implant to a sublimation temperature.

138. The method of claim 137, wherein the sublimation pressure comprises a pressure around the implant of below 610 Pa, below 500 Pa, below 400 Pa, below 300 Pa, below 200 Pa, below 100 Pa, below 50 Pa, below 25 Pa, or below 10 Pa.

139. The method of claim 137, wherein the sublimation temperature comprises a temperature of -5°C, -10°C, -15°C, -20°C, -25°C, -30°C, -35°C, -40°C, -45°C, -50°C, or -55°C.

140. The method of claim 131 , wherein the sublimating comprises dehydrating the implant.

141. The method of claim 131 , wherein the adsorbing comprises heating the implant to an adsorption temperature.

142. The method of claim 141 , wherein the adsorption temperature is higher than the sublimation temperature.

143. The method of claim 141 , wherein the adsorption temperature comprises a temperature above 0°C, 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, or 50°C.

144. The method of claim 130, further comprising storing the lyophilized implant at a storage temperature and for a storage duration.

145. The method of the claim 144, wherein the storage temperature is a temperature of -22°C to 22°C.

146. The method of the claim 144, wherein the storage temperature is a temperature of -10°C to 10°C.

147. The method of the claim 144, wherein the storage temperature is a temperature of 0°C to 10°C.

148. The method of the claim 144, wherein the storage temperature is a temperature of 2°C to 8°C.

149. The method of the claim 144, wherein the storage temperature is a temperature of 4°C.

150. The method of claim 144, wherein the storage temperature is not -80°C.151 . The method of claim 144, wherein the storage duration comprises overnight, 1 day, 2 days, 5 days, 10 days, one month, two months, 6 months, or a year.

152. The method of claim 130, further comprising rehydrating the lyophilized implant by submerging the lyophilized implant in a second media.

153. The method of claim 152, wherein the second media comprises a reprogramming factor.

154. The method of claim 152, wherein the second media comprises phosphate buffered saline.

155. The method of claim 152, wherein the second media comprises a nutrient.

156. The method of claim 155, wherein the nutrient comprises sugar.

157. The method of claim 156, wherein the sugar comprises sucrose, maltose, glucose, raffinose, maltodextrin, trehalose, gum arabic, capsul, lactose, levulose, fructose, ribose, dextrose, isomalt, erythritol, sorbitol, mannitol, xylitol, lactitol, maltitol, pentatol, arabinose, pentose, xylose, galactose, leucrose, stevia, stevioside, rebaudioside A, rubusoside, and mogroside V.

158. The method of claim 152, wherein the second media comprises nutrient at a concentration of 0.01 M to 50M.

159. The method of claim 153, wherein the reprogramming factor comprises a viral vector.

160. The method of claim 159, wherein the viral vector comprises a retroviral vector, an adenoviral vector, or an adeno-associated virus vector.

161. The method of claim 160, wherein the retroviral vector comprises a lentiviral vector or a foamy virus vector.

162. The method of claim 160, wherein the viral vector comprises a pseudotyped viral vector.

163. The method of claim 153, wherein the reprogramming factor comprises a nucleic acid that encodes an expressed molecule.

164. The method of claim 163, wherein the expressed molecule comprises a chimeric antigen receptor (CAR), an engineered T cell receptor (eTCR), or a CAR / TCR hybrid.

165. The method of claim 152, wherein the submerging is for a time period that is less than 5 minutes, less than 10 minutes, less than 30 minutes, less than 1 hour, less than 3 hours, less than 12 hours, or less than 24 hours.

166. The method of claim 152, wherein the second media comprises reprogramming factors at a titer of at least 1 x 103Tll / mL, at least 1 x 104TU / mL, at least 1 x 105TU / mL at least 1 x 106TU / mL, at least 1 x 107TU / mL, at least 1 x 108TU / mL, at least 1 x 109TU / mL, or at least 1 x 1010 TU / mL.

167. A method of treating a solid tumor in a subject in need thereof comprising: implanting the implant of claim 2 into the subject within a proximity to the solid tumor or tumor resection bed, thereby treating the solid tumor in the subject.

168. The method of claim 167, wherein the implanting is within a tumor resection bed.

169. The method of claim 167, wherein the solid tumor comprises an adrenal cancer, a brain cancer, a breast cancer, a cervical cancer, a colon cancer, a colorectal cancer, an ear, nose and throat (ENT) cancer, an endometrial cancer, an esophageal cancer, a gastrointestinal cancer, a glioma, a head and neck cancer, an intestinal cancer, a kidney cancer, a liver cancer,a lung cancer, a lymph node cancer, a melanoma, a neuroblastoma, an ovarian cancer, a pancreatic cancer, a prostate cancer, a rectal cancer, a seminoma, a skin cancer, a stomach cancer, a teratoma, a thyroid cancer, or a uterine cancer, or a metastasis thereof.

170. The method of claim 167, wherein the subject is a mammal.171 . The method of claim 170, wherein the mammal is a human.

172. The method of claim 167, wherein the implanting occurs during a tumor de-bulking surgery.

173. The method of claim 172, wherein the tumor de-bulking surgery is a primary tumor debulking surgery.

174. The method of claim 167, wherein the subject has been pre-screened for an immune cell status before the treating.

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