Methods of generating antibodies that are human from the start

Abstract

Provided herein are methods of producing antibodies in lymph node organoids.

Description

Attorney Docket No.: 144242-0301METHODS OF GENERATING ANTIBODIES THAT ARE HUMAN FROM THE STARTCROSS-REFERENCE TO RELATED PATENT APPLICATIONS

[0001] This application claims the benefit of and priority to US Provisional Application No. 63 / 681,595, filed August 9, 2024, the disclosure of which is incorporated by reference herein in its entirety.SEQUENCE LISTING

[0002] The application contains a Sequence Listing which has been submitted electronically in .XML format and is hereby incorporated by reference in its entirety. Said .XML copy, created on August 6, 2025, is named “144242-0301. xml” and is 24,190 bytes in size. The sequence listing contained in this .XML file is part of the specification and is hereby incorporated by reference herein in its entirety.FIELD

[0003] Provided herein are methods of producing antibodies in lymph node organoids.BACKGROUND

[0004] Immune cell activation, expansion, differentiation, and development is dependent on three-dimensional cell signaling from both surface-fixed and soluble factors. Further, novel antigen discovery, particularly for human antigens, is often prohibitively difficult, time consuming, and erroneous. Therapeutic, antibody, and vaccine testing often relies on expensive multi-species animal testing which in some cases has failed to identify efficacy, off-target effects, and immunogenicity in humans.

[0005] In addition, in-bred animals fail to provide a heterogeneous genetic background, an antigen-experienced immune system, and in many cases direct disease model examination. Moreover, in most therapeutic development processes, ethical or cost considerations are prohibitive to obtaining predictive immune response data across the wide variety of clinical groupings, even in large scale clinical trials.

[0006] What is needed is a method of generating antibodies that are human from the start.- 1 -508002954.1Attorney Docket No.: 144242-0301SUMMARY

[0007] Provided herein are methods of generating antibodies.

[0008] A first aspect provides an in vitro (ex vivo) method of generating (human) antibody(ies), comprising:(i) harvesting donor immune cells;(ii) challenging the donor immune cells with an antigen, wherein the antigen is encapsulated in or conjugated with liposome-based adjuvants, oil-in-water emulsion adjuvants, virus-like particles, TLR-ligands, carrier proteins, carrier polysaccharides and / or phycoerythrin (PE); and(iii) sorting and sequencing resulting antibodies.

[0009] In some embodiments, the method further comprises assembling the harvested donor immune cells into at least one lymph node organoid (LNO), wherein assembly occurs between steps (i) and (ii). In some embodiments, the at least one LNO comprises a 3D scaffold. In some embodiments, the 3D scaffold is 3D printed. In some embodiments, the antigen is presented on the surface of the 3D scaffold.

[0010] In some embodiments, the LNO is maintained for an extended period.

[0011] In some embodiments, the carrier proteins comprise or consist of keyhole limpet hemocyanin (KLH), non-toxic diphtheria toxin mutant (CRM 197), bovine serum albumin (BSA), Tetanus toxoid (TT), non-toxic tetanus toxoid mutant (8MTT), Diphtheria toxoid (DT), Haemophilus protein D (PD), the outer membrane protein complex of serogroup B meningococcus (OMPC), PE, or ovalbumin (OVA), or wherein the carrier polysaccharides comprise or consist of polydextran. In some embodiments, the carrier protein is KLH. In some embodiments, the carrier polysaccharide comprises, consists of, or consists essentially of polydextran or pullulan.

[0012] In some embodiments, the virus-like particles comprise or consist of Qbeta virus-like particle (Qbeta VLP).

[0013] In some embodiments, the method further comprises treating the donor immune cells to remove immunosuppressive cells and cytotoxic cells.

[0014] In some embodiments, treating donor immune cells comprises or consists of administering a lysosomotropic agent, optionally wherein the lysosomotropic agent is L- leucyl-L-leucine methyl ester (LLME), or negative selecting donor immune cells with anti-- 2 -508002954.1Attorney Docket No.: 144242-0301CD56 mAb-, anti-CD8 mAb-, and anti-CD14 mAb-conjugated magnetic beads, or combining administering LLME and negative selection with anti-CD8 mAb-conjugated magnetic beads. In some embodiments, the lysosomotropic agent is LLME.

[0015] In some embodiments, the method comprises isolation of donor total or naive B cells.

[0016] In some embodiments, the method further comprises stimulation of the cells, optionally wherein the stimulation comprises stimulation of the cells with CD40 ligand (CD40L) or toll-like receptor (TLR) ligands, optionally wherein the TLR ligands are selected from the group consisting of GpG ODNs or muramyl dipeptide (MDP).

[0017] In some embodiments, the liposome- or lipoparticle-based adjuvants comprise, consist of, or consist essentially of DOTAP (l,2-dioleoyl-3 -trimethylammonium propane) lipoparticles (or other lipoparticles) / TLR-ligands. In some embodiments, the liposome-based adjuvants comprise, consist of, or consist essentially of DOTAP liposomes (or other liposomes) / TLR-ligands. In some embodiments, the antigen is conjugated with Qbeta VLP.

[0018] In some embodiments, the method further comprises conjugating the antigen(s) with CD40L. In some embodiments, the antigen and CD40L are conjugated through streptavidin, streptavidin-nanoparticle, streptavidin-liposome, or NTA(Ni)-liposome. In some embodiments, the antigen(s) and CD40L are biotinylated. In some embodiments, the antigen(s) and CD40L are conjugated through streptavidin, streptavidin-nanoparticle, or streptavidin-liposome. In some embodiments, the antigen(s) and CD40L are histidine tagged. In some embodiments, the antigen(s) and CD40L are conjugated through NTA(Ni)-liposome.

[0019] In some embodiments, the antigen(s) is conjugated to anti-CD4 mAb, anti- CD3 mAb, or a bispecific anti-CD3 / anti-CD28 mAb through a biotin-streptavidin protocol with streptavidin-nanoparticle. In some embodiments, the method further comprises conjugating the antigen with anti-CD3 or anti-CD4 mAb through streptavidin or streptavidin conjugated nanoparticles. In some embodiments, the antigen(s) is conjugated to, or mixed with, universal T helper epitopes Pan DR-binding epitope (PADRE), Tetanus toxin (TT) (830-843) or chimeric Tetanus-Diphtheria universal T cell helper peptide (TpD).

[0020] In some embodiments, the method further comprises additionally challenging donor immune cells with universal T helper epitopes, e.g., PADRE, TT (830-843) or TpD, primed autologous CD4 T cells. In some embodiments, the method further comprises additionally challenging donor immune cells with the antigen(s) conjugated with class B CpG - 3 -508002954.1Attorney Docket No.: 144242-0301ODN 2006 or class C CpG ODN 2395 through streptavidin-nanoparticle. In some embodiments, the method further comprises additionally challenging donor immune cells with the antigen(s) conjugated with class C CpG ODN HP06T07 through streptavidin- nanoparticle. In some embodiments, the method further comprises adding an immune checkpoint inhibitor, optionally wherein the immune checkpoint inhibitor comprises anti- CTLA-4 and / or anti-PD-1 blocking antibodies, in the cell culture media.

[0021] In some embodiments, the donor immune cells are harvested from umbilical cord blood (UCB). In some embodiments, the donor immune cells are tonsil cells.

[0022] In some embodiments, the donor immune cells are challenged with an antigen foregrounded with DOTAP liposome-antigen complexes and / or Qbeta VLP-antigen conjugates. In some embodiments, the donor immune cells are challenged with an antigen foregrounded with DOTAP / PE, DOTAP / CD3, DOTAP / TL1A, DOTAP-CD19, Qbeta-CD19 peptides, or Qbeta-TNFR2.

[0023] In some embodiments, the method further comprises polarizing T cells in the donor immune cells. In some embodiments, the T cells are polarized to T helper 2 (Th2) cells. In some embodiments, the T cells are polarized to T helper 17 (Thl7) cells.

[0024] In some embodiments, the method further comprises priming T cells with antigen loaded dendritic cells (DCs). In some embodiments, the method further comprises priming helper T cells with antigen-loaded activated B cell.

[0025] In some embodiments, the method further comprises mixing antigen primed Th2 cells generated by DCs with B cells from the donor immune cells, the mixing occurring in the LNO.

[0026] In some embodiments, the method further comprises the generation of Langerhans cells (LCs) from adult monocytes or cord blood pluripotent CD34+cells to be loaded with antigen.

[0027] In some embodiments, the method further comprises the generation of follicular helper T (Tfh) cells in vitro from naive CD4 T cells isolated form adult or cord blood mononuclear cells. In some embodiments, the method further comprises activating Tfh cells using antigen loaded LCs. In some embodiments, the method further comprises mixing antigen-activated Tfh cells with B cells from the same donor, wherein the mixing occurs in the LNO. In some embodiments, the method further comprises fixing and / or embedding the- 4 -508002954.1Attorney Docket No.: 144242-0301 at least one LNO.

[0028] In some embodiments, the antigen is selected from the group consisting of CD3E, TL1 A, PE, CD19 ECD, GIPR ECD, and ACTRIIB. In some embodiments, the antigen is a component of a cell membrane fraction. In some embodiments, the antigen is contained within a cell membrane fragment. In yet other embodiments, the antigen is present in the cell membrane of an intact cell that expresses the antigen.

[0029] In some embodiments, the method is effective for generating antigen-specific cells for about 7 to about 14 days following antigen challenge.

[0030] A second aspect provides an in vitro (ex vivo) method of generating (human) antibody(ies), comprising challenging at least one LNO derived from harvested donor immune cells that were pre-treated to remove immunosuppressive cells and cytotoxic cells, with an antigen; optionally wherein the antigen is encapsulated in or conjugated with liposome-based adjuvants, oil-in-water emulsion adjuvants, virus-like particles, TLR-ligands, carrier proteins, carrier polysaccharides and / or PE.

[0031] In some embodiments, the pre-treatment to remove immunosuppressive cells and cytotoxic cells from the harvested donor immune cells comprises or consists of administering a lysosomotropic agent, optionally wherein the lysosomotropic agent is LLME, or negative selecting donor immune cells with anti-CD56 mAb-, anti-CD8 mAb-, and anti- CD14 mAb-conjugated magnetic beads or combining administering LLME and negative selection with anti-CD8 mAb-conjugated magnetic beads. In some embodiments, the lysosomotropic agent is LLME.

[0032] In some embodiments, the at least one LNO comprises a 3D scaffold. In some embodiments, the 3D scaffold is 3D printed. In some embodiments, the antigen is presented on the surface of the 3D scaffold.

[0033] In some embodiments, the LNO is maintained for an extended period.

[0034] In some embodiments, the carrier proteins comprise or consist of KLH,CRM197, BSA, TT, 8MTT, DT, PD, OMPC, PE, or OVA, or wherein the carrier polysaccharides comprise or consist of polydextran. In some embodiments, the carrier protein is KLH. In some embodiments, the carrier polysaccharide comprises, consists of, or consists essentially of polydextran or pullulan.

[0035] In some embodiments, the virus-like particles comprise or consist of Qbeta- 5 -508002954.1Attorney Docket No.: 144242-0301VLP.

[0036] In some embodiments, the method comprises isolation of donor total or naive B cells.

[0037] In some embodiments, the method further comprises stimulation of the cells, optionally wherein the stimulation comprises stimulation of the cells with CD40L TLR ligands, optionally wherein the TLR ligands are selected from the group consisting of GpG ODNs or MDP.

[0038] In some embodiments, the liposome- or lipoparticle-based adjuvants comprise, consist of, or consist essentially of DOTAP lipoparticles (or other lipoparticles) / TLR-ligands. In some embodiments, the liposome-based adjuvants comprise, consist of, or consist essentially of DOTAP liposomes (or other liposomes) / TLR-ligands. In some embodiments, the antigen is conjugated with Qbeta VLP.

[0039] In some embodiments, the method further comprises conjugating the antigen(s) with CD40L. In some embodiments, the antigen and CD40L are conjugated through streptavidin, streptavidin-nanoparticle, streptavidin-liposome, or NTA(Ni)-liposome. In some embodiments, the antigen(s) and CD40L are biotinylated. In some embodiments, the antigen(s) and CD40L are conjugated through streptavidin, streptavidin-nanoparticle, or streptavidin-liposome. In some embodiments, the antigen(s) and CD40L are histidine tagged. In some embodiments, the antigen(s) and CD40L are conjugated through NTA(Ni)-liposome.

[0040] In some embodiments, the antigen(s) is conjugated to anti-CD4 mAb, anti- CD3 mAb, or a bispecific anti-CD3 / anti-CD28 mAb through a biotin-streptavidin protocol with streptavidin-nanoparticle. In some embodiments, the method further comprises conjugating the antigen with anti-CD3 or anti-CD4 mAb through streptavidin or streptavidin conjugated nanoparticles. In some embodiments, the antigen(s) is conjugated to, or mixed with, universal T helper epitopes PADRE, TT (830-843) or chimeric TpD.

[0041] In some embodiments, the method further comprises additionally challenging donor immune cells with universal T helper epitopes, e.g., PADRE, TT (830-843) or TpD, primed autologous CD4 T cells. In some embodiments, the method further comprises additionally challenging donor immune cells with the antigen(s) conjugated with class B CpG ODN 2006 or class C CpG ODN 2395 through streptavidin-nanoparticle. In some embodiments, the method further comprises additionally challenging donor immune cells with the antigen(s) conjugated with class C CpG ODN HP06T07 through streptavidin-- 6 -508002954.1Attorney Docket No.: 144242-0301 nanoparticle. In some embodiments, the method further comprises adding an immune checkpoint inhibitor, optionally wherein the immune checkpoint inhibitor comprises anti- CTLA-4 and / or anti-PD-1 blocking antibodies, in the cell culture media.

[0042] In some embodiments, the donor immune cells are harvested from UCB. In some embodiments, the donor immune cells are tonsil cells.

[0043] In some embodiments, the donor immune cells are challenged with an antigen foregrounded with DOTAP liposome-antigen complexes and / or Qbeta VLP-antigen conjugates. In some embodiments, the donor immune cells are challenged with an antigen foregrounded with DOTAP / PE, DOTAP / CD3, DOTAP / TL1A, DOTAP-CD19, Qbeta-CD19 peptides, or Qbeta-TNFR2.

[0044] In some embodiments, the method further comprises polarizing T cells in the donor immune cells. In some embodiments, the T cells are polarized to Th2 cells. In some embodiments, the T cells are polarized to Thl7 cells.

[0045] In some embodiments, the method further comprises priming T cells with antigen loaded DCs. In some embodiments, the method further comprises priming helper T cells with antigen-loaded activated B cell.

[0046] In some embodiments, the method further comprises mixing antigen primed Th2 cells generated by DCs with B cells from the donor immune cells, the mixing occurring in the LNO.

[0047] In some embodiments, the method further comprises the generation of LCs from adult monocytes or cord blood pluripotent CD34+cells to be loaded with antigen.

[0048] In some embodiments, the method further comprises the generation of Tfh cells in vitro from naive CD4 T cells isolated form adult or cord blood mononuclear cells. In some embodiments, the method further comprises activating Tfh cells using antigen loaded LCs. In some embodiments, the method further comprises mixing antigen-activated Tfh cells with B cells from the same donor, wherein the mixing occurs in the LNO. In some embodiments, the method further comprises fixing and / or embedding the at least one LNO.

[0049] In some embodiments, the antigen is selected from the group consisting of CD3E, TL1 A, PE, CD19 ECD, GIPR ECD, and ACTRIIB. In some embodiments, the antigen is a component of a cell membrane fraction. In some embodiments, the antigen is contained within a cell membrane fragment. In yet other embodiments, the antigen is present- 7 -508002954.1Attorney Docket No.: 144242-0301 in the cell membrane of an intact cell that expresses the antigen.

[0050] In some embodiments, the method is effective for generating antigen-specific cells for about 7 to about 14 days following antigen challenge.

[0051] A third aspect provides antibodies generated using any of the processes described herein. In some embodiments, the antibodies comprise a VH sequence selected from the group consisting of SEQ ID NO:4 or SEQ ID NO: 8 or the CDRs contained therein. In some embodiments, the antibodies comprise a VL sequence selected from the group consisting of SEQ ID NO: 6 or SEQ ID NO: 10 or the CDRs contained therein. In some embodiments, the antibodies comprise a VH sequence comprising the CDR sequences found in SEQ ID NO: 4 and a VL sequence comprising the CDR sequences found in SEQ ID NO: 6. In some embodiments, the antibodies comprise a VH sequence comprising the CDR sequences found in SEQ ID NO: 8 and a VL sequence comprising the CDR sequences found in SEQ ID NO: 10. In some embodiments, the antibodies comprise a VH sequence comprising SEQ ID NO: 4 and a VL sequence comprising SEQ ID NO: 6. In some embodiments, the antibodies comprise a VH sequence comprising SEQ ID NO: 8 and a VL sequence comprising SEQ ID NO: 10. In some embodiments, the antibodies comprise a VH sequence encoded by a sequence selected from the group consisting of SEQ ID NO: 3 and SEQ ID NO: 7. In some embodiments, the antibodies comprise a VL sequence encoded by a sequence selected from the group consisting of SEQ ID NO: 5 and SEQ ID NO: 9. In some embodiments, the antibodies comprise a VH sequence encoded by SEQ ID NO: 3 and a VL sequence encoded by SEQ ID NO: 5. In some embodiments, the antibodies comprise a VH sequence encoded by SEQ ID NO: 7 and a VL sequence encoded by SEQ ID NO: 9. Vectors comprising any of the nucleic acids described herein, and host cells comprising said vectors are contemplated by the present disclosure.

[0052] A fourth aspect provides a kit comprising an adjuvant, at least one agent for the removal of immunosuppressive cells and cytotoxic cells, and instructions for using said agent to generate LNOs from a population of harvested donor immune cells. In some embodiments, the instructions direct the user to use the agent to remove immunosuppressive cells and cytotoxic cells from the harvested donor immune cells prior to generation of an LNO. In some embodiments, the agent comprises a lysosomotropic agent (e.g., LLME) or agents for negative selection of cells (e.g., anti-CD56 mAb-, anti-CD8 mAb-, and anti-CD14 mAb-conjugated magnetic beads), or a combination thereof. In some embodiments, the kit instructions further include a protocol for generating antibodies from the LNOs using the- 8 -508002954.1Attorney Docket No.: 144242-0301 adjuvant and an antigen of interest. In some embodiments, the kit comprises one or more of liposome-based adjuvants, oil-in-water emulsion adjuvants, virus-like particles, TLR-ligands, CD40L, Nucleotide-binding oligomerization domain-containing protein 2 (N0D2) agonist, carrier proteins, carrier polysaccharides and / or PE and optionally instructions for use thereof.BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Fig. 1 is an exemplary CAD drawing of a 3D scaffold holder designed to contain a 3D printed scaffold within a multiple well tissue culture plate.

[0054] Fig. 2A is a CAD drawing of a perfusion reactor that may be printed using a 3D printer.

[0055] Fig. 2B is an image of a 3D printed perfusion bioreactor capable of holding cell-laden scaffolds under perfusion.

[0056] Figs. 3 A and 3B is an example of generation of PE-specific B cells and antibody secreting cells in LLME treated PBMCs upon in vitro immunization.

[0057] Fig. 4 is a schematic representation of in vitro immunization using immunosuppressive cell / cytotoxic cell-depleted PBMCs.

[0058] Fig. 5 is a schematic representation of immune cell isolation and differentiation.

[0059] Fig. 6 is a schematic representation of DC2 mediated antigen priming of Th2 cells.

[0060] Fig. 7 is a schematic representation of B cell mediated antigen priming of Th2 cells.

[0061] Fig. 8 is a schematic representation of the generation of Tfh cells by antigen priming (top panel) or polyclonal activation (bottom panel).

[0062] Fig. 9 is a schematic representation of the EXIS platform with base reconstitution approach.

[0063] Fig. 10 is a schematic representation of DC2-mediated Th2 priming and assembly of the EXIS platform with a base reconstitution approach.

[0064] Fig. 11 is a schematic representation of B cell-mediated Th2 cell priming.

[0065] Fig. 12 is a schematic representation of the use of antigen primed Tfh cells as described in Example 21.- 9 -508002954.1Attorney Docket No.: 144242-0301

[0066] Fig. 13 is a schematic representation of orchestrating immune cells derived from neonatal human umbilical cord blood (UCB) to generate antigen specific, antibody producing plasmablasts / plasma cells and memory B cells.

[0067] Fig. 14 is a schematic representation of orchestrating immune cells derived from CD34+cell precursors, naive CD4 T cell, and naive B cell fractions of neonatal human umbilical cord blood (UCB) to generate antigen-specific antibody producing plasmablasts / plasma cells and memory B cells.

[0068] Fig. 15 shows representative plots of sorted cells by FACS. Gating strategy: Lymphocytes SingletsLive and non-dump CD19+IgD'PE+.

[0069] Fig. 16 shows representative FACS sorting plots as described in Example 24.

[0070] Figs. 17A-17C show antigen-specific antibody responses induced by in vitro immunization of immunosuppressive cells and cytotoxic cell-depleted PBMCs with antigen and cationic liposome DOTAP:DOPE and CpG ODNs. Fig. 17A is a schematic overview of the in vitro immunization protocol described in detail in Example 26. Fig. 17B shows FACS plots showing the gating strategy and sorting of CD3£-specific B cells. Fig. 17C shows ELISpot analysis of CD3£-specific ASCs from LLME-treated PBMC cultures challenged with either the adjuvant liposome DOTAP:DOPE / CpG ODN alone or in combination with CD3E.

[0071] Figs. 18A-18C show antigen-specific antibody responses induced by in vitro immunization of immunosuppressive cell and cytotoxic cell-depleted PBMCs with antigen and TLR9 ligand CpG ODN2006. Fig. 18A is a schematic overview of the in vitro immunization protocol described in detail in Example 27. Fig. 18B shows FACS plots showing the gating strategy and sorting of TLlA-specific B cells. Fig. 18C shows ELISpot analysis of TLlA-specific IgM and IgG ASCs from LLME-treated PBMC cultures challenged with either CpG ODN 2006 alone or in combination with TL1 A.

[0072] Figs. 19A-19B show the enhancement of antigen-specific antibody responses in vitro by CD40L, DOTAP:Cholesterol / CpG, and antigen-CD40L conjugates. Fig. 19A shows FACS plots showing the gating and sorting of PE-specific B cells. Fig. 19B shows ELISpot analysis of PE-specific antibody-secreting cells (ASCs).

[0073] Figs. 20A-20B show that the N0D2 agonist muramyl dipeptide (MDP) enhances antigen-specific antibody responses in vitro. Fig. 20A shows representative FACS- 10 -508002954.1Attorney Docket No.: 144242-0301 plots showing the sorting of TLlA-specific B cells. Fig. 20B shows flow cytometric analysis of CD19+IgD“ TLlA-specific B cells.

[0074] Figs. 21 A-21B show that oil-in-water nano-emulsion adjuvant AddaS03 enhances antigen-specific antibody responses in vitro. Fig. 21 A shows representative FACS plots showing the sorting of antigen (TL1 A)-specific B cells. Fig. 21B shows flow cytometric analysis of TLlA-specific B cells.

[0075] Fig. 22 shows flow cytometry plots demonstrating that TLR9 ligand CpG ODN 2395, in combination with checkpoint blockade using anti-CTLA-4 mAb, enhances the generation of antigen-specific B cells following in vitro immunization.

[0076] Fig. 23 shows flow cytometry plots demonstrating that TL1 A and CD40L together induce the generation of TLlA-specific B cells in vitro.

[0077] Fig. 24 shows flow cytometry graphs demonstrating that conjugation of antigen with NTA(Ni)-liposomes enhance the generation of antigen-specific B cells upon in vitro immunization.

[0078] Fig. 25 shows flow cytometry plots demonstrating that TLR9 ligand CpG ODN 2006, in combination with checkpoint blockade using anti-PD-1 mAb, enhances the generation of antigen-specific B cells following in vitro immunization.

[0079] Fig. 26 shows flow cytometry plots demonstrating that anionic liposomes (DOPG:DOPC and DOPS:DOPC), in combination with CD40L, enhance CD19-specific antibody responses in vitro.

[0080] Fig. 27 shows flow cytometry plots demonstrating that the universal T cell epitope PADRE and Qbeta VLP enhance CD19-specific antibody responses in vitro.

[0081] Figs. 28A-28B shows that antigen conjugation of antigen with 8MTT enhances antigen-specific antibody responses in vitro. Fig. 28A shows representative flow cytometry plots for CD I 9 IgD ACTRIIB-specific B cells that were analyzed and sorted by flow cytometry. Fig. 28B shows representative flow cytometry plots for CD27+differentiated B cells among the sorted IgD ACTRIIB-specific B cells that were analyzed by flow cytometry.

[0082] Figs. 29A-29C show a cell membrane-based immunization approach that generates antigen-specific, class-switched B cells targeting a multi-pass transmembrane GPCR protein in vitro. Fig. 29A is a schematic illustrating B cell receptor (BCR) engagement- 11 -508002954.1Attorney Docket No.: 144242-0301 using cell membranes derived from GIPR-expressing cells. Fig. 29B is a schematic illustrating the preparation of GIPR-expressing cell membrane fractions and the EXIS immunization approach using membrane-associated antigens. Fig. 29C shows FACS plots of CD19+IgD GIPR-specific B cells and IgG+B cells derived from human PBMCs treated with LLME to generate LNOs, which were stimulated with either the cell membrane fraction from GIPR-expressing HEK293 cells or GIPR ECD protein in the presence of CD40L.

[0083] Fig. 30 shows sensorgrams depicting the binding affinity of in vitro immunization-derived Prellis antibodies P3371 and P3378 against the target antigen TL1 A.DETAILED DESCRIPTION

[0084] Provided herein are methods of generating antibodies. Also provided are methods of using the antibodies, such as therapeutic methods.1. Definitions

[0085] Unless otherwise defined, all terms of art, notations, and other scientific terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a difference over what is generally understood in the art. The techniques and procedures described or referenced herein are generally well understood and commonly employed using conventional methodologies by those skilled in the art, such as, for example, the widely utilized molecular cloning methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. As appropriate, procedures involving the use of commercially available kits and reagents are generally carried out in accordance with manufacturer defined protocols and / or parameters unless otherwise noted.

[0086] As used herein, the singular forms “a,” “an,” and “the” include the plural referents unless the context clearly indicates otherwise.

[0087] The term “about” indicates and encompasses an indicated value and a range above and below that value. In certain embodiments, the term “about” indicates the designated value ± 10%, ± 5%, or ± 1%. In certain embodiments, the term “about” indicates the designated value ± one standard deviation of that value.

[0088] The term “adjuvant” refers to a substance or compound that is added to- 12 -508002954.1Attorney Docket No.: 144242-0301 enhance and improve immune response to an antigen, often by promoting antigen presentation or stimulating immune cell activation. Adjuvant may increase vaccine potency, reduce the amount of antigen needed, speed up immune responses, and broaden antibody repertoires. Adjuvants here may comprise of: molecules targeting pattern recognition receptors (PRRs) such as viral or synthetic RNA (e.g., poly EC, ssRNA40), ODN CpG motif, muramyl dipeptide (MDP), Pam3CSK4 , R848, ssPolyU, lipopolysaccharide (LPS), monophosphorylated lipid A (MPLA), or other TLR ligands, Immunostimulatory complexes (ISCOMs), or nanomaterials based particles, emulsions, such as oil-in water emulsions including AddaS03, AdaVax, and Arava, liposomes, such as NTA(Ni) liposomes, lipoparticles, or micelles to enter the APC and deliver the antigen. Synthetic adjuvants such as of polymer nanoparticles. Virus-like particles (VLP) such as Qbeta phage as antigen carriers and PRR-activator.

[0089] In some embodiments the adjuvant is a TLR ligand. In some embodiments the adjuvant is a NOD2 ligand. In some embodiments the adjuvant is a conjugated liposome. In some embodiments the adjuvant is an oil-in-water solution. In some embodiments the adjuvant is an antigen-anti-CD3 antibody conjugate. In some embodiments the adjuvant is an antigen-anti-CD4 antibody conjugate. In some embodiments the adjuvant is a bacteriophage or a component or VLP thereof. In some embodiments the adjuvant is an anionic liposome. In some embodiments the adjuvant is a universal T helper epitope. In some embodiments the adjuvant is a carrier protein. In some embodiments the adjuvant is a polysaccharide. In some embodiments the adjuvant is an anti-CTLA4 antibody (Tremelimumab, HY-P9918, MedChemExpress), an anti-PD-1 antibody (penpulimab, HY-P99108, MedChemExpress), or an anti-PD-l / CTLA4 antibody (Vudalimab, HY-P99166, MedChemExpress). In some embodiments the adjuvant is an antigen-CD40L (CD40L: CDL-HM440B, KACTUS) nanosphere (CP-01000, Bangs Labs) conjugate. In some embodiments the adjuvant is an antigen-CpGB nanosphere conjugate. In some embodiments the adjuvant is cell-membrane based. In some embodiments the adjuvant is an aluminum salt. In some embodiments the adjuvant is poly(I:C) (tlrl-pic, Invivogen). In some embodiments the adjuvant is LPS (tlrl- b51ps, Invivogen). In some embodiments the adjuvant is R848 (tlrl-r848- 1 , Invivogen). In some embodiments the adjuvant is ssRNA40 (tlrl-lma40, Invivogen). In some embodiments the adjuvant is MDP (tlrl-mdp, Invivogen). In some embodiments the adjuvant is NTA(Ni)- liposome (IMS-2062, Encapsula NanoSciences). In some embodiments the adjuvant is AddaS03 (vac-as03-10, invivogen). In some embodiments the adjuvant is AdaVax (vac-adx-- 13 -508002954.1Attorney Docket No.: 144242-030110, Invivogen). In some embodiments the adjuvant is Qbeta-VLP (Q[3 VLP, Fina Biosolutions). In some embodiments the adjuvant is DOPG:DOPC (CPG-508, Encapsula Nano Sciences). In some embodiments the adjuvant is DOPS:DOPC (CPS-508, Encapsula Nano Sciences). In some embodiments the adjuvant is PADRE (SB054, SB-PEPTIDE). In some embodiments the adjuvant is KLH (protein KLH conjugation with ReadiLink KLH Conjugation Kit, 5502, AAT Bioquest; peptide-KLH conjugates were custom synthesized by CSBio). In some embodiments the adjuvant is CRM197 (Custom Protein-CRM197 conjugation by Fina Biosolutions). In some embodiments the adjuvant is MTT (Custom Protein-8MTT conjugation by Fina Biosolutions). In some embodiments the adjuvant is polydextran (Custom Protein-polydextran conjugation by Fina Biosolutions). In some embodiments the adjuvant is Alhydrogel.

[0090] The term “immunoglobulin” refers to a class of structurally related proteins generally comprising two pairs of polypeptide chains: one pair of light (L) chains and one pair of heavy (H) chains. In an “intact immunoglobulin,” all four of these chains are interconnected by disulfide bonds. The structure of immunoglobulins has been well characterized. See, e.g., Paul, Fundamental Immunology 7th ed., Ch. 5 (2013) Lippincott Williams & Wilkins, Philadelphia, PA. Briefly, each heavy chain typically comprises a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region typically comprises three domains, Cm, Cm, and CH3. Each light chain typically comprises a light chain variable region (VL) and a light chain constant region. The light chain constant region typically comprises one domain, abbreviated CL.

[0091] The term “antibody” describes a type of immunoglobulin molecule and is used herein in its broadest sense. An antibody specifically includes intact antibodies (e.g., intact immunoglobulins), and antibody fragments and antigen binding proteins. Antibodies comprise at least one antigen-binding domain. One example of an antigen-binding domain is an antigen binding domain formed by a VH-VL dimer.

[0092] The VH and VL regions may be further subdivided into regions of hypervariability (“hypervariable regions (HVRs);” also called “complementarity determining regions” (CDRs)) interspersed with regions that are more conserved. The more conserved regions are called framework regions (FRs). Each VH and VL generally comprises three CDRs and four FRs, arranged in the following order (from N-terminus to C-terminus): FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4. The CDRs are involved in antigen binding and- 14 -508002954.1Attorney Docket No.: 144242-0301 confer antigen specificity and binding affinity to the antibody. See Kabat et al., Sequences of Proteins of Immunological Interest 5th ed. (1991) Public Health Service, National Institutes of Health, Bethesda, MD, incorporated by reference in its entirety.

[0093] The light chain from any vertebrate species can be assigned to one of two types, called kappa and lambda, based on the sequence of the constant domain.

[0094] The heavy chain from any vertebrate species can be assigned to one of five different classes (or isotypes): IgA, IgD, IgE, IgG, and IgM. These classes are also designated a, 5, a, y, and p, respectively. The IgG and IgA classes are further divided into subclasses on the basis of differences in sequence and function. Humans express the following subclasses: IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2.

[0095] The term “amino acid” refers to the twenty common naturally occurring amino acids. Naturally occurring amino acids include alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys; C); glutamic acid (Glu; E), glutamine (Gin; Q), Glycine (Gly; G); histidine (His; H), isoleucine (He; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Vai; V).

[0096] The amino acid sequence boundaries of a CDR can be determined by one of skill in the art using any of a number of known numbering schemes, including those described by Kabat et al., supra (“Kabat” numbering scheme); Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948 (“Chothia” numbering scheme); MacCallum et al., 1996, J. Mol. Biol. 262:732-745 (“Contact” numbering scheme); Lefranc et al., Dev. Comp. Immunol., 2003, 27:55-77 (“IMGT” numbering scheme); and Honegge and Pliickthun, J. Mol. Biol., 2001, 309:657-70 (“AHo” numbering scheme), each of which is incorporated by reference in its entirety.

[0097] Table 1 provides the positions of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 as identified by the Kabat and Chothia schemes. For CDR-H1, residue numbering is provided using both the Kabat and Chothia numbering schemes.

[0098] Unless otherwise specified, the numbering scheme used for identification of a particular CDR herein is the Kabat / Chothia numbering scheme. Where the residues encompassed by these two numbering schemes diverge, the numbering scheme is specified as either Kabat or Chothia.- 15 -508002954.1Attorney Docket No.: 144242-0301Table 1. Residues in CDRs according to Kabat and Chothia numbering schemes.* The C-terminus of CDR-H1, when numbered using the Kabat numbering convention, varies between H32 and H34, depending on the length of the CDR.

[0099] The “EU numbering scheme” is generally used when referring to a residue in an antibody heavy chain constant region (e.g., as reported in Kabat et al., supra). Unless stated otherwise, the EU numbering scheme is used to refer to residues in antibody heavy chain constant regions described herein.

[0100] An “antibody fragment” comprises a portion of an intact antibody, such as the antigen binding or variable region of an intact antibody. Antibody fragments include, for example, Fv fragments, Fab fragments, F(ab')2 fragments, Fab' fragments, scFv (sFv) fragments, and scFv-Fc fragments.

[0101] “Fv” fragments comprise a non-covalently-linked dimer of one heavy chain variable domain and one light chain variable domain.

[0102] “Fab” fragments comprise, in addition to the heavy and light chain variable domains, the constant domain of the light chain and the first constant domain (Cm) of the heavy chain. A fab fragment can be composed of one constant and one variable domain of each of the heavy and light chains. Fab fragments may be generated, for example, by papain digestion of a full-length antibody.

[0103] “F(ab')2” fragments contain two Fab' fragments joined, near the hinge region, by disulfide bonds. F(ab')2 fragments may be generated, for example, by pepsin digestion of an intact antibody. The F(ab') fragments can be dissociated, for example, by treatment with B-mercaptoethanol. A Fab’ fragment is a Fab fragment that has been modified with disulfide bonds.

[0104] “Single-chain Fv” or “sFv” or “scFv” antibody fragments comprise a VH domain and a VL domain in a single polypeptide chain. The VH and VL are generally linked by a peptide linker. See Pliickthun A. (1994). Antibodies from Escherichia coli. In- 16 -508002954.1Attorney Docket No.: 144242-0301Rosenberg M. & Moore G.P. (Eds.), The Pharmacology of Monoclonal Antibodies vol. 113 (pp. 269-315). Springer-Verlag, New York, incorporated by reference in its entirety.

[0105] “scFv-Fc” fragments comprise an scFv attached to an Fc domain. For example, an Fc domain may be attached to the C-terminal of the scFv. The Fc domain may follow the VH or VL, depending on the orientation of the variable domains in the scFv (i.e., VH-VL or VL-VH). Any suitable Fc domain known in the art or described herein may be used.

[0106] A “single domain antibody” or “nanobody” is an antibody fragment consisting of a single monomeric variable antibody domain, often a peptide of about 110 amino acids, comprising one variable domain, often of a heavy chain. The single domain antibody often also comprises a common IgG.

[0107] The term “monoclonal antibody” refers to an antibody from a population of substantially homogeneous antibodies. A population of substantially homogeneous antibodies comprises antibodies that are substantially similar and that bind the same epitope(s), except for variants that may normally arise during production of the monoclonal antibody. Such variants are generally present in only minor amounts. A monoclonal antibody is typically obtained by a process that includes the selection of a single antibody from a plurality of antibodies. For example, the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones, yeast clones, bacterial clones, or other recombinant DNA clones. The selected antibody can be further altered, for example, to improve affinity for the target (“affinity maturation”), to humanize the antibody, to improve its production in cell culture, and / or to reduce its immunogenicity in a subject.

[0108] The term “chimeric antibody” refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0109] “Humanized” forms of non-human antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. A humanized antibody is generally a human immunoglobulin (recipient antibody) in which residues from one or more CDRs are replaced by residues from one or more CDRs of a non-human antibody (donor antibody). The donor antibody can be any suitable non-human antibody, such as a mouse, rat, rabbit, chicken, or non-human primate antibody having a desired specificity, affinity, or biological effect. In some instances, selected framework region residues of the recipient- 17 -508002954.1Attorney Docket No.: 144242-0301 antibody are replaced by the corresponding framework region residues from the donor antibody. Humanized antibodies may also comprise residues that are not found in either the recipient antibody or the donor antibody. Such modifications may be made to further refine antibody function. For further details, see Jones et al., Nature, 1986, 321 :522-525; Riechmann et al., Nature, 1988, 332:323-329; and Presta, Curr. Op. Struct. Biol., 1992, 2:593-596, each of which is incorporated by reference in its entirety.

[0110] A “human antibody” is one which possesses an amino acid sequence corresponding to that of an antibody produced by a human or a human cell, or derived from a non-human source that utilizes a human antibody repertoire or human antibody-encoding sequences (e.g., obtained from human sources or designed de novo). Human antibodies specifically exclude humanized antibodies.

[0111] An “isolated antibody” is one that has been separated and / or recovered from a component of its natural environment. Components of the natural environment may include enzymes, hormones, and other proteinaceous or nonproteinaceous materials. In some embodiments, an isolated antibody is purified to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence, for example by use of a spinning cup sequenator. In some embodiments, an isolated antibody is purified to homogeneity by gel electrophoresis (e.g., SDS-PAGE) under reducing or nonreducing conditions, with detection by Coomassie blue or silver stain. An isolated antibody includes an antibody in situ within recombinant cells, since at least one component of the antibody’s natural environment is not present. In some embodiments, an isolated antibody is prepared by at least one purification step.

[0112] In some embodiments, an isolated antibody is purified to at least 80%, 85%, 90%, 95%, or 99% by weight. In some embodiments, an isolated antibody is provided as a solution comprising at least 85%, 90%, 95%, 98%, 99% to 100% by weight of an antibody, the remainder of the weight comprising the weight of other solutes dissolved in the solvent.

[0113] The term “monoclonal antibodies” refers to laboratory -produced molecules designed to target specific proteins or cells in the body. What makes them unique is this specificity; they are designed to bind to a single target with high precision, which allows for tailored therapeutic interventions. Moreover, monoclonal antibodies are produced by identical immune cells, ensuring that both the antibodies and their effects are consistent and reproducible.- 18 -508002954.1Attorney Docket No.: 144242-0301

[0114] The term “antigen” refers to a molecule or substance that is recognized by the immune system as and typically elicits an immune response, such as the production of antibodies or the activation of immune cells.

[0115] The term “antigen-encoding mRNA” or “mRNA antigen” refers to a messenger RNA (mRNA) molecule that encodes an antigen.

[0116] The term “antigen-encoding plasmid DNA” refers to a specially engineered piece of circular DNA (called a plasmid) that contains a gene sequence encoding a specific antigen — the protein or peptide against which an immune response is desired. Antigen-encoding plasmids are the core components of a DNA vaccine.

[0117] “Affinity” refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity, which reflects a 1 : 1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD). Affinity can be measured by common methods known in the art, including those described herein. Affinity can be determined, for example, using surface plasmon resonance (SPR) technology, such as a Biacore® instrument, or using bio-layer interferometry technology, such as an Octet® instrument.

[0118] With regard to the binding of an antibody to a target molecule, the terms “specific binding,” “specifically binds to,” “specific for,” “selectively binds,” and “selective for” a particular antigen (e.g., a polypeptide target) or an epitope on a particular antigen mean binding that is measurably different from a non-specific or non-selective interaction. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule. Specific binding can also be determined by competition with a control molecule that is similar to the target, such as an excess of non-labeled target. In that case, specific binding is indicated if the binding of the labeled target to a probe is competitively inhibited by the excess non-labeled target.

[0119] The term “kd” (sec '), as used herein, refers to the dissociation rate constant of a particular antibody-antigen interaction. This value is also referred to as the koir value.

[0120] The term “ka” (M ' / sec '), as used herein, refers to the association rate constant of a particular antibody-antigen interaction. This value is also referred to as the konvalue.- 19 -508002954.1Attorney Docket No.: 144242-0301

[0121] The term “KD” (M), as used herein, refers to the dissociation equilibrium constant of a particular antibody-antigen interaction. KD = kd / ka.

[0122] The term “KA” (M '), as used herein, refers to the association equilibrium constant of a particular antibody-antigen interaction. KA = ka / kd.

[0123] An “affinity matured” antibody is one with one or more alterations in one or more CDRs or FRs that result in an improvement in the affinity of the antibody for its antigen, compared to a parent antibody which does not possess the alteration(s). In one embodiment, an affinity matured antibody has nanomolar or picomolar affinity for the target antigen. Affinity matured antibodies may be produced using a variety of methods known in the art. For example, Marks et al. (Bio / Technology, 1992, 10:779-783, incorporated by reference in its entirety) describes affinity maturation by VH and VL domain shuffling. Random mutagenesis of CDR and / or framework residues is described by, for example, Barbas et al. (Proc. Nat. Acad. Set. U.S.A., 1994, 91 :3809-3813); Schier et al., Gene, 1995, 169: 147-155; Yelton et al., J. Immunol., 1995, 155: 1994-2004; Jackson et al., J. Immunol., 1995, 154:3310-33199; and Hawkins et al, J. Mol. Biol., 1992, 226:889-896, each of which is incorporated by reference in its entirety.

[0124] When used herein in the context of two or more antibodies, the term “competes with” or “cross-competes with” indicates that the two or more antibodies compete for binding to an antigen. If the presence of the first antibody reduces binding of the second antibody, then the antibodies compete. The term “competes with” also includes combinations of antibodies where one antibody reduces binding of another antibody, but where no competition is observed when the antibodies are added in the reverse order. However, in some embodiments, the first and second antibodies inhibit binding of each other, regardless of the order in which they are added. In some embodiments, one antibody reduces binding of another antibody to its antigen by at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%.

[0125] The term “assembling” refers to bringing together individual components or parts to form a unified structure or entity, often performed to create complex systems or functional units from simpler elements.

[0126] The term “autologous” refers to the utilization of biological material, such as cells or tissues, sourced from the same individual undergoing the treatment. Because the donor and recipient are the same individual, this approach mitigates the risk of immune- 20 -508002954.1Attorney Docket No.: 144242-0301 rejection or adverse reactions. For example, autologous CD4 T cells would imply using the individual’s own CD4 T cells in the described immunotherapeutic method. In this application, it also describes the source of immune cells being one individual donor.

[0127] The term “biotin” refers to a water-soluble B vitamin, also known as vitamin B7, vitamin H, or coenzyme R. It plays a crucial role in various metabolic processes in the body, including the metabolism of carbohydrates, fats, and proteins. Biotin can bind with an extremely high affinity with streptavidin, a protein found in the bacterium Streptomyces avidinii. The binding between biotin and streptavidin is one of the strongest non-covalent interactions known in nature. Biotin-streptavidin is a powerful and widely used in non- covalent interaction between two molecules in biological research. Biotinylated molecules are molecules that have been chemically modified to incorporate biotin into their structure. This modification involves attaching biotin to the molecule, typically through a linker arm, thereby allowing the molecule to be conjugated or bound to biotin. Biotinylated molecules are commonly used in biotechnological applications. In some instances, they can selectively bind to streptavidin, enabling the detection, purification, or immobilization of the target molecule.

[0128] The term “blocking” refers to the use of specific inhibitors, such as monoclonal antibodies, to disrupt or inhibit molecular interactions involved in immune regulation or tumor immune evasion. For example, “anti-CTLA-4” and “anti-PD-1” blocking antibodies interfere with the interactions between CTLA-4 and PD-1 receptors and their respective ligands, in turn preventing the inhibitory signals that suppress T cell activation and function.

[0129] The term “carrier proteins” refers to proteins used to conjugate with antigens to enhance their immunogenicity. Carrier proteins are important components used in vaccines and immunizations to enhance the immune response to antigens. Some carrier proteins may provide T cell epitopes. In some embodiments, the carrier proteins comprise or consist of KLH, PE, CRM197, BSA, TT, DT, PD, OMPC, Pseudomonas aeruginosa exotoxin A (rEPA), Pneumococcal surface proteins (e.g. pneumolysin, PiuA, Sp0418), Group B Streptococcus pill proteins (GBS80, GBS67), or OVA.

[0130] The term “CD3” or “Cluster of Differentiation 3” refers to a complex of surface proteins found on T cells, consisting of CD3y (gamma), CD35 (delta), CD3s (epsilon), and CD3(^ (zeta). CD3 is a co-receptor involved in T cell receptor signaling and- 21 -508002954.1Attorney Docket No.: 144242-0301 activation, particularly for CD8+T cells and CD4+T cells. CD3 is a critical component of the T cell activation machinery and plays a central role in adaptive immunity. Upon antigen recognition by the TCR, CD3 transmits an activation signal from the extracellular space into the T-cell’s interior, initiating a cascade of intracellular events that lead to T-cell activation, proliferation, and differentiation. CD3 serves as a crucial diagnostic marker for identifying T-cells in various immunological assays and tissue samples.

[0131] The term “CD4” or “Cluster of Differentiation 4” refers to an integral membrane glycoprotein that plays an essential role in the immune response and serves multiple functions. In T-cells, CD4 functions primarily as a coreceptor for MHC class II molecule:peptide complex. CD4 interacts simultaneously with the T-cell receptor (TCR) and the MHC class II presented by antigen presenting cells (APCs). In other cells such as macrophages or NK cells, CD4 plays a role in differentiation / activation, cytokine expression, and cell migration. CD4 also participates in the development of T-helper cells in the thymus and triggers the differentiation of monocytes into functional mature macrophages. In this application, CD4 + cells comprise of cells expressing the CD4 receptor, which are in the majority T lymphocytes. Other cell types expressing CD4 are monocytes and macrophages.

[0132] The term “T cells” or “T lymphocytes” refers to a type of white blood cell that plays a central role in adaptive immunity. T cells originate from hematopoietic stem cells in the bone marrow and mature in the thymus (an organ of the immune system and a part of the lymphatic system located in the upper chest, just behind the sternum and in front of the heart). In the thymus, T cells undergo selection and maturation processes to develop functional T cell receptors (TCRs) and self-tol erance (i.e. the ability to recognize and tolerate these T cells as “self’ and not mount an immune response against them). T cells express diverse surface markers, including CD3, CD4, and CD8 that define different T cell subsets and functions. T cells recognize antigens presented by major histocompatibility complex (MHC) molecules on antigen-presenting cells and mediate cellular immunity, including cytotoxicity (a mechanism to recognize and kill infected or abnormal cells), cytokine production, and immunoregulation.

[0133] The term “CD4+T cells”, “CD4 T cells” or “helper T cells” refers to a subset of T lymphocytes that express CD4 protein on their surface and play a crucial role in orchestrating and regulating immune responses. Helper T cells provide essential signals and cytokines that coordinate the activation, differentiation, and effector functions of other immune cells, including B cells, cytotoxic T cells, macrophages, and dendritic cells. Helper - 22 -508002954.1Attorney Docket No.: 144242-0301T cells recognize antigens presented by antigen-presenting cells (APCs) via their T cell receptors (TCRs) and interact with APCs through co-stimulatory molecules and cytokine receptors. Helper T cells provide crucial help to B cells for antibody production and maturation. Helper T cells can differentiate into distinct subsets, such as T helper 1 (Thl), T helper 2 (Th2), T helper 17 (Thl7), and regulatory T cells (Tregs), each with specialized functions in immune regulation, inflammation, and host defense against pathogens.

[0134] The term “naive CD4 T cells” refers to the CD4+T cells that have not encountered their cognate antigen and have not yet differentiated into effector or memory T cell subsets. Naive CD4+T cells circulate in the bloodstream and lymphoid organs, patrolling for antigens presented by antigen-presenting cells (APCs) via major histocompatibility complex (MHC) class II molecules. Upon encountering antigen, naive CD4+T cells undergo activation, proliferation, and differentiation into specialized effector T cell subsets, such as T helper 1 (Thl), T helper 2 (Th2), T helper 17 (Thl7), regulatory T cells (Tregs), or follicular helper T cells (Tfh), depending on the cytokine milieu and costimulatory signals present in the microenvironment.

[0135] The term “Tfh” or “Follicular helper T cell” refers to a specialized subset of CD4+T cells that play a crucial role in providing help to B cells during the germinal center reaction in secondary lymphoid organs. Follicular helper T cells promote germinal center formation, antibody affinity maturation, and B cell differentiation to memory B cells and long-lived plasma cells. Tfh cells are characterized by high expression of CXCR5, PD-1, and ICOS, as well as transcription factor BCL-6. The chemokine receptor CXCR5 expressed by Tfh cells, guides their migration to B cell follicles in lymph nodes and spleen. Tfh cells interact with B cells through cell surface molecules such as CD40 ligand (CD40L) and cytokines like interleukin-21 (IL-21), promoting B cell proliferation, antibody class switching, affinity maturation, and the generation of long-lived plasma cells and memory B cells. Tfh cells are essential for the development of effective antibody responses against pathogens and vaccines and are implicated in autoimmunity and B cell lymphomas.

[0136] The term “Th2” or “T helper 2” refers to a subset of CD4+T cells that play a crucial role in orchestrating immune responses against extracellular parasites, allergens, and helminths (group of parasitic worms that infect humans and other animals). Th2 cells produce cytokines such as IL-4, IL-5, IL-6, IL-9, and IL- 13 that promote antibody production, eosinophil recruitment, and tissue repair. The hallmark of Th2 cells is IL-4 production.- 23 -508002954.1Attorney Docket No.: 144242-0301

[0137] The term “Thl7” or “T helper 17” refers to a subset of CD4+T cells characterized by their production of interleukin- 17 (IL-17) family cytokines, including IL- 17A, IL-17F, IL-21, and IL-22. These cytokines, and subsequently Thl7, are involved in inflammation, immune response, and defense against infection. Thl7 cells interact with other immune cells, such as neutrophils, dendritic cells, and epithelial cells, to regulate immune responses and maintain tissue homeostasis.

[0138] The term “CD8” or “Cluster of Differentiation 8” refers to a type I transmembrane glycoprotein predominantly expressed on cytotoxic T cells (CD8+T cells), NK cells, and some dendritic cells. It plays a crucial role in the immune response by binding to major histocompatibility complex (MHC) class I molecules on antigen-presenting cells (APCs), facilitating the recognition and elimination of virus-infected or abnormal cells.Upon antigen recognition, CD8 acts as a co-receptor with the T-cell receptor (TCR), triggering cytotoxic T cell activation and subsequent release of cytotoxic molecules like perforin and granzymes to modulate immune responses and the direct lysis of infected or malignant cells. This process is essential for immune surveillance and defense against intracellular pathogens and malignant cells. Beyond its role in immunity, CD8 expression can vary across different T cell subsets, influencing their differentiation and effector capabilities. The CD8 molecule itself consists of two chains (alpha and beta) that together form a heterodimer, and its expression is tightly regulated during T cell development and activation.

[0139] The term “CD 14” or “Cluster of Differentiation 14” refers to a glycoprotein expressed on monocytes and macrophages and also on neutrophils and dendritic cells. CD14 exists in two forms: membrane-bound CD14 (mCD14), which anchors to the cell membrane via its transmembrane domain and is essential for LPS recognition on the cell surface and soluble CD14 (sCD14), which lacks the transmembrane domain and is released into the extracellular environment.

[0140] The term “CD28,” “Cluster of Differentiation 28,” or “TP44” describes T-cell specific surface glycoprotein CD28. CD28 is a costimulatory receptor found on T cells and is involved in T-cell activation, the induction of cell proliferation and cytokine production, and promotion of T-cell survival. CD28 binds to CD80 and CD86 (collectively referred to as B7) on antigen-presenting cells. CD28 enhances the production of IL4 and IL10 in T-cells in conjunction with TCR / CD3 ligation and CD40L and is a co-stimulatory protein found on the surface of T cells.- 24 -508002954.1Attorney Docket No.: 144242-0301

[0141] The term “CD34,” “Cluster of differentiation 34” refers to a cell surface marker found on hematopoietic stem cells (HSCs) and endothelial progenitor cells. CD34 is commonly used as a marker for identifying and isolating hematopoietic stem cells from bone marrow, peripheral blood, and cord blood for transplantation and regenerative medicine purposes. CD34 cells possess the capacity for self-renewal and differentiation into mature blood cells, including red blood cells, white blood cells, and platelets, making them valuable in hematopoietic stem cell transplantation and gene therapy.

[0142] The terms “CD40L,” “CD40 ligand,” or “CD 154” refers to a type II transmembrane protein predominantly expressed on activated T cells, but also found on other immune cells such as B cells, macrophages, and dendritic cells. It belongs to the tumor necrosis factor (TNF) superfamily and interacts with its receptor, CD40, primarily expressed on antigen-presenting cells (APCs), such as B cells and dendritic cells. This interaction is crucial for the regulation of immune responses, particularly in the activation and differentiation of B cells, induction of cytokine production, and enhancement of antigen presentation. CD40L-CD40 signaling is involved in various aspects of adaptive immunity, including class-switching and somatic hypermutation of immunoglobulins, germinal center formation, and the establishment of memory T cell responses. Dysfunction or mutations in CD40L or CD40 can lead to immunodeficiencies or autoimmune diseases.

[0143] The term “CD40” or “Cluster of Differentiation 40” refers to a type I transmembrane protein found on antigen-presenting cells and is required for their activation. CD40 binding to CD40L activates antigen presenting cells and induces a variety of downstream effects. Activated CD4+T cells primarily exhibit its CD40 ligand to antigen presenting cells, including dendritic cells, B cells, macrophages on a variety of non-immune cells, including platelets and endothelial cells.

[0144] The term “CD56” or “Cluster of Differentiation 56,” or “NCAM (neural cell adhesion molecule)” refers to a homophilic binding glycoprotein that is expressed on various immune cells. CD56 is a marker commonly used to identify nature killer (NK) cells and certain subsets of T cells in the immune system. It is also involved in cell-cell adhesion, playing a crucial role in the development and maintenance of nervous system. CD56+immune cells generally exhibit strong immunostimulatory and cytotoxic functions, including production of Thl cytokines.

[0145] The term “challenging” refers to presenting a stimulus or substance to an- 25 -508002954.1Attorney Docket No.: 144242-0301 organism or system or cells (such as, for example, without limitation, challenging donor immune cells with an antigen) in order to provoke a response, often to evaluate or enhance its capabilities or reactions.

[0146] The term “CpG oligodeoxynucleotide (ODN)” refer to specific short synthetic single-stranded DNA molecules that contain unmethylated CpG motifs - a cytosine followed by guanine. These molecules function as immunostimulants and are recognized as pathogen- associated molecular patterns (PAMPs) by the immune system. CpG ODNs bind to and active Toll-like receptor 9 (TLR9) to trigger immune responses, including the production of cytokines and enhancement of antigen presentation. There are several classes of CpG ODNs (classes A, B, C, and P) with varying structures and effects on immune cells.

[0147] The term “Class B CpG ODN 2006” refers to a class B CpG ODN that has a full phosphorothioate backbone and contains multiple CpG dinucleotides. The sequence of CpG ODN 2006 is: 5’-TCGTCGTTTTGTCGTTTTGTCGTT-3’ (SEQ ID NO: 22). CpG ODN 2006 strongly activates B cells and induces their proliferation and antibody production. It only weakly stimulates interferon-alpha secretion by plasmacytoid dendritic cells.

[0148] The term “class C CpG ODN 2395” refers to a class C CpG ODN that has a full phosphothioate backbone and a CpG-containing palindromic motif. Class C CpG ODN 2395 is a 22-mer, with the sequence 5’-TCGTCGTTTTCGGCGC:GCGCCG-3’ (SEQ ID NO: 23). CpG ODN 2395 strongly induces B cell stimulation as well as IFN-a production in plasmacytoid dendritic cells. In particular, CpG ODN 2395 is specific for the human and murine Toll-like receptor 9 (TLR9).

[0149] A “conservative substitution” or a “conservative amino acid substitution,” refers to the substitution of one or more amino acids with one or more chemically or functionally similar amino acids. Conservative substitution tables providing similar amino acids are well known in the art. Polypeptide sequences having such substitutions are known as “conservatively modified variants.” By way of example, the following groups of amino acids are considered conservative substitutions for one another.- 26 -508002954.1Attorney Docket No.: 144242-0301Additional conservative substitutions may be found, for example, in Creighton, Proteins: Structures and Molecular Properties 2nd ed. (1993) W. H. Freeman & Co., New York, NY. An antibody generated by making one or more conservative substitutions of amino acid residues in a parent antibody is referred to as a “conservatively modified variant.”

[0150] The term “conjugated” refers to the process of being linked or combined with another molecule or substance to form a complex, often used to describe the attachment of one molecule to another through chemical bonds or interactions.

[0151] The term “cord blood” refers to the blood collected from the umbilical cord and placenta of a newborn infant immediately after birth. Cord blood is a rich source of hematopoietic stem cells (HSCs), progenitor cells, immune cells, and other cell types.

[0152] The term “CTLA-4” or “cytotoxic T-lymphocyte-associated protein 4” is a cell surface receptor expressed on activated T cells and regulatory T cells (Tregs). CTLA-4 acts as a negative regulator of T cell activation by competing with the co-stimulatory receptor CD28 for binding to the ligands CD80 (B7-1) and CD86 (B7-2) on antigen-presenting cells.- 27 -508002954.1Attorney Docket No.: 144242-0301By binding to CD80 / CD86, CTLA-4 inhibits T cell activation and reduces immune responses, contributing to immune tolerance and homeostasis. Anti-CTLA-4 therapy blocks CTLA-4, thereby enhancing T cell activation and unleashing anti-tumor immune responses.

[0153] The term “culture” in the context of immunotherapy refers to the process of proliferating and maintaining cells, tissues, or microorganisms in an artificial environment outside their natural habitat, typically in a laboratory setting (in vitro). Cell culture techniques are widely used in biomedical research, drug discovery, and vaccine development.

[0154] The term “donor” refers to an individual, organism, or entity that provides something, such as cells, tissues, or substances, for use in a specific context, often for the benefit of another organism or system.

[0155] The term “DOTAP”, or N-[l-(2,3-dioleoyloxy)propyl]-N,N,N- trimethylammonium chloride, refers to a cationic lipid commonly used in liposome formulations. As with all cationic molecules, DOTAP carries a positive electric charge due to the loss of electrons, which makes it attracted to negatively charged particles or surfaces. DOTAP is designed to aid in the uptake and transport of nucleic acids or other substances into cells.

[0156] The term “DOTAP liposomes” refers to cationic liposomes comprising of the positively charged lipid DOTAP. DOTAP liposomes often combined DOTAP with neutral lipids, such as DOPE (l,2-dioleoyl-sn-glycero-3-phosphoethanolamine) or DOPC (1,2- dioleoyl-sn-glycero-3-phosphocholine). DOTAP liposomes are commonly used for drug and gene delivery due to their ability to form complexes with negatively charged molecules, such as DNA, RNA, or some proteins, facilitating cellular uptake. DOTAP liposomes have shown significant potential as immunostimulatory agent and adjuvant in vaccine development.

[0157] The term “embedding” refers to the method of encasing biological specimens, such as cells or tissues, in a solid support medium. Embedding preserves the spatial organization and structural integrity of specimens, allowing thin sections to be cut for histological examination, immunostaining, or electron microscopy. Embedding media provide mechanical support and protect specimens from damage during sectioning, handling, and staining processes, ensuring high-quality imaging and accurate interpretation of biological structures and cellular morphology. Exemplary methods of embedding are provided in Examples 2 and 3.

[0158] The term “emulsion” refers to a mixture of two or more immiscible liquids,- 28 -508002954.1Attorney Docket No.: 144242-0301 such as oil and water, stabilized by an emulsifying agent, often used in pharmaceuticals.

[0159] “The term “encapsulates” or “encapsulated” refers to containment of a substance within a capsule or vesicle, often as enclosed or surrounded by a membrane or protective layer.

[0160] The term “epitope” means a portion of an antigen capable of specific binding to an antibody. Epitopes frequently consist of surface-accessible amino acid residues and / or sugar side chains and may have specific three-dimensional structural characteristics, as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents. An epitope may comprise amino acid residues that are directly involved in the binding, and other amino acid residues, which are not directly involved in the binding.

[0161] The term “fixing” or “fixation” refers to a process in biological sample preparation that involves preserving cellular structures, proteins, and other biomolecules in their native state to prevent degradation, denaturation, or loss of cellular integrity. Fixation helps to immobilize cellular components, stabilize protein-protein interactions, and maintain the structural integrity of cells for subsequent analysis by microscopy, immunostaining, or molecular assays.

[0162] The term “foreground” or “foregrounded” refers to the process of enhancing the presentation or visibility of a specific antigen or immunogen to the immune system. This could involve conjugating (coupling) the antigen with adjuvants (substances that enhance an immune response), carrier molecules, or targeting moieties (components of a therapeutic agent designed to specifically recognize and bind to a particular target in the body) to improve antigen uptake, processing, and exhibition by antigen-presenting cells. Thus, foregrounding antigens are meant to improve the efficacy of antigen presentation by APC, by incubating the APC of choice with the antigen for a period of time to allow for antigen processing and epitopes display prior to mixing with responding cells.

[0163] “Harvesting” involves the process of gathering or collecting biological material, such as cells or tissues, from a variety of sources for further use or analysis. Harvesting can include, for example, without limitation, the collection of cells or tissues from donor organisms. It can also involve gathering material from other sources, such as cell cultures or engineered tissues. For example, harvesting donor immune cells would entail the collection of immune cells from a donor organism for subsequent applications in therapeutics- 29 -508002954.1Attorney Docket No.: 144242-0301 or research. It also refers to the process of collecting cells after the formation of the immunized organoid in vitro for downstream analysis.

[0164] The term “immune cells” refers to a variety of cell types integral to the immune system’s function, including, for example, without limitation, lymphocytes (such as B cells, T cells, and natural killer (NK) cells), phagocytes (such as neutrophils, monocytes, mast cells, and macrophages), granulocytes (such as neutrophils, basophils, and eosinophils) as well as mast cells, dendritic cells (DCs), and certain subsets of epithelial cells.

[0165] Leukocytes, also known as white blood cells, represent a significant subset of immune cells and include both lymphocytes and phagocytes. These white blood cells are vital for protecting the body against pathogens and foreign invaders. They develop from stem cells found in the bone marrow and can be divided into two main types: lymphocytes and phagocytes. Leukocytes are crucial for immune surveillance, recognizing antigens, and orchestrating immune responses.

[0166] The term “mononuclear cells” refer to a heterogeneous population of white blood cells (leukocytes) characterized by a single round nucleus. Mononuclear cells include lymphocytes (T cells, B cells, and NK cells), monocytes (which eventually differentiate into macrophages or DCs), dendritic cells, and pluripotent CD34+cells from umbilical cord blood. These cells play critical roles in immune surveillance, host defense, and antigen presentation. Mononuclear cells are commonly isolated from peripheral blood, bone marrow, or lymphoid tissues for research, diagnostic, and therapeutic applications in immunology, hematology, and cell-based therapies.

[0167] The term “B cell” refers to a type of lymphocyte involved in the adaptive immune response, particularly in humoral immunity mediated by antibody production. B cells develop from hematopoietic stem cells in the bone marrow and mature into antibodysecreting plasma cells upon encountering specific antigens. B cells express unique antigen receptors known as B cell receptors (BCRs), which recognize and bind to specific epitopes on pathogens or antigens. Upon activation by antigens, TLR ligands, and T cell-derived signals, B cells undergo proliferation, differentiation, immunoglobulin somatic hypermutation, and class switching to produce diverse classes and subclasses of antibodies (immunoglobulins) with different effector functions, such as neutralization, opsonization, and complement activation. B cells also serve as antigen-presenting cells (APCs) and play critical roles in antigen processing, presentation, and immune regulation.- 30 -508002954.1Attorney Docket No.: 144242-0301

[0168] The term “DCs” or “dendritic cells” refers to specialized antigen-presenting cells (APCs) that play a central role in initiating and regulating adaptive immune responses. DCs are named for their distinctive shape and branching structure, which resembles the dendrites of neurons. DCs are strategically distributed throughout the body in peripheral tissues, lymphoid organs, and mucosal surfaces, where they capture antigens, process them into antigenic peptides, and present them to T cells in the context of major histocompatibility complex (MHC) molecules. DCs express high levels of co-stimulatory molecules and cytokines that provide essential signals for T cell activation, differentiation, and effector function. As recited herein, monocyte-derived dendritic cells (MDDC) are referred to also as DC, while MDDC polarized by cytokines in DC2 and / or DC 17 are also referred generically as DC.

[0169] The term “LC” or “Langerhans cells” refers to a specialized subset of dendritic cells (DCs) found in the epidermis and mucosal epithelia, where they function as sentinels of the skin and mucosal barrier against pathogens and environmental antigens. Langerhans cells express unique cell surface markers, including CD la and Langerin (CD207) and possess characteristic dendritic processes that extend between adjacent keratinocytes in the epidermis. Langerhans cells capture antigens and microbial pathogens through receptor-mediated endocytosis and phagocytosis and migrate to regional lymph nodes to present processed antigens to T cells and initiate adaptive immune responses. Langerhans cells play essential roles in skin immunity, tolerance induction, and immune surveillance against infectious agents and skin cancer. As recited herein, LC also refers to Langerhans cells derived from cord blood CD34+cells or adult monocytes generated in vitro.

[0170] The term “Natural Killer (NK)” cells refer to type of cytotoxic lymphocyte that plays a critical role in the innate immune response by identifying and eliminating virus- infected cells and tumor cells without prior sensitization.

[0171] The term “granulocytes” refer to a white blood cell with secretory granules in its cytoplasm. Each type is differentiated by the color the granules exhibit when subjected to a compound dye.

[0172] The term “basophils” refer to a type of granulocytic white blood cell characterized by large cytoplasmic granules that stain deep blue or purple with basic dyes such as hematoxylin. Basophils play a crucial role in the immune system by releasing histamine and other mediators during allergic reactions and inflammatory processes,- 31 -508002954.1Attorney Docket No.: 144242-0301 contributing to vasodilation and increased vascular permeability. They also participate in the defense against parasitic infections.

[0173] The term “eosinophils” refer to a type of granulocytic white blood cell identified by their bilobed nucleus and large granules that stain bright red or pink with eosin, an acidic dye. They typically make up 1-3% of the white blood cell count. Eosinophils are primarily involved in combating multicellular parasites and certain infections. They also regulate inflammatory responses, particularly in allergic conditions such as asthma and hay fever, by releasing toxic granules and cytokines that modulate the activity of other immune cells.

[0174] The term “neutrophils” refer to a type of phagocytic granulocyte that constitute the first line of defense against bacterial infections. They engulf and destroy pathogens through phagocytosis and release antimicrobial substances to eliminate invading microorganisms.

[0175] The term “macrophages” refer to a type of tissue-resident phagocytic cells derived from monocytes. They play essential roles in host defense by engulfing and digesting pathogens, clearing cellular debris, and modulating immune responses through cytokine secretion.

[0176] The term “mast cells” refer to a cell that plays a vital role in the body’s immune system, particularly in allergic reactions and defense against pathogens. They are found in various tissues throughout the body, especially in the skin, lungs, and digestive tract. Mast cells contain granules rich in histamine and other inflammatory mediators, which they release upon activation.

[0177] The term “monocytes” refers to a type of leukocyte, specifically a mononuclear phagocyte, that circulates in the bloodstream and serves as a precursor to tissue macrophages and dendritic cells in the peripheral tissues. Monocytes originate from hematopoietic stem cells in the bone marrow and circulate in the bloodstream before migrating into tissues where they differentiate into tissue-specific macrophages or dendritic cells. Monocytes play a crucial role in innate immunity by phagocytosing pathogens, debris, and dead cells. They also contribute to adaptive immunity by presenting antigens to T cells and secreting cytokines that regulate immune responses. Monocytes are integral to immune surveillance, inflammation, tissue repair, and maintaining homeostasis within the body.

[0178] The term “immunosuppressive cells” refers to all cells which inhibit or- 32 -508002954.1Attorney Docket No.: 144242-0301 suppress the activity of the immune system, often leading to a decreased immune response. Exemplary immunosuppressive cells include Regulatory T cells (Tregs). These cells are a subset of CD4 T cells characterized by expression of CD4, CD25(high), and / or Foxp3.

[0179] The term “cytotoxic cells” refers to immune cells capable of directly killing other cells. The main types are cytotoxic T cells (CD8+T cells) of the adaptive immune system and natural killer (NK) cells of the innate immune system. Cytotoxic cells destroy target cells by releasing cytotoxic molecules like perforin and granzymes, leading to programmed cell death (apoptosis).

[0180] The term “z z vitro" or “ex vivo” refers to any experimental procedures conducted outside of a living organism’s natural environment, typically within controlled laboratory conditions, wherein biological samples or tissues are removed from the organism and studied or manipulated in isolation, while retaining their physiological relevance and functionality. As used herein, the terms 'in vitro’ and 'ex vivo’ shall have similar senses.

[0181] The term “keyhole limpet hemocyanin” or “KLEI” refers to a large, oxygencarrying protein derived from the hemolymph of the keyhole limpet (Megathura creniilalci). commonly used as an immunogenic carrier protein to enhance immune responses to attached antigens.

[0182] The term “liposome” refers to a small artificial vesicle composed of lipids, typically used as a vehicle for the delivery of drugs or other substances in medical or research applications. Lipids are a class of molecules including fats, oils, and cholesterol, essential for energy storage and cell membrane structure. Their amphipathic nature, with hydrophilic heads and hydrophobic tails, allows them to form bilayers in aqueous environments. These bilayers can spontaneously curve into liposomes (spherical structures).

[0183] The term “lipoparticle” or “lipid nanoparticle” refers to a nanoparticle composed of a core-shell structure comprising a polymeric core and a lipid shell. They combine the biomimetic properties of long-circulating vesicles with the mechanical advantages of nanoparticles. In some embodiments, the polymeric nanoparticle core is enveloped by single or multiple pegylated lipid layers (shell). The polymer core may comprise poly lactic-co-glycolic acid or PLGA which is coated by a lipid membrane. Lipoparticles may be used as carriers for drug delivery comprising enhanced drug encapsulation, loading efficiency, and cellular internalization, allowing for the transport of both hydrophilic and hydrophobic drugs. Lipoparticles can incorporate various molecules- 33 -508002954.1Attorney Docket No.: 144242-0301 like proteins, radioactive substances, magnetic materials, or fluorophores. This hybrid structure allows lipoparticles to be versatile tools for applications like drug delivery, protein transport, and cellular uptake studies. Their core-shell design provides both stability and biocompatibility.

[0184] The term “LLME” refers to L-leucyl-L-leucine methyl ester, as used herein, refers to a dipeptide compound commonly used to selectively induce apoptosis (programmed cell death) in immune cells, such as NK cells, monocytes, and some T cell subsets. LLME displays immunomodulatory properties. The cell type-specific toxicity effect of LLME involves LLME being converted to a membranolytic compound inside lysosomes, leading to cell death in susceptible cell types.

[0185] The term “loaded” refers to the process of antigen uptake, processing, and presentation by antigen-presenting cells (APCs) - such as dendritic cells (DCs), macrophages, and B cells - in immunology and cell biology. Loaded antigens are peptides derived from internalized or endocytosed pathogens, proteins, or apoptotic cells that are bound to major histocompatibility complex (MHC) molecules on the surface of APCs. As described herein, loading of APC with antigen may also comprise, consist of, or consist essentially of delivering antigen-adjuvant formulations to APCs prior to co-culture with other immune cells. Antigen loading enables APCs to present antigenic peptides to T cells via MHC class I or MHC class II molecules, triggering antigen-specific T cell activation, proliferation, and effector functions. Loaded antigens play a critical role in initiating adaptive immune responses, including T cell-mediated immunity and antibody production.

[0186] The term “lymph node organoid” or “LNO” refers to a three-dimensional (3D) tissue structure engineered to mimic the organization and function of lymph nodes, typically composed of multiple cell types arranged in a spatially organized manner to replicate the physiological environment of lymphoid tissue.

[0187] The term “3D” refers to objects or structures that have three dimensions - length, width, and height - allowing for realistic representation and manipulation in physical or virtual space.

[0188] The term “3D printed” or “3D printing” refers to the production or manufacturing of objects using additive manufacturing techniques. These techniques build up layers of material to create three-dimensional objects based on digital designs, enabling precise control over shape and structure.- 34 -508002954.1Attorney Docket No.: 144242-0301

[0189] The term “maintaining” refers to the ongoing preservation or sustenance of a particular condition, state, or structure, including organoids, over a period of time, often involving regular care or intervention to ensure stability or functionality.

[0190] The term “magnetic beads” refers to small, spherical particles containing magnetic material, often coated with specific molecules such as antibodies or ligands, used for cell separation, purification, or labeling in biological and biomedical applications.

[0191] The term “media” refers to the liquid or gel-like formulation containing essential nutrients, growth factors, salts, buffering agents, and other supplements necessary for the growth, survival, and propagation of cultured cells. Cell culture media are tailored to specific cell types and applications, ensuring that the media can provide all components necessary to support cellular metabolism, protein synthesis, and cell signaling pathways.

[0192] The term “nanoparticle” refers to small particles with dimensions in the nanometer scale, typically ranging from 1 to 100 nanometers, often used in various fields due to their unique properties and versatile applications. These nanoparticles can be conjugated with biotinylated antibodies, peptides, nucleic acids, or other molecules of interest, enabling targeted delivery, detection, or manipulation of biological substances.

[0193] The term “negative selection” refers to the process of isolating specific cell populations by removing unwanted cells from a heterogeneous mixture, typically achieved through targeted labeling and subsequent removal of undesired cells using magnetic beads or other separation techniques.

[0194] The term “NTA(Ni)-liposomes” or “nitrilotriacetic acid (NT A) nickel (Ni)- liposomes” refer to liposomes containing nickel- nitrilotriacetic acid (Ni-NTA) groups on the surface. NTA (Ni)-liposomes typically contain a mixture of lipids, including a small percentage (usually 1-3%) of lipids with Ni-NTA headgroups. The Ni-NTA groups on the liposome surface can reversibly bind to histidine residues, typically at the N- or C-terminus of proteins, allowing these proteins to anchor to the liposomes, and facilitate protein display and presentation to immune cells.

[0195] The term “PADRE” or “Pan DR-binding epitope” refers to a synthetic peptide sequence designed to bind to a broad range of major histocompatibility complex (MHC) class II molecules. Originally developed to enhance the immunogenicity of vaccines by promoting T-cell responses. PADRE consists of 13 amino acids (AKFVAAWTLKAAA (SEQ ID NO: 1)) that are highly conserved across MHC class II molecules, allowing for broad recognition - 35 -508002954.1Attorney Docket No.: 144242-0301 by T-helper cells irrespective of individual MHC haplotypes. This characteristic makes PADRE a valuable tool in vaccine research and development, where it is utilized to improve the efficacy and potency of vaccines against infectious diseases, cancer, and other conditions by stimulating robust and diverse T-cell-mediated immune responses.

[0196] The term “TT (830-843)” or “Tetanus Toxin (830-843)” refers to a specific peptide fragment derived from Tetanus Toxoid. It serves as a universal human tetanus toxin T cell epitope. It induces T-cell activation and is used as a helper peptide in vaccinations.

[0197] The term “TpD” or “Tetanus-Diphtheria universal T cell helper peptide” refers to a synthetic chimeric peptide designed to act as a universal T cell helper epitope in vaccines. TpD consists of 32 amino acids (ILMQYIKANSKFIGIPMGLPQSIALSSLMVAQ (SEQ ID NO: 2)) constructed by combining fragments from the tetanus toxoid and diphtheria toxoid, separated by an internal cleavage site. This design allows TpD to activate a broad range of CD4+T cells across a diverse human population. These activated T cells then provide help to B cells, enhancing the production of antibodies against the specific antigen included in a vaccine with TpD.

[0198] The term “PD-1” or “programmed cell death protein 1” refers to a cell surface receptor primarily expressed on activated T cells, B cells, and myeloid cells. PD-1 interacts with its ligands, PD-L1 (programmed death-ligand 1) and PD-L2 (programmed death-ligand 2), which are expressed on various cell types, including tumor cells and antigen-presenting cells. Binding of PD-1 to PD-L1 / PD-L2 delivers inhibitory signals that suppress T cell activation, therefore contributing to immune evasion by tumors and tolerance to self-antigens.

[0199] The term “phycoerythrin” or “PE” refers to a light-sensitive protein pigment found in certain algae and cyanobacteria, commonly used as a fluorescent label in biological research and diagnostics for detecting and visualizing specific molecules or cells.

[0200] The term “pluripotent” describes stem cells that have the potential to differentiate into multiple cell types of the body. Examples include embryonic stem cells and induced pluripotent stem cells (iPSCs).

[0201] The term “polarizing” in immunology refers to directing the differentiation and functional specialization of T cells towards specific effector subsets, such as Thl (T helper 1), Th2 (T helper 2), Thl7 (T helper 17), or regulatory T cells (Tregs). Polarization of T cells occur in response to cytokine signals present in the local microenvironment (cytokines are small proteins involved in communication between immune cells). Differentiated T cell - 36 -508002954.1Attorney Docket No.: 144242-0301 subsets exhibit distinct cytokine profiles, surface markers, and effector functions tailored to combat specific pathogens or mediate immune responses in different tissues or disease states.

[0202] The term “priming” refers to the initial activation and programming of immune cells, such as T cells, B cells, and antigen-presenting cells (APCs), by exposure to specific antigens, pathogens, or immunomodulatory signals. Priming induces a series of cellular and molecular events that initiate adaptive immune responses, including antigen recognition, T cell receptor (TCR) signaling, co-stimulation, cytokine secretion, and differentiation into effector or memory cell subsets. Antigen presentation by APCs, such as dendritic cells (DCs), macrophages, and B cells, is crucial for priming naive T cells and initiating antigen-specific immune responses. Priming establishes immunological memory and provides the basis for protective immunity upon re-exposure to the same antigen or pathogen.

[0203] The term “Qbeta VLP” or “Qbeta virus-like particle” refers to a selfassembling nanostructure derived from bacteriophage Qbeta coat protein utilized as a carrier platform in vaccine development. Qbeta VLPs form icosahedral particles approximately 30 nm in diameter and mimic the structure of bacteriophage Qbeta. Qbeta VLPs can encapsulate a variety of RNA molecules, including bacterial RNA, which can activate TLR7. Qbeta VLPs are highly immunogenic due to their particulate nature and ability to display antigen in a repetitive, high-density, format. Antigen or other molecules can be chemically coupled to the surface of Qbeta CLPs allowing for customized vaccine design. By leveraging Qbeta VLPs as carriers, vaccine formulations aim to enhance the immune system’s recognition and response to specific antigens, thereby improving the efficacy of vaccination strategies.

[0204] The term “scaffold” refers to a supportive framework or structure upon which other components can be assembled or attached, often used in tissue engineering and regenerative medicine to provide mechanical support and guide the growth of cells or tissues.

[0205] The term “sequencing” refers to the process of determining the order of nucleotides or amino acids in a molecule, typically referring to DNA, RNA, or protein sequences, which provides valuable information about the genetic or structural composition of the molecule.

[0206] The term “sorting” refers to the process of isolating and categorizing specific components or entities based on defined criteria, such as affinity, size, shape, molecular properties, or some other property, often performed to separate desired materials from a- 37 -508002954.1Attorney Docket No.: 144242-0301 mixture.

[0207] As used herein, the term “subject” means a mammalian subject. Exemplary subjects include, but are not limited to humans, monkeys, dogs, cats, mice, rats, cows, horses, camels, llamas, alpacas, avians, goats and sheep. In certain embodiments, the subject is a human. In some embodiments, the subject has cancer, an autoimmune disease or condition, and / or an infection that can be treated with an antibody provided herein. In some embodiments, the subject is a human that is suspected to have cancer, an autoimmune disease or condition, and / or an acute infection and chronic infection.

[0208] The term “TLR ligand” refers to a molecule that bind to Toll-like receptors (TLRs) on immune cells and triggers signaling pathways that activate the innate immune response and promote inflammation. TLR ligands are typically conserved molecular structures found in pathogens but not in host cells. Recognition of TLR ligands by TLRs triggers signaling cascades that activates innate immune responses and link to adaptive immunity. The specificity of TLR ligand interactions allows the immune system to tailor responses to different types of pathogens.

[0209] The term “TNF” or “Tumor Necrosis Factor” is a cytokine, a type of protein involved in cell signaling, that plays a key role in regulating the immune response. Produced mainly by macrophages, TNF is crucial for defending against infections and cancer by promoting inflammation, inducing fever, and aiding in the destruction of abnormal cells.

[0210] The term “TNFR2” or “tumor necrosis factor receptor 2” refers to a cell surface receptor belonging to the tumor necrosis factor (TNF) receptor superfamily. TNFR2, a receptor for this protein, is expressed on various cell types, including immune cells, endothelial cells, and some tumor cells. TNFR2 mediates diverse biological functions, including cell survival, proliferation, immune regulation, and inflammation in response to binding with its ligands, TNF-a and TNF-[3. TNFR2 signaling influences immune cell activation, differentiation, and contributing to subsequent immune responses, autoimmune diseases, and cancer progression.

[0211] The term “tonsil cells” refers to cells derived from the tonsils, which are lymphoid organs located at the back of the throat. Tonsils are composed of lymphoid tissue containing B cells, T cells, dendritic cells, macrophages, and other immune cells. Tonsils play a crucial role in immune surveillance and defense against pathogens, particularly in the upper respiratory tract (which includes the nose, nasal cavity, pharynx, and larynx). Tonsil - 38 -508002954.1Attorney Docket No.: 144242-0301 cells, particularly T cells and antigen-presenting cells, can be utilized in studies of immune responses, infection, and immunotherapy.

[0212] The term “treating” refers to subjecting cells, for example, without limitation, donor immune cells, to specific conditions, interventions, or manipulations aimed at altering their behavior, function, or properties.

[0213] The term “virus-like particle” or “VLP” refers to a nanoscale structure that mimics the shape and surface features of viruses but lacks genetic material, often used in vaccines to stimulate an immune response against specific viral pathogens. Commonly used VLPs in vaccine development include HBV, HPV, Qbeta, CuMV, AP205, CCMV, MS2, PP7, RHDS, and CPV VLPs.2. Method of Generating Antibodies

[0214] Provided herein are methods of generating antibodies.

[0215] A first aspect provides an in vitro (ex vivo) method of generating (human) antibody(ies), comprising, consisting of, or consisting essentially of:(i) harvesting donor immune cells;(ii) challenging the donor immune cells with an antigen, an antigen-encoding mRNA, or antigen-encoding plasmid DNA, wherein the antigen, antigen-encoding mRNA, or antigen-encoding plasmid DNA is encapsulated in or conjugated with liposome-based adjuvants, lipoparticle-based adjuvants, oil-in-water emulsion adjuvants, virus-like particles (VLPs), TLR ligands, carrier proteins, carrier polysaccharides, PE, dextran, outer membrane vesicle (OMV), and / or other nanoparticles (e.g., lipid nanoparticles, polymer nanoparticles); and(iii) sorting and sequencing resulting antibodies.

[0216] In some embodiments, the method further comprises assembling the harvested donor immune cells into at least one LNO, wherein assembly occurs between steps (i) and (ii). In some embodiments, the at least one LNO comprises a 3D scaffold. In some embodiments, the 3D scaffold is 3D printed. In some embodiments, the antigen is presented on the surface of the 3D scaffold. In some embodiments, the 3D scaffold is prepared as described herein. In some embodiments, the 3D scaffold may be prepared and tested according to the general procedures described in PCT Pub. Nos. WO 2023 / 114782 and / or WO 2023 / 114785, the full disclosures of each of which are incorporated herein by reference.- 39 -508002954.1Attorney Docket No.: 144242-0301

[0217] In some embodiments, the LNO is maintained for an extended period. In some embodiments, an extended period comprises, consists of, or consists essentially of between about 1 and 30 days, about 2 and 21 days, about 5 and 18, about 10 and 15, or between about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about or 30 days.

[0218] In some embodiments, the carrier proteins comprise, consists of, or consist essentially of KLH, CRM197, TT, DT, PD, OMPC, BSA, Pseudomonas aeruginosa exotoxin A (rEPA), Pneumococcal surface proteins (e.g. pneumolysin, PiuA, Sp0418), Group B Streptococcus pill proteins (GBS80, GBS67), or OVA. In some embodiments, the carrier protein is KLH. In some embodiments, the carrier protein is CRM197. In some embodiments, the carrier protein is TT, including non-toxic TT mutant 8MTT. In some embodiments, the carrier protein is DT. In some embodiments, the carrier protein is PD. In some embodiments, the carrier protein is OMPC. In some embodiments, the carrier protein is BSA. In some embodiments, the carrier protein is PE. In some embodiments, the carrier protein is rEPA. In some embodiments, the carrier protein is Pneumococcal surface proteins (e.g., pneumolysin, PiuA, Sp0418). In some embodiments, the carrier protein is Group B Streptococcus pill proteins (GBS80, GBS67). In some embodiments, the carrier protein is OVA.

[0219] In some embodiments, the virus-like particles comprises, consists, or consists essentially of Qbeta VLP.

[0220] In some embodiments, the carrier polysaccharide comprises, consists of, or consists essentially of polydextran or pullulan. In some embodiments, the carrier polysaccharide is polydextran. In some embodiments, the carrier polysaccharide is pullulan.

[0221] In some embodiments, the method further comprises treating the donor immune cells to remove immunosuppressive cells and cytotoxic cells.

[0222] In some embodiments, treating donor immune cells comprises or consists of administering a lysosomotropic agent, optionally wherein the lysosomotropic agent is LLME, or negative selecting donor immune cells with anti-CD56 mAb-, anti-CD8 mAb-, and anti- CD14 mAb-conjugated magnetic beads, or combining administering LLME and negative selection with anti-CD8 mAb-conjugated magnetic beads. In some embodiments, the- 40 -508002954.1Attorney Docket No.: 144242-0301 lysosomotropic agent is LLME.

[0223] In some embodiments, the method comprises isolation of donor total or naive B cells.

[0224] In some embodiments, the method further comprises stimulation of the cells, optionally wherein the stimulation comprises stimulation of the cells with CD40L TLR ligands, optionally wherein the TLR ligands are selected from the group consisting of GpG ODNs or MDP.

[0225] In some embodiments, the liposome- or lipoparticle-based adjuvants comprise, consist of, or consist essentially of DOTAP lipoparticles (or other lipoparticles) / TLR-ligands. In some embodiments, the liposome-based adjuvants comprise, consist of, or consist essentially of DOTAP liposomes (or other liposomes) / TLR-ligands. In some embodiments, antigen(s) is conjugated to liposome through streptavidin-liposome, maleimide-liposome, or NTA(Ni)-liposome. In some embodiments, antigen(s) is conjugated to lipoparticles through streptavidin-liposome, maleimide-lipoparticle, or NTA(Ni)-liposome. In some embodiments, the antigen(s) is conjugated with Qbeta VLP. In some embodiments, the oil-in-water emulsion adjuvant comprises squalene, DL-a-tocopherol, and polysorbate 80. In some embodiments, the oil-in-water emulsion adjuvants comprise AddaS03.

[0226] In some embodiments, the method further comprises conjugating the antigen(s) with CD40L. In some embodiments, the antigen(s) and CD40L are conjugated through streptavidin, streptavidin-nanoparticle, streptavidin-liposome, or NTA(Ni)-liposome. In some embodiments, the antigen(s) and CD40L are biotinylated. In some embodiments, the antigen(s) and CD40L are conjugated through streptavidin, streptavidin-nanoparticle, or streptavidin-liposome. In some embodiments, the antigen(s) and CD40L are histidine tagged. In some embodiments, the antigen(s) and CD40L are conjugated through NTA(Ni)-liposome.

[0227] In embodiments, the antigen is conjugated to anti-CD4 mAb, anti-CD3 mAb, or a bispecific anti-CD3 / anti-CD28 mAb through a biotin-streptavidin protocol with streptavidin-nanoparticle. In embodiments, the antigen is conjugated to anti-CD4 mAb through a biotin-streptavidin protocol with streptavidin-nanoparticle. In embodiments, the antigen is conjugated to anti-CD3 mAb through a biotin-streptavidin protocol with streptavidin-nanoparticle. In embodiments, the antigen is conjugated to a bispecific anti- CD3 / anti-CD28 mAb through a biotin-streptavidin protocol with streptavidin-nanoparticle. In some embodiments, the antigen(s) is conjugated to or mixed with universal T helper- 41 -508002954.1Attorney Docket No.: 144242-0301 epitopes PADRE, TT (830-843) or chimeric TpD.

[0228] In some embodiments, the method further comprises additionally challenging donor immune cells with universal T helper epitope primed, e.g., PADRE, TT (830-843) or TpD, autologous CD4 T cells. In some embodiments, the method further comprises additionally challenging donor immune cells with the antigen(s) conjugated with a CpG oligodeoxynucleotide (ODN). In some embodiments, the method further comprises additionally challenging donor immune cells with the antigen(s) conjugated with class B CpG ODN 2006 or class C CpG ODN 2395 through streptavidin-nanoparticle. In some embodiments, the method further comprises additionally challenging donor immune cells with the antigen(s) conjugated with class C CpG ODN HP06T07 through streptavidin- nanoparticle. In some embodiments, the method further comprises adding an immune checkpoint inhibitor, optionally wherein the immune checkpoint inhibitor comprises anti- CTLA-4 and / or anti-PD-1 blocking antibodies, in the cell culture media. In some embodiments, the method further comprises challenging donor immune cells with MDP. In some embodiments, the method further comprises challenging donor immune cells with the non-toxic Tetanus toxoid mutant 8MTT, optionally wherein the 8MTT is conjugated to an antigen.

[0229] In some embodiments, the donor immune cells are harvested from UCB. In some embodiments, the donor immune cells are tonsil cells.

[0230] In some embodiments, the donor immune cells are challenged with an antigen foregrounded with DOTAP liposome-antigen complexes and / or Qbeta VLP-antigen conjugates. In some embodiments, the donor immune cells are challenged with an antigen foregrounded with DOTAP / antigen, DOTAP / PE, DOTAP / CD3, DOTAP / TL1A, DOTAP- CD19, Qbeta-CD19 peptides, or Qbeta- TNFR2.

[0231] In some embodiments, the method further comprises polarizing T cells in the donor immune cells. In some embodiments, the T cells are polarized to Th2 cells. In some embodiments, the T cells are polarized to Thl7 cells.

[0232] In some embodiments, the method further comprises priming T cells with antigen loaded DCs. In some embodiments, the method further comprises priming helper T cells with antigen-loaded activated B cell. In some embodiments, the method further comprises mixing antigen primed Th2 cells generated by DCs with B cells from the donor immune cells, the mixing occurring in the LNO.- 42 -508002954.1Attorney Docket No.: 144242-0301

[0233] In some embodiments, the method further comprises the generation of LCs from adult monocytes or cord blood pluripotent CD34+cells to be loaded with antigen.

[0234] In some embodiments, the method further comprises the generation of Tfh cells in vitro from naive CD4 T cells isolated form adult or cord blood mononuclear cells. In some embodiments, the method further comprises activating, or licensing, Tfh cells using antigen loaded LCs. In some embodiments, the method further comprises mixing antigen licensed Tfh cells with B cells from the same donor, the mixing occurring in the LNO. In some embodiments, the method further comprises fixing and / or embedding the at least one LNO.

[0235] In some embodiments, the antigen is selected from group comprising, consisting of, or consisting essentially of a protein, a protein fragment, a peptide, antigenencoding mRNA, or antigen-encoding plasmid DNA. In some embodiments, the antigen is a protein. In some embodiments, the antigen is a protein fragment. In some embodiments, the antigen is a peptide. In some embodiments, the antigen is an mRNA encoding an antigen. In some embodiments, the antigen is a plasmid DNA encoding an antigen. In some embodiments, the antigen is selected from the group consisting of CD3E (DGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDE DHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMD (SEQ ID NO: 11)), TL1A (LKGQEFAPSHQQVYAPLRADGDKPRAHLTVVRQTPTQHFKNQFPALHWEHELGLA FTKNRMNYTNKFLLIPESGDYFIYSQVTFRGMTSECSEIRQAGRPNKPDSITVVITKVT DSYPEPTQLLMGTKSVCEVGSNWFQPIYLGAMFSLQEGDKLMVNVSDISLVDYTKE DKTFFGAFLL (SEQ ID NO: 12)), PE (PE is composed of a, P, and y subunits, with the most common structure being (aP)6y. A subunit:MKSVITTTISAADAAGRYPSTSDLQSVQGNIQRAAARLEAAEKLGSNHEAVVKEAGDACFSK YGYLKNPGEAGENQEKINKCYRDIDHYMRLINYTLVVGGTGPLDEWGIAGAREVYRTLNLP SAAYIAAFVFTRDRLCIPRDMSAQAGVEFCTALDYLINSLS (SEQ ID NO: 13), P subunit: MLDAFSRVVVNSDSKAAYVSGSDLQALKTFINDGNKRLDAVNYIVSNSSCIVSDAISGMICEN PGLITPGGNCYTNRRMAACLRDGEIILRYVSYALLAGDASVLEDRCLNGLKETYIALGVPTNS TVRAVSIMKAAAVCFISNTASQRKVEVIEGDCSALASEVASYCDRVVAAVS (SEQ ID NO: 14), y subunit:MDSPAFAVTGMFAAAKVGVTSFTGATEPVSTRRRTSGNVTMAVDAFQKKFQTFGKIGVDYS RPKKLASYKRGGFDAASVEYPNAPSFAGKYSIAPCGQPSGASKILMKYDEYCAKGVLQVFKR- 43 -508002954.1Attorney Docket No.: 144242-0301NAVPFGVYTTKCTEGTVAGQAQEKRVFNRTMAFRQAQKPVNVRLAEQYEARRKCFILANG CSREEDQFKSMPVSAATFLAGKHESLGTCFRVVTPSNIAEDYIASGVRMQLVAKSNSTGVYG VGSCMEGFAKGDAEARRVAALAAEYRALQQSPAAVTGRQYESSRMAVKLYAQNCSHEQEQ LYKWPATAAAFCRY (SEQ ID NO: 15)), CD19 ECD (PEEPLVVKVEEGDNAVLQCLKGTSDGPTQQLTWSRESPLKPFLKLSLGLPGLGIHMR PLAIWLFIFNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGL GCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSQDLT MAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMET GLLLPRATAQDAGKYYCHRGNLTMSFHLEITARPVLWHWLLRTGGWK (SEQ ID NO: 16)), GIPR ECD (RAETGSKGQTAGELYQRWERYRRECQETLAAAEPPSGLACNGSFDMYVCWDYAA PNATARASCPWYLPWHHHVAAGFVLRQCGSDGQWGLWRDHTQCENPEKNEAFLD QRLILERLQ (SEQ ID NO: 17)) and ACTRIIB (SGRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVK KGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPP TAPTLLT (SEQ ID NO: 18)). In some embodiments, the antigen is administered with CD40L. In some embodiments, the antigen is contained within a nanodisc. In some embodiments, the antigen is a component of a cell membrane fraction. In some embodiments, the antigen is contained within a cell membrane fragment. In yet other embodiments, the antigen is present in the cell membrane of an intact cell that expresses the antigen. In some embodiments, the method further comprises a step of isolating a membrane fraction comprising the antigen from one or more cells.

[0236] In some embodiments, the method is effective for generating antigen-specific cells for about 7 to about 14 days post antigen challenge.

[0237] A second aspect provides an in vitro (ex vivo) method of generating (human) antibody(ies), comprising challenging at least one LNO derived from harvested donor immune cells that were pre-treated to remove immunosuppressive cells and cytotoxic cells, with an antigen; optionally wherein the antigen is encapsulated in or conjugated with liposome-based adjuvants, oil-in-water emulsion adjuvants, virus-like particles, TLR-ligands, carrier proteins, carrier polysaccharides and / or PE.

[0238] In some embodiments, the pre-treatment to remove immunosuppressive cells and cytotoxic cells from the harvested donor immune cells comprises or consists of administering a lysosomotropic agent, optionally wherein the lysosomotropic agent is LLME,- 44 -508002954.1Attorney Docket No.: 144242-0301 or negative selecting donor immune cells with anti-CD56 mAb-, anti-CD8 mAb-, and anti- CD14 mAb-conjugated magnetic beads or combining administering LLME and negative selection with anti-CD8 mAb-conjugated magnetic beads. In some embodiments, the lysosomotropic agent is LLME.

[0239] In some embodiments, the at least one LNO comprises a 3D scaffold. In some embodiments, the 3D scaffold is 3D printed. In some embodiments, the antigen is presented on the surface of the 3D scaffold. In some embodiments, the 3D scaffold is prepared as described herein. In some embodiments, the 3D scaffold may be prepared and tested according to the general procedures described in PCT Pub. Nos. WO 2023 / 114782 and / or WO 2023 / 114785, the full disclosures of each of which are incorporated herein by reference.

[0240] In some embodiments, the LNO is maintained for an extended period. In some embodiments, an extended period comprises, consists of, or consists essentially of between about 1 and 30 days, about 2 and 21 days, about 5 and 18, about 10 and 15, or between about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about or 30 days.

[0241] In some embodiments, the carrier proteins comprise, consists of, or consist essentially of KLH, CRM197, TT, DT, PD, OMPC, BSA, rEPA, Pneumococcal surface proteins (e.g. pneumolysin, PiuA, Sp0418), Group B Streptococcus pill proteins (GBS80, GBS67), or OVA. In some embodiments, the carrier protein is KLH. In some embodiments, the carrier protein is CRM197. In some embodiments, the carrier protein is TT, including non-toxic TT mutant 8MTT. In some embodiments, the carrier protein is DT. In some embodiments, the carrier protein is PD. In some embodiments, the carrier protein is OMPC. In some embodiments, the carrier protein is BSA. In some embodiments, the carrier protein is PE. In some embodiments, the carrier protein is rEPA. In some embodiments, the carrier protein is Pneumococcal surface proteins (e.g., pneumolysin, PiuA, Sp0418). In some embodiments, the carrier protein is Group B Streptococcus pill proteins (GBS80, GBS67). In some embodiments, the carrier protein is OVA.

[0242] In some embodiments, the virus-like particles comprises, consists, or consists essentially of Qbeta VLP.

[0243] In some embodiments, the carrier polysaccharide comprises, consists of, or- 45 -508002954.1Attorney Docket No.: 144242-0301 consists essentially of polydextran or pullulan. In some embodiments, the carrier polysaccharide is polydextran. In some embodiments, the carrier polysaccharide is pullulan.

[0244] In some embodiments, the method comprises isolation of donor total or naive B cells.

[0245] In some embodiments, the method further comprises stimulation of the cells, optionally wherein the stimulation comprises stimulation of the cells with CD40L TLR ligands, optionally wherein the TLR ligands are selected from the group consisting of GpG ODNs or MDP.

[0246] In some embodiments, the liposome- or lipoparticle-based adjuvants comprise, consist of, or consist essentially of DOTAP lipoparticles (or other lipoparticles) / TLR-ligands. In some embodiments, the liposome-based adjuvants comprise, consist of, or consist essentially of DOTAP liposomes (or other liposomes) / TLR-ligands. In some embodiments, antigen(s) is conjugated to liposome through streptavidin-liposome, maleimide-liposome, or NTA(Ni)-liposome. In some embodiments, antigen(s) is conjugated to lipoparticles through streptavidin-liposome, maleimide-lipoparticle, or NTA(Ni)-liposome. In some embodiments, the antigen(s) is conjugated with Qbeta VLP. In some embodiments, the oil-in-water emulsion adjuvant comprises squalene, DL-a-tocopherol, and polysorbate 80. In some embodiments, the oil-in-water emulsion adjuvants comprise AddaS03.

[0247] In some embodiments, the method further comprises conjugating the antigen(s) with CD40L. In some embodiments, the antigen(s) and CD40L are conjugated through streptavidin, streptavidin-nanoparticle, streptavidin-liposome, or NTA(Ni)-liposome. In some embodiments, the antigen(s) and CD40L are biotinylated. In some embodiments, the antigen(s) and CD40L are conjugated through streptavidin, streptavidin-nanoparticle, or streptavidin-liposome. In some embodiments, the antigen(s) and CD40L are histidine tagged. In some embodiments, the antigen(s) and CD40L are conjugated through NTA(Ni)-liposome.

[0248] In embodiments, the antigen is conjugated to anti-CD4 mAb, anti-CD3 mAb, or a bispecific anti-CD3 / anti-CD28 mAb through a biotin-streptavidin protocol with streptavidin-nanoparticle. In embodiments, the antigen is conjugated to anti-CD4 mAb through a biotin-streptavidin protocol with streptavidin-nanoparticle. In embodiments, the antigen is conjugated to anti-CD3 mAb through a biotin-streptavidin protocol with streptavidin-nanoparticle. In embodiments, the antigen is conjugated to a bispecific anti- CD3 / anti-CD28 mAb through a biotin-streptavidin protocol with streptavidin-nanoparticle.- 46 -508002954.1Attorney Docket No.: 144242-0301In some embodiments, the antigen(s) is conjugated to or mixed with universal T helper epitopes PADRE, TT (830-843) or chimeric TpD.

[0249] In some embodiments, the method further comprises additionally challenging donor immune cells with universal T helper epitope primed, e.g., PADRE, TT (830-843) or TpD, autologous CD4 T cells. In some embodiments, the method further comprises additionally challenging donor immune cells with the antigen(s) conjugated with a CpG oligodeoxynucleotide (ODN). In some embodiments, the method further comprises additionally challenging donor immune cells with the antigen(s) conjugated with class B CpG ODN 2006 or class C CpG ODN 2395 through streptavidin-nanoparticle. In some embodiments, the method further comprises additionally challenging donor immune cells with the antigen(s) conjugated with class C CpG ODN HP06T07 through streptavidin- nanoparticle. In some embodiments, the method further comprises adding an immune checkpoint inhibitor, optionally wherein the immune checkpoint inhibitor comprises anti- CTLA-4 and / or anti-PD-1 blocking antibodies, in the cell culture media. In some embodiments, the method further comprises challenging donor immune cells with MDP. In some embodiments, the method further comprises challenging donor immune cells with the non-toxic Tetanus toxoid mutant 8MTT, optionally wherein the 8MTT is conjugated to an antigen.

[0250] In some embodiments, the donor immune cells are harvested from UCB. In some embodiments, the donor immune cells are tonsil cells.

[0251] In some embodiments, the donor immune cells are challenged with an antigen foregrounded with DOTAP liposome-antigen complexes and / or Qbeta VLP-antigen conjugates. In some embodiments, the donor immune cells are challenged with an antigen foregrounded with DOTAP / antigen, DOTAP / PE, DOTAP / CD3, DOTAP / TL1A, DOTAP- CD19, Qbeta-CD19 peptides, or Qbeta- TNFR2.

[0252] In some embodiments, the method further comprises polarizing T cells in the donor immune cells. In some embodiments, the T cells are polarized to Th2 cells. In some embodiments, the T cells are polarized to Thl7 cells.

[0253] In some embodiments, the method further comprises priming T cells with antigen loaded DCs. In some embodiments, the method further comprises priming helper T cells with antigen-loaded activated B cell. In some embodiments, the method further comprises mixing antigen primed Th2 cells generated by DCs with B cells from the donor- 47 -508002954.1Attorney Docket No.: 144242-0301 immune cells, the mixing occurring in the LNO.

[0254] In some embodiments, the method further comprises the generation of LCs from adult monocytes or cord blood pluripotent CD34+cells to be loaded with antigen.

[0255] In some embodiments, the method further comprises the generation of Tfh cells in vitro from naive CD4 T cells isolated form adult or cord blood mononuclear cells. In some embodiments, the method further comprises activating, or licensing, Tfh cells using antigen loaded LCs. In some embodiments, the method further comprises mixing antigen licensed Tfh cells with B cells from the same donor, the mixing occurring in the LNO. In some embodiments, the method further comprises fixing and / or embedding the at least one LNO.

[0256] In some embodiments, the antigen is selected from group comprising, consisting of, or consisting essentially of a protein, a protein fragment, a peptide, antigenencoding mRNA, or antigen-encoding plasmid DNA. In some embodiments, the antigen is a protein. In some embodiments, the antigen is a protein fragment. In some embodiments, the antigen is a peptide. In some embodiments, the antigen is an mRNA encoding an antigen. In some embodiments, the antigen is a plasmid DNA encoding an antigen. In some embodiments, the antigen is selected from the group consisting of CD3E, TL1A, PE, CD19 ECD, GIPR ECD, and ACTRIIB. In some embodiments, the antigen is administered with CD40L. In some embodiments, the antigen is contained within a nanodisc. In some embodiments, the antigen is a component of a cell membrane fraction. In some embodiments, the antigen is contained within a cell membrane fragment. In yet other embodiments, the antigen is present in the cell membrane of an intact cell that expresses the antigen. In some embodiments, the method further comprises a step of isolating a membrane fraction comprising the antigen from one or more cells.

[0257] In some embodiments, the method is effective for generating antigen-specific cells for about 7 to about 14 days post antigen challenge.

[0258] A third aspect provides antibodies generated using any of the processes described herein. In some embodiments, the antibodies comprise a VH sequence selected from the group consisting of SEQ ID NO:4 or SEQ ID NO: 8 or the CDRs contained therein. In some embodiments, the antibodies comprise a VL sequence selected from the group consisting of SEQ ID NO: 6 or SEQ ID NO: 10 or the CDRs contained therein. In some embodiments, the antibodies comprise a VH sequence comprising the CDR sequences found - 48 -508002954.1Attorney Docket No.: 144242-0301 in SEQ ID NO: 4 and a VL sequence comprising the CDR sequences found in SEQ ID NO: 6. In some embodiments, the antibodies comprise a VH sequence comprising the CDR sequences found in SEQ ID NO: 8 and a VL sequence comprising the CDR sequences found in SEQ ID NO: 10. In some embodiments, the antibodies comprise a VH sequence comprising SEQ ID NO: 4 and a VL sequence comprising SEQ ID NO: 6. In some embodiments, the antibodies comprise a VH sequence comprising SEQ ID NO: 8 and a VL sequence comprising SEQ ID NO: 10. In some embodiments, the antibodies comprise a VH sequence encoded by a sequence selected from the group consisting of SEQ ID NO: 3 and SEQ ID NO: 7. In some embodiments, the antibodies comprise a VL sequence encoded by a sequence selected from the group consisting of SEQ ID NO: 5 and SEQ ID NO: 9. In some embodiments, the antibodies comprise a VH sequence encoded by SEQ ID NO: 3 and a VL sequence encoded by SEQ ID NO: 5. In some embodiments, the antibodies comprise a VH sequence encoded by SEQ ID NO: 7 and a VL sequence encoded by SEQ ID NO: 9. Vectors comprising any of the nucleic acids described herein, and host cells comprising said vectors are contemplated by the present disclosure.3. Liposomes

[0259] Liposomes are spherical vesicle structures composed of a uni- or multi- lamellar lipid bilayer surrounding internal aqueous compartments and a relatively impermeable outer lipophilic phospholipid bilayer. Liposomes may be anionic, neutral or cationic. Liposomes are biocompatible, nontoxic, can deliver both hydrophilic and lipophilic drug molecules, protect their cargo from degradation by plasma enzymes, and transport their load across biological membranes and the blood brain barrier (BBB) (see, e.g., Spuch and Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 pages, 2011. doi: 10.1155 / 2011 / 469679 for review).

[0260] Liposomes can be made from several different types of lipids; however, phospholipids are most commonly used to generate liposomes as carriers (e.g., adjuvant carriers). Liposomes may comprise, without limitation, DOTAP, DOTMA, DOTIM, DOPE, DOPC, DOPG, DOPS and / or DDAB. Methods for preparation of multilamellar vesicle lipids are known in the art (see for example U.S. Pat. No. 6,693,086, the teachings of which relating to multilamellar vesicle lipid preparation are incorporated herein by reference). In some embodiments, liposomes are anionic liposomes including DOPG:DOPC and / or DOPS:DOPC. Although vesicle formation can be spontaneous when a lipid film is mixed with an aqueous solution, it can also be expedited by applying force in the form of shaking by - 49 -508002954.1Attorney Docket No.: 144242-0301 using a homogenizer, sonicator, or an extrusion apparatus (see, e.g., Spuch and Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 pages, 2011. doi: 10.1155 / 2011 / 469679 for review). Extruded lipids may be prepared by extruding through filters of decreasing size, as described in Templeton et al., Nature Biotech, 15:647-652, 1997, the teachings of which relating to extruded lipid preparation are incorporated herein by reference.

[0261] In some embodiments, liposomes are prepared to encapsulate or conjugate antigens for challenging donor immune cells. Liposomes may comprise, consist of, or consist essentially of one or more lipids, an antigen, and an adjuvant e.g., CpG-ODN, R848, or LPS). In some embodiments, the liposome comprises, consists of, or consists essentially of an antigen / DOTAP liposome, antigen / DOTAP liposome / CpG-ODN, antigen / DOTAP liposome / R848, or antigen / DOTAP liposome / LPS. In some embodiments, the DOTAP liposome comprises, consists of, or consists essentially of DOTAP (N-[l-(2,3-Di oleoyl oxy )propyl]-N,N,N trimethyl-ammonium methyl-sulfate) only, DOTAP / Cholesterol, DOTAP / DOPE (l,2-dioleoyl- w-glycero-3 -phosphoethanolamine), DOTAP / DOPC (1,2- Dioleoyl-sn-glycero-3-phosphocholine), or DOTAP and other helper lipids with variant ratios. In some embodiments, cationic liposomes, such as DOTAP liposomes or DOTAP liposome and CpG ODN, R848, or LPS as adjuvants are used in methods described herein. Antigen / DOTAP liposomes antigen / DOTAP liposome / CpG ODN, antigen / DOTAP liposome / R848, or antigen / DOTAP liposome / LPS complexes are generated by mixing antigen with DOTAP liposome, DOTAP liposome and CpG ODN, DOTAP liposome and R848, or DOTAP liposome and LPS. In some embodiments, the liposome is a DOPG:DOPC or DOPS:DOPC anionic liposome.4. Methods of generating Lymph Node Organoids

[0262] A fifth aspect provides a method of printing or generating a LNO for use in methods described herein. LNOs comprise, consist of, or consist essentially of a 3D structure that provides one or more of the following, e.g., support for cells (e.g., all or specific cell types), presenting one or more proteins on the surface and within the scaffold, one or more cell types, providing nutrients oxygen and / or removing waste, and creating different compartments for different cell types. A LNO may be created in multiple ways, for example, a scaffold may be casted, formed using electrospinning, or 3D printed. In some embodiments, the LNO may be prepared and tested according to the general procedures described in PCT Pub. Nos. WO 2023 / 114782 and / or WO 2023 / 114785, the full disclosures - 50 -508002954.1Attorney Docket No.: 144242-0301 of each of which are incorporated herein by reference.

[0263] The scaffold material is chosen for desired properties. Scaffold material may be chosen from the list comprising, consisting of, or consisting essentially of naturally- derived hydrogels, synthetic hydrogels, peptides, or proteins. The method may comprise polymerization of monomeric units of biological materials by heat. The solution of polymers may be cured together with cells disposed within by energy sources, such as heat, laser, and mechanical means inherent to polymers. For example, a non-thixotropic material with cell deposited within maintains a liquid form when under pressure. Once the pressure is removed, the nonthixotropic material becomes solid. In other cases, the method may comprise polymerization of a photopolymerizable material by a laser light source. The organ and / or the organoid may be two-dimensional or three-dimensional. The organ and / or the organoid may be a lymph node.

[0264] The scaffold material may be made biologically active. To make the material biologically active, it may be decorated with peptides and / or proteins (e.g., bioactive molecules) to activate one or more desired functions (e.g., cellular functions). The bioactive molecules may be incorporated into the scaffold in several ways. In some embodiments, the scaffold material matrix properties may be tuned by varying the length of the hydrogel molecules, in order to trap molecules of a certain size and / or electromagnetic properties. The bioactive molecules or the active parts of the bioactive molecules may also be attached to the scaffold material itself and bound to the structure in that way. The process can also be achieved by having a chemical tag molecule exposed on the surface, and in a postprocessing step binding the desired bioactive molecules to the surface. For example, peptides and / or proteins may be added and bound to scaffold material (e.g., hydrogel molecules) to have them decorating the surface. In some embodiments, the bioactive molecule may be added to the ink solution. In some embodiments, secondary biomolecules are added to the scaffold after printing the desired scaffold (e.g., a secondary biomolecule that may bind to the bioactive molecule within the scaffold.

[0265] The polymer of at least a portion of 3D LNO may form a network. The polymer may be collagen, hyaluronic acid and other glycosaminoglycans, poly-dl-lactic-co- glycolic acid (PLGA), poly-I-lactic acid (PLLA), polyglycolic acid (PGA), alginate, gelatin, agar, or any derivative thereof, or a combination thereof. The polymer may comprise an extracellular matrix component. Non-limiting examples of extracellular matrix components used to create 3D LNOs may include proteoglycans such as heparan sulfate, chondroitin- 51 -508002954.1Attorney Docket No.: 144242-0301 sulfate, and keratan sulfate, non-proteoglycan polysaccharide such as hyaluronic acid, collagen, and elastin, fibronectin, laminin, nidogen, or any combination thereof. These extracellular matrix components may be functionalized with acrylate, diacrylate, methacrylate, cinnamoyl, coumarin, thymine, or other side-group or chemically reactive moiety to facilitate cross-linking induced directly by multi-photon excitation or by multiphoton excitation of one or more chemical doping agents. In some cases, photopolymerizable macromers and / or photopolymerizable monomers may be used in conjunction with the extracellular matrix components to create cell-containing structures. Non-limiting examples of photopolymerizable macromers may include polyethylene glycol (PEG) acrylate derivatives, PEG methacrylate derivatives, and polyvinyl alcohol (PVA) derivatives. In some instances, collagen used to create a cell containing structure may be fibrillar collagen such as type I, II, III, V, and XI collagen, facit collagen such as type IX, XII, and XIV collagen, short chain collagen such as type VIII and X collagen, basement membrane collagen such as type IV collagen, type VI collagen, type VII collagen, type XIII collagen, or any combination thereof.

[0266] The polymer of at least a portion of the 3D LNO may contain other polymerizable monomers that are synthesized and not native to mammalian tissues, comprising a hybrid of biologic and synthetic materials. An example mixture may comprise about 0.4% w / v collagen methacrylate plus the addition of about 50% w / v polyethylene glycol diacrylate (PEGDA). Photoinitiators to induce polymerization may be reactive in the ultraviolet (UV), infrared (IR), or visible light range. Examples of two such photo initiators are Eosin Y (EY) and triethanolamine (TEA), that when combined may polymerize in response to exposure to visible light (e.g., wavelengths of about 390 to 700 nanometers). Non-limiting examples of photoinitiators may include azobisisobutyronitrile (AIBN), benzoin derivatives, benziketals, hydroxyalkylphenones, acetophenone derivatives, trimethylolpropane triacrylate (TPT), acryloyl chloride, benzoyl peroxide, camphorquinone, benzophenone, thioxanthones, and 2-hydroxy-l-[ 4-(hydroxyethoxy)phenyl ]-2-methyl-l- propanone. Hydroxyalkylphenones may include 4-(2- hydroxyethylethoxy)-phenyl-(2- hydroxy-2-methyl propyl) ketone (Irgacure® 295), 1-hidroxycyclohexyl-l -phenyl ketone (Irgacure® 184) and 2,2- dimethoxy-2-phenylacetophenone (Irgacure® 651). Acetophenone derivatives may include 2,2-dimethoxy-2-phenylacetophenone (DMP A). Thioxanthones may include isopropyl thioxanthone.

[0267] The shape of the 3D LNO may be selected from the list comprising,- 52 -508002954.1Attorney Docket No.: 144242-0301 consisting, or consisting essentially of spherical, oval, ovate, ovoid, square, rectangular, cuboid, cylindrical, hemispherical, any polygonal shape, free-form, and tear-drop shape.

[0268] The printed bioink materials may be selected to be oxygen permeable. By allowing for oxygen permeability, any scaffold or other printed polymers may better facilitate for oxygen to reach cells contained in the engineered LNO. For example, the polymer can be a polyethylene glycol that is crosslinked, which is highly oxygen permeable. Additionally, polymer may be selected to comprise a specific density, for example, to prevent free floating cells or excessive mobility of cells, while still maintaining the organoid structure. For example, polymer density that form active and functional organoids may be between about 0. 75-5% by weight of protein concentration (collagen or non-thixotropic protein) and about 0.5% to 5% by weight of synthetic hydrogel. The protein concentration of the polymer can comprise a protein concentration of at least about 0.25%, 0.5%, 0.75%, 1%, 1.25%, 1.5%, 1.75%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 9%, or 10% by weight of the polymer, or more. The polymer can comprise an amount of synthetic hydrogel of at least about 0.25%. 0.5%, 0.75%, 1%, 1.25%, 1.5%, 1.75%, 2%, 2.5%, 3%, 3.5%, 4%. 4.5%, 5%, 6%, 7%, 8%, 9%, or 10% by weight, or more. It is important to use and maintain proper density range of the polymers used to construct functional LNOs, which require a polymer density that is dense enough to maintain organoid structure in a liquid media bath but also are of a low enough density that supports cell motility during an immune response to stimulations. Immune cells are highly motile cells within living lymph nodes and their independent motility is necessary for developing an immune response to stimulations, such as exposure to antigens.

[0269] The polymers may comprise functional chemical moiety groups that may be used to conjugate to additional molecules. The functional chemical moiety groups may be able to react with other molecules and form bonds thereby conjugating a molecule to the polymer. The functional chemical moiety groups may be used to perform click chemistry reactions. The functional chemical moiety groups may comprise an azide, azido group, alkyne, alkene, carbonyl, thiol, or other reactive functional group. The functional chemical moiety groups may be distributed selectively in throughout the scaffold. The polymers may be conjugated to other molecules prior to polymerization, during polymerization, or after polymerization. The polymers may be conjugated to streptavidin. The polymers may be conjugated to biotin, or other biotinylated molecules. The biotinylated molecules may be biotinylated polypeptides. The other molecules conjugated to the polymers may be- 53 -508002954.1Attorney Docket No.: 144242-0301 distributed selectively in throughout the scaffold. The molecules may be selectively coated on only a part of the scaffold via immersing the scaffold in the desired biomolecule solution. In some embodiments, one or more proteins may be coated selectively on the scaffold surface via this method.

[0270] In some embodiments, the printed bioink or resin is prepared by a solution that comprises, consists of, or consists essentially of one or more of the following: a hydrogel (e.g., PEG-DA mean molecular weight 700 Da, 834 mM), a photo initiator (Eosin Y, 27 mM), a co-initiator (Triethanolamine (TEO A) at 48 mM, and / or Diphenyliodonium chloride at 0.76 mM), and PBS (40% by weight). In some embodiments, bioactive proteins or peptides are added to the resin, for example, as unbound and trapped by the PEG-matrix (e.g., fibronectin and / or streptavidin), and / or b) bound to PEG-DA and / or crosslinked to the matrix (PEG-RGD). In some embodiments, the bioactive proteins or peptides may be attached to the scaffold, for example, by incorporating a tag (e.g., streptavidin) to the ink or resin and attaching the bioactive protein or peptide using a binder to the tag (e.g., biotin).

[0271] In some embodiments, the scaffolds may be seeded with a plurality of cells. In some embodiments, the scaffold may be seeded with the plurality cells in a well plate (e.g., a 24-well plate) or a bioreactor.

[0272] In some embodiments, a 3D scaffold holder may be designed to contain the 3D printed scaffold (e.g., within a multiple well tissue culture plate). In some embodiments, the 3D scaffold holder and the 3D scaffold are placed into a 24-well plate and plurality of cells processed and prepared in a desired medium are added directly to the scaffold. In some embodiments, the plurality of cells is allowed to settle, e.g., for about 10 minutes. In some embodiments, the desired media is added to the multiple well tissue culture plate so as to fully submerge the scaffold. In some embodiments, the cells are cultured as needed.

[0273] In some embodiments, the cells are seeded in the scaffold by a bioreactor (e.g., a perfusion bioreactor). In some embodiments, the scaffold holder and scaffold may be placed within a bioreactor (e.g., a perfusion bioreactor) and seeded with a plurality of cells for extended culture under conditions (e.g., perfusion conditions) to facilitate nutrient exchange and waste removal across the scaffold walls. In some embodiments, a perfusion bioreactor may be designed that is capable of holding cell-laden scaffolds under perfusion. The perfusion bioreactor may be printed using a 3D printer (e.g., formlabs; BIOMED Clear Resin) following manufacturer protocol for part printing and post-printing processing.- 54 -508002954.1Attorney Docket No.: 144242-0301

[0274] In some embodiments, a scaffold is embedded within a gelatin for imaging or staining. In some embodiments, the prior to embedding the scaffold, an image (e.g., a brightfield image) is taken of the scaffold. In some embodiments, the gelatin is a solution of Porcine Gelatin Type A (Sigma Aldrich, CAS: 9000-70-8). In some embodiments, the scaffold is removed from a bracket in which it is seeded and placed in, for example, a 48-well plate. In some embodiments, sufficient gelatin solution may be added to each well to completely embed the scaffold. In some embodiments, the gelatin solution may be added by touching the pipette tip to the upper surface of the media and continuously moving around the well wall while keeping the pipette tip in constant contact with the well wall. In some embodiments, the gelatin solution may be incubated for about 10-30 minutes or about 15-20 minutes. In some embodiments, the incubation occurs at about 37 °C. Subsequently, the 48- well plate may be cooled by placing the 48-well plate on ice (e.g., ice and water), where water is sufficiently touching the plate bottom uniformly, for about 15 to 30 minutes. In some embodiments, once the gelatin solidifies, most of the remaining liquid from the well may be removed. In some embodiments, a thin needle may be used to remove the gelatin from the well. In some embodiments, a biopsy punch may be used to remove the gelatin from the well. In some instances, the scaffold may be completely suspended for lightsheet imaging. The embedded scaffold as previously prepared may be re-embedded in another layer of gelatin. A thin layer of gelatin may be added to a 24-well plate and allowed to cool for 2-5 minutes. The previously embedded scaffold may be placed on the thing later of gelatin within the 24-well plate.

[0275] In some embodiments, an LNO is embedded within a gelatin for imaging or staining. In some embodiments, the prior to embedding the LNO, an image (e.g., a brightfield image) is taken of the LNO. In some embodiments, the gelatin is a solution of Porcine Gelatin Type A (Sigma Aldrich, CAS: 9000-70-8). In some embodiments, sufficient gelatin solution may be added to each well to completely embed the LNO. In some embodiments, the gelatin solution may be added by touching the pipette tip to the upper surface of the media and continuously moving around the well wall while keeping the pipette tip in constant contact with the well wall. In some embodiments, the gelatin solution may be incubated for about 10-30 minutes or about 15-20 minutes. In some embodiments, the incubation occurs at about 37 °C. Subsequently, the well plate (e.g., 24-well plate) may be cooled by placing the well plate on ice (e.g., ice and water), where water is sufficiently touching the plate bottom uniformly, for about 15 to 30 minutes. In some embodiments, once- 55 -508002954.1Attorney Docket No.: 144242-0301 the gelatin solidifies, most of the remaining liquid from the well may be removed. In some embodiments, 4.0% PFA (Thermo PIERCE™ 16% Formaldehyde (w / v)) solution in lx PBS with Ca++and Mg++may be added to each well to ensure the gelatin block is fully submerged. In some embodiments, the gelatin block may be incubated for 30 minutes at room temperature on a shaker. In some embodiments, the 4.0% PFA solution is removed and discarded, and the gelatin block is washed 3x (20 minutes) with lx PBS on a shaker. The sample may be stained with primary and / or secondary antibodies.

[0276] LNOs may be made in a glass bottom well plate and imaged directly on the imbedding plate. However, the LNO may be made in a different well and retrieved and transferred for imaging. A 27G needle may be used to slowly dislodge the gelatin from the well wall. Alternatively, a biopsy punch may be used to remove the gelatin from the well. The scaffold embedded within the gelatin may be removed using a curved spatula. The scaffold within the gelatin may be further supported with a second spatula during removal. The gelatin block may be stored in 2x antibiotic-antimycotic solution prepared in lx PBS by way of submerging the gelatin block in the solution, covered, and stored at 4 °C. Brightfield images are taken before final storage.5. Pharmaceutical Compositions, and Methods of Administration

[0277] A sixth aspect provides a pharmaceutical composition comprising one or more of the antibodies generated by the methods set forth herein. Any of the antibodies provided herein can be provided in any appropriate pharmaceutical composition and be administered by any suitable route of administration. Suitable routes of administration include, but are not limited to, the inhalation, intra-arterial, intradermal, intramuscular, intraperitoneal, intravenous, nasal, parenteral, pulmonary, intravesical, and subcutaneous routes.

[0278] The pharmaceutical composition may comprise one or more pharmaceutical excipients. Any suitable pharmaceutical excipient may be used, and one of ordinary skill in the art is capable of selecting suitable pharmaceutical excipients. Accordingly, the pharmaceutical excipients provided below are intended to be illustrative, and not limiting. Additional pharmaceutical excipients include, for example, those described in the Handbook of Pharmaceutical Excipients, Rowe et al. (Eds.) 6th Ed. (2009), incorporated by reference in its entirety.

[0279] In some embodiments, the pharmaceutical composition comprises an antifoaming agent. Any suitable anti-foaming agent may be used. In some embodiments, the- 56 -508002954.1Attorney Docket No.: 144242-0301 anti-foaming agent is selected from an alcohol, an ether, an oil, a wax, a silicone, a surfactant, and combinations thereof. In some embodiments, the anti-foaming agent is selected from a mineral oil, a vegetable oil, ethylene bis stearamide, a paraffin wax, an ester wax, a fatty alcohol wax, a long chain fatty alcohol, a fatty acid soap, a fatty acid ester, a silicon glycol, a fluorosilicone, a polyethylene glycol-polypropylene glycol copolymer, polydimethylsiloxanesilicon dioxide, ether, octyl alcohol, capryl alcohol, sorbitan trioleate, ethyl alcohol, 2-ethyl- hexanol, dimethicone, oleyl alcohol, simethicone, and combinations thereof.

[0280] In some embodiments, the pharmaceutical composition comprises a cosolvent. Illustrative examples of cosolvents include ethanol, poly(ethylene) glycol, butylene glycol, dimethylacetamide, glycerin, and propylene glycol.

[0281] In some embodiments, the pharmaceutical composition comprises a buffer. Illustrative examples of buffers include acetate, borate, carbonate, lactate, malate, phosphate, citrate, hydroxide, diethanolamine, monoethanolamine, glycine, methionine, guar gum, and monosodium glutamate.

[0282] In some embodiments, the pharmaceutical composition comprises a carrier or filler. Illustrative examples of carriers or fillers include lactose, maltodextrin, mannitol, sorbitol, chitosan, stearic acid, xanthan gum, and guar gum.

[0283] In some embodiments, the pharmaceutical composition comprises a surfactant. Illustrative examples of surfactants include t / -alpha tocopherol, benzalkonium chloride, benzethonium chloride, cetrimide, cetylpyridinium chloride, docusate sodium, glyceryl behenate, glyceryl monooleate, lauric acid, macrogol 15 hydroxystearate, myristyl alcohol, phospholipids, polyoxyethylene alkyl ethers, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, polyoxylglycerides, sodium lauryl sulfate, sorbitan esters, and vitamin E polyethylene(glycol) succinate.

[0284] In some embodiments, the pharmaceutical composition comprises an anticaking agent. Illustrative examples of anti-caking agents include calcium phosphate (tribasic), hydroxymethyl cellulose, hydroxypropyl cellulose, and magnesium oxide.

[0285] Other excipients that may be used with the pharmaceutical compositions include, for example, albumin, antioxidants, antibacterial agents, antifungal agents, bioabsorbable polymers, chelating agents, controlled release agents, diluents, dispersing agents, dissolution enhancers, emulsifying agents, gelling agents, ointment bases, penetration enhancers, preservatives, solubilizing agents, solvents, stabilizing agents, and sugars. Specific - 57 -508002954.1Attorney Docket No.: 144242-0301 examples of each of these agents are described, for example, in the Handbook of Pharmaceutical Excipients, Rowe et al. (Eds.) 6th Ed. (2009), The Pharmaceutical Press, incorporated by reference in its entirety.

[0286] In some embodiments, the pharmaceutical composition comprises a solvent. In some embodiments, the solvent is saline solution, such as a sterile isotonic saline solution or dextrose solution. In some embodiments, the solvent is water for injection.

[0287] In some embodiments, the pharmaceutical compositions are in a particulate form, such as a microparticle or a nanoparticle. Microparticles and nanoparticles may be formed from any suitable material, such as a polymer or a lipid. In some embodiments, the microparticles or nanoparticles are micelles, liposomes, or polymersomes. In certain embodiments, a composition provided herein is a pharmaceutical composition or a single unit dosage form. Pharmaceutical compositions and single unit dosage forms provided herein comprise a prophylactically or therapeutically effective amount of one or more prophylactic or therapeutic antibodies.

[0288] Further encompassed herein are anhydrous pharmaceutical compositions and dosage forms comprising an antibody, since water can facilitate the degradation of some antibodies.

[0289] Anhydrous pharmaceutical compositions and dosage forms provided herein can be prepared using anhydrous or low moisture containing ingredients and low moisture or low humidity conditions. Pharmaceutical compositions and dosage forms that comprise lactose and at least one active ingredient that comprises a primary or secondary amine can be anhydrous if substantial contact with moisture and / or humidity during manufacturing, packaging, and / or storage is expected.

[0290] An anhydrous pharmaceutical composition should be prepared and stored such that its anhydrous nature is maintained. Accordingly, anhydrous compositions can be packaged using materials known to prevent exposure to water such that they can be included in suitable formulary kits. Examples of suitable packaging include, but are not limited to, hermetically sealed foils, plastics, unit dose containers (e.g., vials), blister packs, and strip packs.5.1. Parenteral Dosage Forms

[0291] In some embodiments, parenteral dosage forms are provided. Parenteral- 58 -508002954.1Attorney Docket No.: 144242-0301 dosage forms can be administered to subjects by various routes including, but not limited to, subcutaneous, intravenous (including bolus injection), intramuscular, and intra-arterial. Because their administration typically bypasses subjects’ natural defenses against contaminants, parenteral dosage forms are typically, sterile or capable of being sterilized prior to administration to a subject. Examples of parenteral dosage forms include, but are not limited to, solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, and emulsions.

[0292] Suitable vehicles that can be used to provide parenteral dosage forms are well known to those skilled in the art. Examples include, but are not limited to: Water for Injection USP; aqueous vehicles such as, but not limited to, Sodium Chloride Injection, Ringer’s Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, and Lactated Ringer’s Injection; water miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and polypropylene glycol; and non-aqueous vehicles such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

[0293] Excipients that increase the solubility of one or more of the antibodies disclosed herein can also be incorporated into the parenteral dosage forms.5.2. Dosage and Unit Dosage Forms

[0294] In human therapeutics, the doctor will determine the dosology which she considers most appropriate according to a preventive or curative treatment and according to the age, weight, condition and other factors specific to the subject to be treated.

[0295] The amount of the antibody or composition which will be effective in the prevention or treatment of a disorder, or one or more symptoms thereof, will vary with the nature and severity of the disease or condition, and the route by which the antibody is administered. The frequency and dosage will also vary according to factors specific for each subject depending on the specific therapy (e.g., therapeutic or prophylactic agents) administered, the severity of the disorder, disease, or condition, the route of administration, as well as age, body, weight, response, and the past medical history of the subject. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0296] In certain embodiments, exemplary doses of a composition include milligram- 59 -508002954.1Attorney Docket No.: 144242-0301 or microgram amounts of the antibody per kilogram of subject or sample weight (e.g., about 10 micrograms per kilogram to about 50 milligrams per kilogram, about 100 micrograms per kilogram to about 25 milligrams per kilogram, or about 100 microgram per kilogram to about 10 milligrams per kilogram). In some embodiments, the dosage of the antibody provided herein, based on weight of the antibody, administered to prevent, treat, manage, or ameliorate a disorder, or one or more symptoms thereof in a subject is 0.1 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 10 mg / kg, or 15 mg / kg or more of a subject’s body weight. In some embodiments, the dosage of the composition or a composition provided herein administered to prevent, treat, manage, or ameliorate a disorder, or one or more symptoms thereof in a subject is 0.1 mg to 200 mg, 0.1 mg to 100 mg, 0.1 mg to 50 mg, 0.1 mg to 25 mg, 0.1 mg to 20 mg, 0.1 mg to 15 mg, 0.1 mg to 10 mg, 0.1 mg to 7.5 mg, 0.1 mg to 5 mg, 0.1 to 2.5 mg, 0.25 mg to 20 mg, 0.25 to 15 mg, 0.25 to 12 mg, 0.25 to 10 mg, 0.25 mg to 7.5 mg, 0.25 mg to 5 mg, 0.25 mg to 2.5 mg, 0.5 mg to 20 mg, 0.5 to 15 mg, 0.5 to 12 mg, 0.5 to 10 mg, 0.5 mg to 7.5 mg, 0.5 mg to 5 mg, 0.5 mg to 2.5 mg, 1 mg to 20 mg, 1 mg to 15 mg, 1 mg to 12 mg, 1 mg to 10 mg, 1 mg to 7.5 mg, 1 mg to 5 mg, or 1 mg to 2.5 mg.

[0297] The dose can be administered according to a suitable schedule, for example, once, two times, three times, or for times weekly. It may be necessary to use dosages of the antibody outside the ranges disclosed herein in some cases, as will be apparent to those of ordinary skill in the art. Furthermore, it is noted that the clinician or treating physician will know how and when to interrupt, adjust, or terminate therapy in conjunction with subject response.

[0298] Different therapeutically effective amounts may be applicable for different diseases and conditions, as will be readily known by those of ordinary skill in the art. Similarly, amounts sufficient to prevent, manage, treat or ameliorate such disorders, but insufficient to cause, or sufficient to reduce, adverse effects associated with the antibodies provided herein are also encompassed by the herein described dosage amounts and dose frequency schedules. Further, when a subject is administered multiple dosages of a composition provided herein, not all of the dosages need be the same. For example, the dosage administered to the subject may be increased to improve the prophylactic or therapeutic effect of the composition or it may be decreased to reduce one or more side effects that a particular subject is experiencing.

[0299] In some embodiments, treatment or prevention can be initiated with one or more loading doses of an antibody or composition provided herein followed by one or more - 60 -508002954.1Attorney Docket No.: 144242-0301 maintenance doses.

[0300] In some embodiments, a dose of an antibody or composition provided herein can be administered to achieve a steady-state concentration of the antibody in blood or serum of the subject. The steady-state concentration can be determined by measurement according to techniques available to those of skill or can be based on the physical characteristics of the subject such as height, weight and age.

[0301] In some embodiments, administration of the same composition may be repeated and the administrations may be separated by at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or 6 months. In some embodiments, administration of the same prophylactic or therapeutic agent may be repeated and the administration may be separated by at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or 6 months.6. Kits

[0302] In some embodiments, an antibody prepared according to the methods provided herein is provided in the form of a kit, i.e., a packaged combination of reagents in predetermined amounts with instructions for performing a procedure. In some embodiments, the procedure is a diagnostic assay. In other embodiments, the procedure is a therapeutic procedure. In other embodiments, the procedure is any of the experimental methods described herein, including the experiments described in Examples 1-39, and the kit comprises any of the cells, plates, antigens, adjuvants, or other reagents described herein for the performance of any of the experiments. In some embodiments, the kit comprises instructions for using any of the cell cultures or LNOs described herein for the generation of antibodies. In some embodiments, the kit comprises an antibody. In some embodiments, the antibodies comprise a VH sequence selected from the group consisting of SEQ ID NO:4 or SEQ ID NO: 8 or the CDRs contained therein. In some embodiments, the antibodies comprise a VL sequence selected from the group consisting of SEQ ID NO: 6 or SEQ ID NO: 10 or the CDRs contained therein. In some embodiments, the antibodies comprise a VH sequence comprising the CDR sequences found in SEQ ID NO: 4 and a VL sequence comprising the CDR sequences found in SEQ ID NO: 6. In some embodiments, the antibodies comprise a VH sequence comprising the CDR sequences found in SEQ ID NO: 8 and a VL sequence comprising the CDR sequences found in SEQ ID NO: 10. In some embodiments, the antibodies comprise a VH sequence comprising SEQ ID NO: 4 and a VL sequence comprising SEQ ID NO: 6. In some embodiments, the antibodies comprise a VH - 61 -508002954.1Attorney Docket No.: 144242-0301 sequence comprising SEQ ID NO: 8 and a VL sequence comprising SEQ ID NO: 10. In some embodiments, the antibodies comprise a VH sequence encoded by a sequence selected from the group consisting of SEQ ID NO: 3 and SEQ ID NO: 7. In some embodiments, the antibodies comprise a VL sequence encoded by a sequence selected from the group consisting of SEQ ID NO: 5 and SEQ ID NO: 9. In some embodiments, the antibodies comprise a VH sequence encoded by SEQ ID NO: 3 and a VL sequence encoded by SEQ ID NO: 5. In some embodiments, the antibodies comprise a VH sequence encoded by SEQ ID NO: 7 and a VL sequence encoded by SEQ ID NO: 9. Vectors comprising any of the nucleic acids described herein, and host cells comprising said vectors may also be included in kits of the present disclosure.

[0303] A fourth aspect provides a kit comprising an adjuvant, at least one agent for the removal of immunosuppressive cells and cytotoxic cells, and instructions for using said agent to generate LNOs from a population of harvested donor immune cells. In some embodiments, the instructions direct the user to use the agent to remove immunosuppressive cells and cytotoxic cells from the harvested donor immune cells prior to generation of a LNO. In some embodiments, the agent comprises a lysosomotropic agent (e.g., LLME) or agents for negative selection of cells (e.g., anti-CD56 mAb-, anti-CD8 mAb-, and anti-CD14 mAb- conjugated magnetic beads, or a combination thereof. In some embodiments, the kit instructions further describe protocols for generating antibodies from the LNOs using the adjuvant and an antigen of interest. In some embodiments, the kit comprises one or more of liposome-based adjuvants, oil-in-water emulsion adjuvants, virus-like particles, TLR ligands, CD40L, NOD2 agonist, carrier proteins, carrier polysaccharides and / or phycoerythrin (PE).

[0304] In some embodiments, the kit further comprises a solvent for the reconstitution of the antibody(ies) prepared according to methods herein. In some embodiments, the antibody is provided in the form of a pharmaceutical composition.EXAMPLESExample 1: Production of Lymph Node Organoids

[0305] In this example, the production of three-dimensional LNOs is described. Except where noted otherwise, the LNOs described in any of Examples 2-39 were made using the methods described herein.

[0306] LNOs comprise, consist of, or consist essentially of a 3D structure that provides one or more of the following, e.g., support for cells (e.g., all or specific cell types),- 62 -508002954.1Attorney Docket No.: 144242-0301 presenting one or more proteins on the surface and within the scaffold, one or more cell types, providing nutrients oxygen and / or removing waste, and creating different compartments for different cell types.

[0307] A LNO may be created in various ways. For example, the LNO structure may be casted, formed using electrospinning, or 3D printed. Moreover, LNO scaffold may be prepared and tested according to the general procedures described in PCT Pub. Nos. WO 2023 / 114782 and / or WO 2023 / 114785, the full disclosures of each of which are incorporated herein by reference.

[0308] The scaffold material is chosen for its desired properties. There are several alternatives such as naturally-derived hydrogels, synthetic hydrogels, peptides, proteins or combinations thereof. To make the material biologically active, one can decorate it with peptides and / or proteins (hereafter bioactive molecules) to activate one or many desired cell functions. The bioactive molecules can be incorporated into the scaffold in several ways: The scaffold material matrix properties can be tuned by varying the length of the hydrogel molecules, in order to trap molecules of a certain size and / or electromagnetic properties. The bioactive molecules or the active parts of the bioactive molecules can also be attached to the scaffold material itself and bound to the structure in that way. The process can also be achieved by having a chemical tag molecule exposed on the surface, and in a postprocessing step binding the desired bioactive molecules to the surface.

[0309] For example, inorganic hydrogel Polyethylene Glycol Diacrylate (PEG-DA), with an average molecular weight of 700 Da., is mixed with co-initiator Triethanolamine (TEO A), Diphenyliodonium chloride (DPI) and Eosin Y (EY) as a photo-initiator using Phosphate-buffered saline as solvent. The concentration of these could be 834 mM PEG-DA, 48 mM TEOA, 0.76 mM DPI and 27 mM EY.

[0310] Proteins and / or peptides may be added and bound to hydrogel molecules to have them decorating the surface. In some embodiments, 10 pg / mL human fibronectin may be added to the ink solution. In some embodiments, 10 pg / mL streptavidin to the ink solution. In some embodiments, 100 pg / mL PEG-RGD may be added to the ink solution. In some embodiments, 0.5 pg / mL of CD40L may be added to the ink solution. In some embodiments, 1.0 pg / mL of Intercellular Adhesion Molecule 1 (ICAM1) may be added to the ink solution. In some embodiments, 1.0 pg / mL of Vascular Cell Adhesion Molecule 1 (VCAM1) may be added to the ink solution. In some embodiments, more than 1 proteins and / or peptides may be- 63 -508002954.1Attorney Docket No.: 144242-0301 added to the ink solution.

[0311] One could also add secondary biomolecules after printing the desired scaffold. For example, one could add 1 pg / mL of biotin- AF647 to the scaffold which will bind to the Streptavidin already incorporated into the scaffold.

[0312] A Computer-Aided Design (CAD) model is used to create structures of a desired shape. The CAD-model may be processed by a print planning software that produces print instructions for a 3D printer. The 3D printer may then print the structure using the desired ink described above. The 3D printer may be based on the methods described in WO2023114782A1 and the references therein.

[0313] The scaffold may be seeded with cells, as described herein, to produce LNOS.Example 2: Embedding scaffolds to stain for imaging

[0314] In this example, an exemplary method of preparing the scaffolds for imaging is described.

[0315] A 6-10 wt% solution of Porcine Gelatin Type A (Sigma Aldrich, CAS: 9000- 70-8) to a desired volume. A hotplate is preheated to 60 °C. To a beaker, the gelatin is added, followed by addition of PBS to produce to the 6-10 wt% solution. The solution is gently stirred (e.g., to reduce or eliminate the creation of bubbles) at 200 rpm until gelatin is fully dissolved, e.g., approximately 20 minutes. The gelatin may be covered with parafilm while stirred to reduce evaporation. The solution may be sterile filtered using centrifuge filter tubes, e.g., 0.22 pm pore centrifuge tubes (Millipore; 50 mL process volume Steriflip- GP). The centrifuge filter tubes are pre-heated while in the packaging at 37 °C for at least 1 hour. The gelatin solution is filtered at about 60 °C, and may be re-heated if temperature cools and viscosity increase. The gelatin solution may be stored in sterile conditions at 4 °C. For usage, the gelatin may be thawed in a 37 °C water bath.

[0316] Prior to embedding the scaffold, a brightfield image may be taken for comparison. The pH of the gelatin solution is adjusted to about 7.1-7.4. 1.0 N and 0.1 N NaOH may be used to neutralize the gelatin solution. The scaffold is removed from the bracket in which it is seeded and placed, for example, in a new 48-well plate. By maintaining constant contact with the well wall in a swirling motion around the well, 400 pL of gelatin solution is added to each well, or sufficient gelatin solution to completely embed the scaffold. The gelatin solution may be added by touching the pipette tip to the upper surface of the media and continuously moving around the well wall while keeping the pipette tip in constant - 64 -508002954.1Attorney Docket No.: 144242-0301 contact with the well wall. The scaffold within the gelatin solution may be incubated for 15- 20 minutes at 37 °C. Subsequently, the 48-well plate is cooled by placing the 48-well plate on ice (e.g., ice and water), where water is sufficiently touching the plate bottom uniformly, for about 15 to 30 minutes. Once the gelatin solidifies, most of the remaining liquid from the well may be removed. A thin needle may be used to slowly dislodge the gelatin from the well wall. Alternatively, a biopsy punch may be used to remove the gelatin from the well. The scaffold embedded within the gelatin may be removed using a curved spatula. The scaffold within the gelatin may be further supported with a second spatula during removal.

[0317] In some instances, the scaffold may be completely suspended for lightsheet imaging. The embedded scaffold as previously prepared may be re-embedded in another layer of gelatin. A thin layer of gelatin may be added to a 24-well plate and allowed to cool for 2-5 minutes. The previously embedded scaffold may be placed on the thing later of gelatin within the 24-well plate.

[0318] The embedded scaffold is placed in a larger well and lx PBS may be added to maintain hydration. If present, lx PBS is removed from the larger well and 4% formaldehyde (Thermo PIERCE™ 16% Formaldehyde (w / v)) solution in lx PBS with Ca++and Mg++may be added to each well to ensure the gelatin block is fully submerged. The gelatin block may be incubated for 30 minutes at room temperature on a shaker. The 4.0% formaldehyde solution is removed and discarded, and the gelatin block is washed 3x (20 minutes) with lx PBS on a shaker. The gelatin block may be stored in 2x antibiotic- antimycotic solution prepared in lx PBS by way of submerging the gelatin block in the solution, covered, and stored at 4 °C. Brightfield images are taken before final storage.Example 3: Embedding LNOs to stain for imaging

[0319] In this example, an exemplary method of embedding LNOs in a gelatin solution for imaging is described.

[0320] A 6-10 wt% solution of Porcine Gelatin Type A (Sigma Aldrich, CAS: 9000- 70-8) to a desired volume. A hotplate is preheated to 60 °C. To a beaker, the gelatin is added, followed by addition of PBS to produce to the 6-10 wt% solution. The solution is gently stirred (e.g., to reduce or eliminate the creation of bubbles) at 200 rpm until gelatin is fully dissolved, e.g., approximately 20 minutes. The gelatin may be covered with parafilm while stirred to reduce evaporation. The solution may be sterile filtered using centrifuge filter tubes, e.g., 0.22 pm pore centrifuge tubes (Millipore; 50 mL process volume Steriflip-- 65 -508002954.1Attorney Docket No.: 144242-0301GP). The centrifuge filter tubes are pre-heated while in the packaging at 37 °C for at least 1 hour. The gelatin solution is filtered at about 60 °C, and may be re-heated if temperature cools and viscosity increase. The gelatin solution may be stored in sterile conditions at 4 °C. For usage, the gelatin may be thawed in a 37 °C water bath.

[0321] Prior to embedding the LNO, a brightfield image of the LNO may be taken. The pH of the gelatin solution is adjusted to about 7.1-7.4. 1.0 N and 0.1 N NaOH may be used to neutralize the gelatin solution. By maintaining constant contact with the well wall in a swirling motion around the well, 200 pL of gelatin solution is added to each well. The gelatin solution may be added by touching the pipette tip to the upper surface of the media and continuously moving around the well wall while keeping the pipette tip in constant contact with the well wall. The LNO within the gelatin solution may be incubated for 15-20 minutes at 37 °C. Subsequently, the 24-well plate is cooled by placing the 24-well plate on ice (e.g., ice and water), where water is sufficiently touching the plate bottom uniformly, for about 15 to 30 minutes. Once the gelatin solidifies, most of the remaining liquid from the well may be removed. 4.0% formaldehyde (Thermo PIERCE™ 16% Formaldehyde (w / v)) solution in lx PBS with Ca++and Mg++is added to each well to ensure the gelatin block is fully submerged. The gelatin block may be incubated for 30 minutes at room temperature on a shaker. The 4.0% formaldehyde solution is removed and discarded, and the gelatin block is washed 3x (20 minutes) with lx PBS on a shaker. The sample may be stained with primary and / or secondary antibodies.

[0322] LNOs may be made in a glass bottom well plate and imaged directly on the imbedding plate. However, the LNO may be made in a different well and retrieved and transferred for imaging. A 27G needle may be used to slowly dislodge the gelatin from the well wall. Alternatively, a biopsy punch may be used to remove the gelatin from the well. The scaffold embedded within the gelatin may be removed using a curved spatula. The scaffold within the gelatin may be further supported with a second spatula during removal. The gelatin block may be stored in 2x antibiotic-antimycotic solution prepared in lx PBS by way of submerging the gelatin block in the solution, covered, and stored at 4 °C. Brightfield images are taken before final storage.Example 4: Cell seeding in scaffolds

[0323] In this example, an exemplary method of cell seeding in scaffolds is described. Cell seeding may occur in a multiple well tissue culture plate (e.g., a 24-well plate) or a bioreactor.- 66 -508002954.1Attorney Docket No.: 144242-0301

[0324] A 3D scaffold holder may be designed to contain the 3D printed scaffold within a multiple well tissue culture plate (e.g., a 24-well plate) (Figure 1). The 3D scaffold holder and the scaffold may be printed using a 3D printer (e.g., formlabs) following manufacturer protocol for part printing and post-printing processing. All printed parts are stored at 4 °C until further use.

[0325] The cells may be seeded in a multiple well tissue culture plate. The scaffold holder and scaffold are placed into a 24-well plate. Any excess liquid from inside the scaffold holder or scaffold is removed, for example, using a fiber-free cotton swab (QTIP). Cells are processed and prepared in the desired medium, as described in the Examples herein. Cells may be resuspended such that a desired cell number is contained at a volume of <50 pL. The resuspended cells are added directly to the scaffold and are allowed to settle for 10 minutes. The remaining scaffold volume is filled with the desired medium. 1,600 pL of media is added to the 24-well plate so as to fully submerge the scaffold. The cells are cultured as needed.

[0326] The cells may be seeded in a bioreactor (e.g., a perfusion bioreactor). The scaffold holder and scaffold may be placed within a bioreactor (e.g., a perfusion bioreactor) and seeded with cells for extended culture under conditions (e.g., perfusion conditions) to facilitate nutrient exchange and waste removal across the scaffold walls.

[0327] A perfusion bioreactor may be designed that is capable of holding cell-laden scaffolds under perfusion (Figure 2A-2B). The perfusion bioreactor may be printed using a 3D printer (e.g., formlabs; BIOMED Clear Resin) following manufacturer protocol for part printing and post-printing processing. The printed perfusion bioreactor parts may be disinfected by submerging in 70% ethanol for 24 hours followed by a 2x wash with lx PBS and stored at 4 °C until use.Example 5: Liposome

[0328] In this example, an exemplary method of preparing liposomes for challenging donor immune cells is described.

[0329] Liposomes are prepared to encapsulate or conjugate antigens for challenging donor immune cells. Liposomes may comprise, consist, or consist essentially of antigen, one or more lipids, and an adjuvant (e.g., CpG ODNs). In some embodiments, antigen / DOTAP liposome and antigen / DOTAP liposome / CpG ODN complexes are used. In some embodiments, cationic liposomes, such as DOTAP liposome - DOTAP (N-[l-(2,3-- 67 -508002954.1Attorney Docket No.: 144242-0301Di oleoyl oxy )propyl]-N,N,N trimethyl-ammonium methyl-sulfate): Cholesterol (1 : 1 molar ratio) or DOTAP:DOPE (l,2-dioleoyl- w-glycero-3 -phosphoethanolamine) (1 : 1 molar ratio), or DOTAP and CpG ODN (short synthetic single-stranded DNA molecules containing unmethylated oligodeoxynucleotides) as adjuvants are used in method described herein. Antigen / DOTAP liposome or antigen / DOTAP / CpG ODN complexes are generated by mixing antigen with DOTAP liposome, or DOTAP liposome and CpG ODN.

[0330] Immune donor immune cells may be challenged with an antigen that is encapsulated in, or conjugated with, liposome-based adjuvants. Liposomes may comprise one or more lipids. In some embodiments, liposomes comprise antigen / DOTAP. In some embodiments, the liposomes comprise antigen / DOTAP liposome / CpG ODN complexes. Briefly, after DOTAP liposome and antigen solution reach room temperature, vials of DOTAP liposome are shaken vigorously to ensure a homogenous mixture before opening. Next, DOTAP liposome is diluted in Phosphate-buffered saline (PBS) to a final concentration of 1.0 pg / pL. CpG ODNs are diluted in PBS to a final concentration of 100 pM. Antigen is diluted in PBS to a final concentration of 1.0-5.0 pg / 10 pL. The PBS used in preparing liposomes should not contain serum.

[0331] To make antigen / DOTAP complex, DOTAP liposome is mixed with an equal volume of antigen solution for a 1 : 1 ratio. The 1 : 1 v / v DOTAP liposome: antigen solution is homogenized by pipetting up and down several times to ensure homogenous mix, followed by incubation at room temperature for 20-30 minutes. After incubation, 0.5 volume of antigen solution is added to the solution, followed by homogenization by pipetting up and down several times, and incubation at room temperature for 10 minutes.

[0332] To make antigen / DOTAP liposome / CpG complex, 1.0 volume of DOTAP liposome is first mixed with an 0.5 volume of CpG ODN solution for a 1.0:0.5 v / v ratio. The 1.0:0.5 v / v DOTAP liposome:CpG ODN solution is homogenized by pipetting up and down several times, followed by incubation at room temperature for 20 minutes. After incubation, 0.5 volume of antigen solution is added to the solution, followed by homogenization by pipetting up and down several times, and incubation at room temperature for 10 minutes.Example 6: Antigen / CD40L-nanoparticle conjugates

[0333] In this example, antigen / CD40L-nanoparticle conjugates are described.

[0334] 20 pL of Streptavidin coated microsphere are washed 2 times with 200 pLPBS using Vivaspin MWCO 300kDa (12,000x g for 5 minutes each). The microspheres are - 68 -508002954.1Attorney Docket No.: 144242-0301 suspended in 400 pL PBS. Subsequently, 10 pg of biotinylated CD40L and 10 pg biotinylated antigen with 200 pL washed microsphere, or 10 pg biotinylated CD40L with 100 pl washed microsphere. Incubate at room temperature for 30 minutes with gentle mixing. The particles are washed 2 times with 450 pL PBS. Resuspend the particles in 25 pL PBS.Example 7: Antigen / CpG-B-nanoparticle conjugates

[0335] In this example, antigen / CpG ODN2006-nanoparticle conjugates are described.

[0336] 50 pL of Streptavidin coated microsphere is washed 2 times with 500 pL PBS using Vivaspin MWCO 300 kDa (12,000x g for 5 minutes each). The microspheres are suspended in 1,000 pL PBS-5% BSA. 25 pg biotinylated PE or 17 pg biotinylated TNFR2 and 25 pg biotinylated CpG ODN2006 is mixed with 500 pL of washed microsphere and incubated at room temperature for 30-60 minutes with gentle mixing. After incubation, the particles are 2 times with 500 pL PBS using Vivaspin MWCO 300 kDa. PBS is added to final volume 25.0 pL. 2.0 pL of conjugated microsphere are used for 1.0 mL of culture.Example 8: Antigen / universal epitope conjugates

[0337] In this example, antigen / CD40L-nanoparticle conjugates are described.

[0338] Antigen and universal T helper epitopes are conjugated through biotinstreptavidin interaction. Biotinylated antigen, biotinylated Pan DR-binding epitope (PADRE) peptide and streptavidin are mixed at 2:2: 1 molar ratio and incubated at room temperature for 30-60 minutes with gentle mixing. After incubation, the particles are washed 2 times with 500 pL PBS using Vivaspin MWCO 50 kDa. PBS is added to final volume 25 pl.Example 9: Removal of immunosuppressive cells and cytotoxic cells

[0339] In this example, methods for removal of immunosuppressive cells and cytotoxic cells from a sample are described. In some embodiments, immunosuppressive cells and cytotoxic cells are removed using LLME, depletion with antibody-conjugated magnetic beads, or a combination thereof.

[0340] Human peripheral blood mononuclear cells (PBMCs) are strongly suppressed by immunosuppressive cells and cytotoxic cells including natural killer (NK) cells, cytotoxic T cells and monocytes: no sensitization to antigens occurs if PBMCs are used when directly isolated from human blood. Treatment of PBMCs with LLME removes these immunosuppressive cells and cytotoxic cells, and thus enables the in vitro sensitization with- 69 -508002954.1Attorney Docket No.: 144242-0301 any antigen. Then, LLME treated cells are immunized with antigens in the presence of cytokines (IL2, IL-4, and IL-21), class B CpG ODN2006 and subsequent tissue culture for a week in 24-well transwell plate. Compared to the control, PBMCs immunized in vitro demonstrates induced antigen-specific antibody producing cells (Figures 3A-3B and Figure 4).

[0341] The immunosuppressive cells and cytotoxic cells may be removed using LLME. Frozen human peripheral blood mononuclear cells (PBMCs) (Catalog #70025, STEMCELL Technologies Inc) are thawed with ThawSTAR® CFT2 Automated Thawing System (Catalog #100-0650, STEMCELL Technologies Inc). The PBMCs are subsequently suspended with 20 mL prewarmed (37 °C) RPMI1640 / 10% human serum (HS) (complete RPMI) media. Cells are pelleted by centrifugation at 300x g for 10 minutes, and the supernatant is aspirated off. PBMCs are resuspended with 20 mL RPMI 1640 / 10% HS media supplemented with DNase I (0.025 mg / mL). Cells are mixed gently and incubated at RT for about 10 minutes. The sample is subsequently passed through 40 pm cell strainer. Cells may be counted, for example, with LUNA-FX7 cell counter. Cells are pelleted by centrifugation at 300x g for 10 minutes, and the supernatant is aspirated off. PBMCs are then resuspended with RPMI 1640 / 10% HS media with 0.25 mM LLME and cell density is adjusted to 1.0 * 107cells / mL. Cells are incubated at room temperature for 20 minutes. Cells are pelleted by centrifugation at 300 / g for i 0 minutes, and the supernatant is aspirated off. Cells are resuspended with RPMI 1640 / 10% HS media and centrifuge at 300 * g for 10 minutes, and the supernatant is aspirated off. Cells are resuspended with RPMI 1640 / 10% HS media and centrifuged at 300 x g for 10 minutes, and the supernatant is aspirated off.

[0342] The immunosuppressive cells and cytotoxic cells may be depleted with antibody-conjugated magnetic beads. PBMCs are resuspended with MACS separation buffer (Cat # 130-091-221, Miltenyi Biotec) at a density of 5.0 xlO6cells / mL. 0.5 pL each of anti- CD56 microbeads (Cat # 130-097-042, Miltenyi Biotec), anti-CD8 microbeads (Cat # 130- 045-201, Miltenyi Biotec), anti-CD14 microbeads (Cat # 130-050-201, Miltenyi Biotec) and anti-CD25 microbeads (Cat # 130-092-983, Miltenyi Biotec) per 1.0 xlO6PBMCs are added. The mixed cells and microbeads are loaded on LD Columns (Cat # 130-042-901, Miltenyi Biotec), and the flowthrough is collected. The cells are pelleted by centrifugation at 300 g for 10 minutes, and the supernatant is aspirated off.

[0343] Immunosuppressive cells and cytotoxic cells may be removed by combined LLME treatment and antibody-conjugated magnetic beads. LLME treated PBMCs (as - 70 -508002954.1Attorney Docket No.: 144242-0301 described above) are resuspended with MACS separation buffer (Cat # 130-091-221, Miltenyi Biotec) at a density of 5.0 xlO6cells / mL. CD8 microbeads (Cat # 130-045-201, Miltenyi Biotec) are added to the suspension. The suspension is loaded on LD columns (Cat # 130-042-901, Miltenyi Biotec) and flowthrough is collected. Cells are pelleted by centrifugation at 300 * g for 10 minutes, and the supernatant is aspirated off.Example 10: Immunization of samples

[0344] In this example, methods of immunization of samples with removed or depleted immunosuppressive cells and cytotoxic cells is described.

[0345] PBMCs treated as described in Example 9 may be resuspended with RPMI 1640 / 10% HS media supplemented with IL-2 (10 ng / mL), IL-4 (10 ng / mL) and IL-21 (50 ng / mL) at density of 5.0 x 106cells / 300 pL. Anti-CTLA-4 mAb or bispecific anti-CTLA- 4 / PD-l mAb blocking antibodies (20 pg / mL) may be added in the cell culture media. 300 pL of depleted cell suspension is added to the top chamber of the transwell of a 24-well transwell plate. 600 pL RPMI 1640 / 10% HS media supplemented with IL-2 (10 ng / mL), IL-4 (10 ng / mL) and IL-21 (50 ng / mL) is added to the bottom chamber. Anti-CTLA-4 or bispecific anti-CTLA-4 / PD-l blocking antibodies (20 pg / mL) may be added in the cell culture media. 20 pl DOTAP / antigen, or DOTAP / antigen / CpG ODN2216 complex, prepared as described herein, may be added to the top chamber of the transwell. The cells may be cultured for 3 days at 37 °C with 5.0% CO2. 20 pl of DOTAP / antigen, or DOTAP / antigen / CpG ODN2006 complex is added to the top chambers of each transwell. 2 pl of antigen / CD40L-nanoparticle conjugates may be added to the top chambers of each transwell. The cells may be cultured for 4 days at 37 °C with 5.0% CO2. 2.0 pl of antigen / CpG ODN2006-nanoparticle conjugates may be added to the top chambers of each transwell and cultured for 5 more days at 37 °C with 5.0% CO2. Cells are subsequently collected and used for fluorescence- activated cell sorting (FACS) sorting of antigen-specific B cells and enzyme-linked immunospot (ELISpot) analysis for antigen-specific antibody secreting cells (ASCs).Example 11: Two-step in vitro immunization of samples

[0346] In this example, a two-step method of immunization of samples with removed or depleted immunosuppressive cells and cytotoxic cells is described. The first step may comprise primary in vitro immunization and the second step may comprise secondary in vitro immunization.

[0347] For primary in vitro immunization, at day 0, frozen PBMCs are thawed.- 71 -508002954.1Attorney Docket No.: 144242-0301Immunosuppressive cells and cytotoxic cells may be removed, for example, by LLME treatment, anti-CD56 / anti-CD8 / anti-CD14 antibodies-conjugated magnetic beads, or combined LLME treatment and antibody-conjugated magnetic beads, as described in the Examples herein. The cells are challenged with antigen-PADRE conjugates, antigen- PADRE-Qbeta VLP conjugates, or antigen-PADRE / DOTAP liposomes, and cultured for 3 days in RPMI 1640 / 10% HS media supplemented with IL-2 (10 ng / mL), IL-4 (10 ng / mL) and IL-21 (50 ng / mL). After culturing, the cells are re-challenge with antigen-PADRE conjugates, antigen-PADRE-Qbeta VLP conjugates, or antigen-PADRE / DOTAP liposomes, and culture for a further 4 days in RPMI 1640 / 10% HS media supplemented with IL-2 (10 ng / mL), IL-4 (10 ng / mL) and IL-21 (50 ng / mL). At day 7, CD19+B cells are isolated by positive selection with anti-CD19 magnetic micro beads.

[0348] At day 0 of T cell priming, CD4+T cells are isolated from PBMCs by negative selection with magnetic beads. Autologous PBMCs are irradiated with X-ray (4,000 rad). CD4+T cells (1.0 xlO6cells / mL) are stimulated with PADRE (10 pg / mL) and irradiated PBMCs (2.0 xlO6cells / mL), and cultured in RPMI 1640 / 10% HS media. The CD4+T cells are restimulated twice, at day 3 and day 5, by adding PADRE (10 pg / mL) and PBMCs (2.0 x 106cells / mL). At day 7, CD4+T cells are isolated from the cultured cells by positive selection with magnetic micro beads.

[0349] For secondary in vitro immunization, the primary immunized CD19+B cells (1.0 x 105cells / mL) are cocultured with the PADRE primed CD4+T cells (1.0 x 105cells / mL) in the presence of antigen-PADRE conjugates, antigen-PADRE-Qbeta VLP conjugates, or antigen-PADRE / DOTAP liposomes, and CD40L (100 pg / mL) and cultured for 5 days in RPMI 1640 / 10% HS media supplemented with IL-2 (10 ng / mL), IL-4 (10 ng / mL) and IL-21 (50 ng / mL). The cells are collected and used for FACS sorting of antigen-specific B cells and ELISpot analysis for antigen-specific ASCs.Example 12: Induction of B cell intrinsic antibody response

[0350] In this example, an exemplary method of induction of B cell intrinsic antibody response is described.

[0351] PBMCs are thawed and B cells are isolated by negative selection with antibody-conjugated magnetic beads. Isolated B cells are cocultured in RPMI 1640 / 10% HS media supplemented with IL-2 (10 ng / mL), IL-4 (10 ng / mL), IL-21 (50 ng / mL) and CpG ODN2006 (0.1 pM). The isolated B cells are challenged at day 0 and day 3 with antigens,- 72 -508002954.1Attorney Docket No.: 144242-0301 antigen-Qbeta conjugates, or antigen / DOTAP. After 7 days of culturing, the cells are collected and used for FACS sorting of antigen-specific B cells and ELISpot analysis for antigen-specific ASCs.Example 13: ELISpot

[0352] In this example, antibody secreting cells (ASCs) within the challenged cells described herein may be evaluated by ELISpot assays.

[0353] 15 pL / well of sterile 35% ethanol is added onto Multiscreen (ELISpot) PVDF membrane 96-well plate. The ethanol is immediately poured off and the 96-well plate is washed 4x with 200 pL / well lx PBS. The plates are coated overnight at 4 °C with antigen (5 pg / mL in PBS) for the detection of antigen-specific IgM or IgG, or with anti-IgM or anti-IgG capture antibody for the detection of total IgM or IgG. The plates are washed 6x with PBS (200 pL / well) and patted on paper towel to remove excess liquid. 100 pL / well of blocking solution (RPMI 1640 media with 10% FBS) is added and incubated at room temperature for 2 hours. Cells in 100 pL RPMI 1640 media with 10% FBS per well are added. The plate is incubated for 12-18 hours at 37 °C with 5% CO2. The plate cannot be disturbed during the incubation. Plates are washed 6x with PBS-0.05% Tween-20 (200 pL / well). Biotinylated anti-IgM / IgG / IgA, anti-IgM or anti-IgG detection antibody (100 pL / well) is added and incubated at room temperature for 2 hours. The plates are subsequently washed 6x with PBS- 0.05% Tween-20 (200 pL / well). Streptavidin-HRP (diluted 1 : 1000) is added and incubated at room temperature for 1 hour. After incubation, the plates are washed 6x with PBS-0.05% Tween-20 (200 pL / well). TMB (tetramethylbenzidine) or AEC (3-amino-9-ethylcarbazole) peroxidase substrate are added and incubated at room temperature for 13 minutes. The plates are scanned with ImmunoSpot® Analyzer (Cellular Technology Limited) and analyze the spots with the BioSpot™ software.Example 14: Immune cell preparation

[0354] In this example, methods for preparing immune cells from peripheral blood mononuclear cells (PBMCs) are described.

[0355] Immune cells, as described herein, may be produced from PBMCs. In some embodiments, the PBMCs may be from a subject. In some embodiments, the PBMCs may be commercially sourced PBMCs. For example, commercially sourced frozen PBMCs are thawed under standard conditions. After cells are thawed, immune cell subpopulations are isolated, for example, by paramagnetic microbeads separation (for example, Miltenyi),- 73 -508002954.1Attorney Docket No.: 144242-0301 according to manufacturers’ instructions. Figure 5 provides an exemplary representation of immune cell preparation for generating the cell types or cell precursors described below.

[0356] The following provides exemplary production of dendritic cells (DCs) from PBMCs. PBMCs are incubated with paramagnetic microbeads loaded with anti-human CD14 mAb and subsequently purified by a paramagnetic purification column (for example, a Miltenyi column), according to manufacturers’ instructions. Purified CD14+cells are subsequently cultured for, e.g., 5 days in supplemented StemCells dendritic cells differentiation medium (ImmunoCult™-ACF Dendritic Cell Differentiation Supplement, StemCell Technologies) to obtain immature monocyte-derived dendritic cells (iMDDC). These cells are here referred to as iDCs. iDCs are characterized, for example, by means of flow cytometry for expression of HL A Class II molecules, CD80, CD83, CD86, and DC- SIGN (Dendritic Cell-Specific Intercellular adhesion molecule-3 -Grabbing Non-integrin). These cells are then loaded with antigen to allow for antigen processing and display of immune epitopes.

[0357] The following provides exemplary production of DC2 phenotype cells. iDC are generated as described above and loaded with antigen. Antigen loaded iDC are subsequently matured in a cytokine cocktail to polarize them to a DC2 phenotype.

[0358] The following provides exemplary production of CD4+T cells from PBMCs. naive CD4+T cells may be isolated by negative selection from, for example, frozen human PBMC by paramagnetic beads and subsequently purified by a paramagnetic purification column (for example, a Miltenyi column), according to manufacturers’ instructions. In some embodiments, CD4+T cells are isolated by negative selection using column- free magnetic cell separation system (e.g., EasySep™ Magnetic Cell Separation system, StemCell Technologies).

[0359] The following provides exemplary production of B cells from PBMCs. Naive B lymphocytes are isolated by negative selection from, for example, frozen human PBMC by paramagnetic beads and subsequently purified by a paramagnetic purification column (for example, a Miltenyi column) according to manufacturers’ instructions. In some embodiments, total B cell populations, e.g., naive and memory B cells, are isolated by either negative or positive selection, as described above.Example 15: DC-mediated antigen primed Th2 cells

[0360] In this example, DC loaded with antigen and polarized to a DC2 phenotype are- 74 -508002954.1Attorney Docket No.: 144242-0301 cocultured with autologous, naive CD4 T cells at a 1 : 10 ratio and incubated for up to 14 days to generate antigen primed, polarized Th2 CD4 T cells. A representation of this process is depicted in Figure 6. The quality of Th2 cells thus obtained is verified for expression of cell surface markers by flow cytometry and measurement of cytokines secreted in the cell culture milieu by Meso Scale Discovery (MSD) platform. Th2 hallmark cytokines are IL-4, IL- 10, IL-5, IL-9, IL-13, and undetectable IL-12, and Interferon gamma (INF-y).Example 16: B cell priming of Th2 Cells

[0361] As an alternative to DC2 mediated Th2 cell polarization, Th2 cells may be generated by incubating naive CD4 T cells with a commercially available Th2 polarization kit (ImmunoCult™ Human Th2 Differentiation Supplement, StemCell Technologies) that comprises human recombinant IL-4 and mouse anti-human interferon gamma. Naive CD4 T cells are incubated for up to 9 days in supplement containing medium and then evaluated for phenotype by flow cytometry. Autologous, naive B cells are then activated by 0.1 pg / mL trimeric CD40L and 1000 U / mL IL-4 for 2 days to generated activated B cells to act as antigen presenting cells. Activated B cells are then pulsed with antigen for 2 hours at 37 °C and subsequently mixed with Th2 cells at a 1 : 10 ratio and cocultured for 7 days to generate antigen primed Th2 cells. A representation of this process is depicted in Figure 7.Example 17: Generation and priming of Tfh cells

[0362] Tfh cells are generated by either antigen loaded activated B cells or by polyclonal activation. As shown in Figure 8, isolated naive B cells are loaded with Ag-Qbeta VLP and incubated with isolated naive CD4 T cells at a 1 :4 ratio for up to 7 days in medium containing Activin A (50 ng / mL), IL-12 (5 ng / mL), and IL-7 (4 ng / mL). After the incubation, cells are examined by flow cytometry for expression of surface key markers PD- 1, CXCR5, and CD40L, and for intracellular expression of BCL-6 and IL-21 (Figure 8, top panel). Alternatively, naive CD4 T cells are cultured in medium containing anti CD3 / CD28 polyclonal activator (ImmunoCult™ Human CD3 / CD28 T Cell Activator) according to manufacturers’ instructions, Activin A (50 ng / mL), IL-12 (5 ng / mL), and IL-7 (4 ng / mL) for up to 7 days (Figure 8, bottom panel). Cell phenotype is determined by flow cytometry as described above.

[0363] As described herein, methods of orchestrating different immune cell components to induce B cells differentiation in producing antibod(ies) of the IgG type against a specific antigen are provided. The antigen used for immunization and its formulations are- 75 -508002954.1Attorney Docket No.: 144242-0301 herein generically referred to as Antigen or Antigen-Qbeta VLP conjugate. These designations encompass different forms and combinations of antigen and adjuvants as described herein. This platform, and the subsequently described modifications, are here referred to as EXIS platform.Example 18: DC antigen presenting cells

[0364] iDCs generated from isolated monocytes (CD14+cells) as described in the examples above are incubated with antigen overnight at 37 °C and then matured with ImmunoCult™ Dendritic Cell Maturation Supplement (Cat. #10989, StemCell Technologies) for up to 48 hours. Ag-loaded DCs are then incubated with CD4 T cells and antigen pulsed B cells in a transwell plate at a ratio of 10% DCs to 30% CD4 T cells to 60% B cells for a total number of 2 x 106cells / well in complete RPMI 1640 media supplemented with IL-2 (2.0 ng / mL), IL-4 (2.0 ng / mL), and either one of BAFF (1.0 pg / mL), or IL-21 (50 ng / mL). Cocultures are incubated for 7 day and then cells are collected and stained with antigen specific tetramer for cell sorting on FACS. Tetramer positive B cells are collected at 1 cell / well in a 96-well plate in RNA preserving buffer for Next Generation Sequencing (NGS) (Figure 9). In a modification of this process, naive B cells and naive CD4 T cells may be isolated by magnetic beads negative selection and used in lieu of total CD4 T cells or total B cells, maintaining the remainder of the described protocol.Example 19: DC2 cells used as antigen presenting cells

[0365] In this example, DC2 are used as antigen presenting cells and antigen primed Th2 cells are used prior to formation of the EXIS platform.

[0366] DC generated as in the examples above are incubated with antigen overnight at 37 °C and then matured with DC2 polarizing cytokines TNF-a (final cone. 50 ng / mL), IL- ip (final cone. 25 ng / mL), IL-6 (final cone. 1,000 U / mL, and prostaglandin E2 (PGE2) (final cone. 1.0 pM) for up to 48 hours. Antigen-loaded DC2 are then incubated for up to 9 days with naive CD4 T cells to generate primed Th2 cells.

[0367] Naive B cells are incubated (e.g., pulsed) with antigen for 2 hours prior to being co-cultured with the antigen primed Th2 cells for 7 days in a transwell plate at the ratio of 40% Th2 cells to 60% B cells. The culture media is supplemented with IL-2 (10 ng / mL), IL-4 (10 ng / mL), and IL-21 (50 ng / mL). After the incubation period, cells are collected and stained with antigen specific tetramer for cell sorting on FACS. Tetramer positive B cells are collected at 1 cell / well in a 96-well plate in RNA preserving buffer for next generation- 76 -508002954.1Attorney Docket No.: 144242-0301 sequencing (NGS). (Figure 10).Example 20: B cells used as antigen presenting cells and antibody producing cells

[0368] In another modification of the above protocol, purified B cells are used as both antigen presenting cells and antibody producing cells (Figure 11).

[0369] Naive CD4 T cells are isolated and cultured in Th2 differentiation medium for 7 days. Naive B cells are activated with CD40L (100 ng / mL) and IL-4 (1000 U / mL) for 2 days. Activated B cells are then pulsed with antigen for 2 hours and cocultured with Th2 CD4 T cells for 7 days.

[0370] Antigen primed Th2 cells are cocultured for 7 days in a transwell plate with freshly isolated naive B cells pulsed with antigen at the ratio of 40% Th2 cells to 60% pulsed B cells. The culture media is supplemented with IL-2 (10 ng / mL), IL-4 (10 ng / mL) and IL-21 (50 ng / mL). After incubation, cells are collected and stained with antigen specific tetramer for cell sorting on FACS. Tetramer positive B cells are collected at 1 cell / well in a 96-well plate in RNA preserving buffer for NGS.Example 21: Use of antigen primed Tfh cells

[0371] In this example, Tfh cells derived from naive CD4 T cells are used in lieu of either naive CD4 T or Th2 cells in the EXIS platform (Figure 12). Tfh cells licensed by interaction with antigen loaded B cells are cocultured with naive B cells in Activin A (50 ng / mL), IL-12 (5 ng / mL), and IL-7 (4 ng / mL) supplemented culture medium for 7 days. Cells are then collected and cocultured with antigen pulsed naive B cells in a transwell plate. The cocultures are incubated for 7 days then cells are collected and stained with antigen specific tetramer for cell sorting on FACS. Tetramer positive B cells are collected at 1 cell / well in a 96-well plate in RNA preserving buffer for NGS.Example 22: Human neonatal umbilical cord blood (UCB) as a source of immune cells for in vitro immunization

[0372] In this example, human neonatal umbilical cord blood (UCB) is demonstrated as a source of immune cells for in vitro immunization.

[0373] Without wishing to be bound by theory, due to their relatively short tolerogenic exposure compared to adult B cells, UCB derived B cells may be more likely to be primed to respond to self-antigen(s). UCB comprises a major fraction of mature B cells that are phenotypically most similar (but not identical) to adult naive B cells, respond faster and more efficiently to autologous T cell stimulation, are transcriptionally prepared to- 77 -508002954.1Attorney Docket No.: 144242-0301 respond to CpG ODNs, anti-Ig Ab, and anti-Ig Ab / CD40L stimulation, and have response capabilities in both T-independent and T-dependent immunity. It has also been shown that both T and B cells from UCB can readily be activated to undergo expansion, maturation, and polarization, while B cells are competent as the adult counterpart to generate IgM, IgG and IgA. Therefore, the use of immune cells derived from cord blood may be beneficial in successfully immunize against both xeno- and self-antigens in vitro. In this modification of the EXIS platform (described above), immune and pluripotent cells derived from UCB are utilized to elicit an antigenic response and production of antibody.

[0374] Intradermal (ID) immunization has shown its potency in humans for the induction of both humoral and cellular responses. The skin possesses unique and specialized DC populations, dermal dendritic cells (DDCs) and Langerhans cells (LCs). It has been recently shown that LCs are pivotal for the formation of germinal centers, where they first induce the formation of Tfh cells and then, likely licensed by the Tfh cells, migrate to the B cell area to initiate B cell responses. As shown below, Langerhans cells may be derived in vitro from UCB CD34+pluripotent stem cells. Therefore, the use of Langerhans cells as antigen presenting cells to elicit the formation of a functional germinal center comprising of Tfh to induce a strong B cell response to antigen and production of antibodies is described. The production of the immune cell types and their orchestration to generate antigen specific (IgG) antibody production are described in the examples below.

[0375] In this example, the production of the immune cell types and their orchestration to generate antigen specific (IgG) antibody production are described.

[0376] Immune cell preparation.

[0377] Fresh UCB is obtained from a commercial vendor and immediately processed to obtain mononuclear cells (MNCs) by standard Ficoll hypaque gradient separation. MNC fraction so obtained contains pluripotent stem cell CD34+, naive and pre-naive B cells and naive T cells.

[0378] Langerhans cells

[0379] To obtain Langerhans cells, CD34+pluripotent stem cells are isolated from UCB mononuclear cells (MNCs) by positive selection using paramagnetic beads and exclusion magnetic columns from Miltenyi according to manufacturer’s instructions. Following separation, CD14+cells are cultured at 37 °C in synthetic medium, for example X- VIVO 15, supplemented with c-Kit ligand / SCF (20 ng / mL), FKT-3 ligand (50 ng / mL), GM- - 78 -508002954.1Attorney Docket No.: 144242-0301CSF (1,000 U / mL), and TGF-[3 (10 ng / mL) for 6 days. After 6 days, the culture medium is replenished with synthetic medium (e.g., X-VIVO 15) supplemented with GM-CSF (1,000 U / mL), TNF-alpha (10 ng / mL), and TGF-[3 (10 ng / mL), and cultured for a further 5 days. Langerhans cells thus obtained are characterized by flow cytometry for expression of biomarkers CD la, HLA-DR, and langerin (CD207). Langerhans cells are then matured by exposure to IL-lb (2 ng / mL), IL-6 (1,000 U / mL), TNF-a (10 ng / mL), and prostaglandin E2 (PGE2) (5 mM).

[0380] Naive CD4 T cells

[0381] Naive CD4 T cells are isolated form UCB by magnetic beads via negative selection according to manufacturers’ instructions.

[0382] Tfh cells

[0383] Tfh cells are derived from UCB naive CD4 T cells isolated by magnetic beads negative selection and cocultured with antigen pulsed LC for up to 4 days in medium containing IL-15 and IL-7 to generated antigen primed Tfh cells. Tfh cells are characterized by the expression of programmed cell death protein 1 (PD-1), C-X-C chemokine receptor type 5 (CXCR5), B-cell lymphoma 6 (BCL-6), inducible T-cell costimulatory receptor (ICOS), and CD45RO markers constellation.

[0384] B cells

[0385] Naive B cells are isolated from UCB by magnetic beads negative selection and cultured for 3 days in RPMI 1640 media supplemented with IL-2 (50 ng / mL), IL-6 (50 ng / mL), IL- 10 (50 ng / mL), and IL- 15 (25 ng / mL) induce a mature naive B cell phenotype in the fraction of pre-naive B cells present in the UCB B cell population. After incubation, naive B cells are pulsed with an antigen of choice for 2 hours at 37 °C prior to use in LNO formation.Example 23: Orchestration of immune cells to generate antigen specific, antibody producing plasma cells and memory B cells

[0386] In this example, two methods of orchestrating immune cells derived from neonatal human umbilical cord blood (UCB), as described in Example 21, to elicit production of antibodies are described. In particular, a first method comprises the use of monocyte, B cell, and CD4+T cell fractions of UCB (Figure 13), while an alternative method comprises the use of CD34+cell precursors, naive CD4 T cell, and naive B cell fractions of UCB- 79 -508002954.1Attorney Docket No.: 144242-0301(Figure 14).

[0387] In a first exemplary method, immune cell fractions prepared as described in Example 21 are orchestrated to elicit production of antibodies as follows. In this example, monocytes are cultured in hrIL-4 (1000 U / mL) and GM-CSF (1000 U / mL) for 5 days to generate immature dendritic cell (DC). Isolated naive B cells are cultured for 3 days in RPMI1640 supplemented IL-2 (50 ng / mL), IL-6 (50 ng / mL), IL-10 (50 ng / mL), and IL-15 (25 ng / mL). After culturing, the B cells are loaded with the antigen of choice (e.g., as described herein) and cocultured for 3 days with isolated naive CD4 T and DC in the trans well of a 24-well plate in complete RPMI 1640 supplemented with IL-2 (50 ng / mL), IL-6 (50 ng / mL), IL- 10 (50 ng / mL), and IL- 15 (25 ng / mL). Subsequently, a second antigen stimulation is performed again in complete RPMI 1640 media supplemented with IL-6 (50 ng / mL), IL-15 (25 ng / mL), and IFNa-2b (105 U / mL). Cocultures are incubated for a further 3 days and then cells are collected and stained with antigen specific tetramer for cell sorting on FACS. Tetramer positive B cells are collected at 1 cell / well in a 96-well plate in RNA preserving buffer for NGS.

[0388] In a second exemplary method, immune cell fractions described above are orchestrated to elicit production of antibodies (Figure 14) as follows. In this example, the antigen is loaded on Langerhans cells (LCs) derived from CD34+cell precursors and antigen- loaded LC are then cocultured with naive CD4 T cells for 4 days at a 1 :4 ratio (LC: CD4 T). In parallel, naive B cells are differentiated into matured naive B cells by culture in medium supplemented with IL-2 (50 ng / mL), IL-6 (50 ng / mL), IL- 10 (50 ng / mL), and IL- 15 (25 ng / mL) for 3 days and then pulsed with antigen for 2 hours. LC licensed Tfh cells and antigen pulsed B cells are then cocultured in a 24-well transwell plate at a 40% : 60% T:B cell ratio for a total number of 2 x 106cells / well in complete RPMI 1640 supplemented with IL-2 (50 ng / mL), IL-6 (50 ng / mL), IL-10 (50 ng / mL), and IL-15 (25 ng / mL). After 3 days, antigen is added again in complete RPMI 1640 media supplemented with IL-6 (50 ng / mL), IL- 15 (25 ng / mL), and IFNa~2b (105 U / mL). Cocultures are incubated for 7 days and then cells are collected and stained with antigen specific tetramer for cell sorting on FACS. Tetramer positive B cells are collected at 1 cell / well in a 96-well plate in RNA preserving buffer for NGS.Example 24: Orchestration of immune cells to generate antigen specific, antibody producing plasma cells and memory B cells

[0389] In this example, in vitro immunization of immune cells derived from UCB - 80 -508002954.1Attorney Docket No.: 144242-0301 according to Example 22, as shown in Figure 13, was performed using antigen conjugated DOTAP liposome. In this example, the antigen used is Phycoerythrin (PE). To make antigen / DOTAP complex, DOTAP liposome is mixed with an equal volume of antigen solution for a 1 : 1 ratio. The 1 : 1 DOTAP liposome: antigen solution is homogenized by pipetting up and down several times to ensure homogenous mix, followed by incubation at room temperature for 20-30 minutes. After incubation, 0.5 volume of antigen solution is added to the solution, followed by homogenization by pipetting up and down several times, and incubation at room temperature for 10 minutes.

[0390] Immune cells derived from UCB were assembled as depicted in Example 22, as shown in Figure 13, and cocultured for 7 days in 24-well transwell plate. Briefly, cells were restimulated once with antigen at day 3 of the coculture period. After 7 days, cells were harvested and stained with PE tetramer to detect antigen specific B cells, as well as anti-IgG, -IgD, -IgM to identify class-switch recombination (memory) B cells. A representative plot of sorted cells by FACS is shown in Figure 15. The relative percentages of positive cells for selected cell surface markers are listed in Table 2.Table 2.Example 25: In vitro immunization using human Tumor Necrosis Factor Receptor 2 conjugated with Qbeta VLP

[0391] Adult B cells were generated as described in Example 14, and pulsed with Qbeta- TNFR2 overnight prior to coculture with autologous naive T cells, for 7 days in 24- well transwell plate, as described in Example 20. Antigen primed Th2 cells were cocultured for 7 days in a 24-well transwell plate with freshly isolated naive B cells pulsed with antigen at the ratio of 40% Th2 cells to 60% pulsed B cells. The culture media was supplemented with IL-2 (10 ng / mL), IL-4 (10 ng / mL), and IL-21 (50 ng / mL). After incubation, cells were collected and stained with antigen specific tetramer for cell sorting on FACS. Tetramer positive B cells were collected at 1 cell / well in a 96-well plate in RNA preserving buffer for NGS.- 81 -508002954.1Attorney Docket No.: 144242-0301

[0392] Cells were restimulated once with antigen at day 3 of the coculture period.After coculturing for 7 days, cells were harvested and stained with TNFR2 tetramer to detect antigen specific B cells, as well as anti-IgG, -IgD, -IgM to determine class-switch recombination. A representative plot of sorted cells by FACS is shown in Figure 16. The relative percentages of positive cells for selected cell surface markers are listed in Table 3.Table 3.Example 26: Antigen-specific antibody responses induced by in vitro immunization of immunosuppressive cell- and cytotoxic cell-depleted PBMCs with antigen and cationic liposome DOTAP:DOPE and CpG ODNs

[0393] As shown in Fig. 17A, human peripheral blood mononuclear cells (PBMCs) were depleted of immunosuppressive cells and cytotoxic cells (CD8+T cells, monocytes, and NK cells) using LLME. PBMCs were resuspended in serum-free RPMI 1640 medium at a density of 5 * 106cells / mL and treated with 2.5 mM LLME for 20 minutes at room temperature. Cells were then washed three times with RPMI 1640 supplemented with 10% heat-inactivated human serum (HS), centrifuging at 300 * g for 10 minutes each to remove LLME and debris. Antigen / liposome complexes were prepared by mixing antigen with DOTAP:DOPE liposomes and gently shaking the mixture at room temperature for 30 minutes. For immunization, LLME-treated PBMCs were resuspended in complete RPMI 1640 / 10% HS media, IL-2 (10 ng / mL), IL-4 (10 ng / mL), and IL-21 (50 ng / mL), and cultured in 24-well transwell plates at a density of 5 * 106cells / mL per well. Cells were immunized with antigen / DOTAP:DOPE complexes containing 3 pg antigen and 5 pg DOTAP:DOPE in the presence of 0.5 pM CpG ODN 2216. On day 3, 50% of the medium was replaced with fresh cytokine-supplemented medium, and cells were re-challenged with the same amount of antigen / DOTAP:DOPE complex and 0.5 pM CpG ODN 2006. On day 7, the cells were harvested and analyzed. Antigen-specific CD3 CD I4 CD I 6 CD I 9 live B cells, which were stained with PE-conjugated antigen tetramers, were sorted by FACS. Antigen-specific antibody-secreting cells (ASCs) were assessed using ELISpot.

[0394] Fig. 17B is a series of graphs showing the results of FACs sorting as shown in- 82 -508002954.1Attorney Docket No.: 144242-0301Fig. 17A. Fig. 17B shows that (left graph) CD3E antigen-specific B cells were isolated from immunosuppressive cell / cytotoxic cell-depleted PBMCs challenged with DOTAP:DOPE. The gating strategy used for the sorting was Lymphocytes / Live / CD47 CD8 CD14“CD16“CD567CD19+ / IgD“ / HSA7CD3£-PE+. HSA was used to exclude cells non-specifically stained with the CD3e tetramer. Fig 17B (right graph) shows the number of actual sorted cells from the sorter. This is further confirmed via ELISpot analysis, shown in Fig. 17C, where CD3£-binding antigen secreting cells (ASCs) were detected in CD3E stimulated LNOs but not in control LNOs with no CD3E stimulation. Accordingly, the method was shown to be effective for the selection and the isolation of CD3£-specific B cells generated using the LLME depletion / LNO stimulation and sort strategy.Example 27: Antigen-specific antibody responses induced by in vitro immunization of cytotoxic cell-depleted PBMCs with antigen and TLR9 ligand CpG ODN2006

[0395] As shown in Fig. 18A, human peripheral blood mononuclear cells (PBMCs) were depleted of cytotoxic cells using LLME. For immunization, LLME-treated PBMCs were resuspended in complete RPMI 1640 / 10% HS Media supplemented with IL-2 (10 ng / mL), IL-4 (10 ng / mL), and IL-21 (50 ng / mL), and cultured in 24-well transwell plates at a density of 5 * 106cells / mL per well. Cells were immunized with antigen (3 pg / mL) in the presence of 0.5 pM class B CpG ODN 2006. On day 3, 50% of the medium was replaced with fresh cytokine-supplemented medium, and cells were re-challenged with the antigen (3 pg / mL) and CpG ODN 2006 (0.5 pM). On day 7, antigen-binding B cells were harvested and analyzed. Live CD3 CD I4 CD I6 CD I 9 cells were stained with PE-conjugated antigen tetramers and sorted by FACS. Antigen-specific antibody-secreting cells (ASCs) were identified using ELISpot.

[0396] Similar to the results described in Example 26, Fig. 18B demonstrates the sorting-based selection of TLlA-specific B cells isolated from immunosuppressive cells / cytotoxic cell-depleted PBMCs challenged with CPG ODN2006. Fig. 18B (left graph) shows that when the LNOs are not challenged with the TL1 A antigen, no TL1 A signal is detected using the TL1 A tetramer. In contrast, LNOs challenged with the TL1 A antigen (middle and right graphs) exhibit a strong signal in the TL1 A channel along the X axis. The middle graph shows the full sorting profile of TLlA-specific cells sorted using the gating strategy : Lymphocytes / Live / CD3-CD 14 CD 16-CD567CD 19+ / IgD7HS A7TL 1 A-PE+. The right graph shows the actual number of sorted cells. ELISPot results in Fig. 18C further- 83 -508002954.1Attorney Docket No.: 144242-0301 confirm the presence of TLlA-binding IgM or IgG antibody-secreting cells (ASCs) in LNOs challenged with TL1 A (right), while unchallenged LNOs (left) show no TL1 A-specific response. These findings demonstrate that the LLME depletion and LNO stimulation followed by sorting is an effective strategy for selecting and isolating TL1 A-specific B cells.Example 28: Enhancement of antigen-specific antibody responses in vitro by CD40L, DOTAP:Cholesterol / CpG, and antigen-CD40L conjugates

[0397] Using the experimental strategy shown in Fig. 17A and described in Example 26, it was further demonstrated that stimulation of immunosuppressive cell / cytotoxic cell- depleted PBMCs with the model antigen phycoerythrin (PE), in combination with stimulants such as CD40L, DOTAP:Cholesterol / CpG, and PE-CD40L conjugates, led to the generation of PE-specific B cells. These B cells were sorted using a strategy similar to that described in Examples 26 and 27. Human PBMCs were depleted of immunosuppressive cells and cytotoxic cells using LLME and cultured for 7 days in RPMI 1640 / 10% HS media supplemented with IL-2 (10 ng / mL), IL-4 (10 ng / mL), and IL-21 (50 ng / mL). Cells were stimulated under one of the following conditions: unstimulated (Nil), CD40L alone, PE alone, PE plus CD40L, PE formulated with DOTAP:Cholesterol / CpG (PE / DOTAP:Chol / CpG), PE / DOTAP:Chol / CpG plus CD40L, or PE / DOTAP:Chol / CpG plus a PE-CD40L conjugate.

[0398] Fig. 19A shows that in the absence of PE stimulation, there is a very low signal in the PE channel (first two graphs from left to right). With PE alone a modest signal above background is observed (third graphs from left to right). Notably, upon stimulation with the aforementioned adjuvants, a substantial increase in PE-positive B cells is detected (Fig. 19A fourth through seventh graphs from left to right). These results were confirmed via ELISpot analysis for PE-specific B cells, shown in Fig. 19B. A minimal signal was observed for “Nil” and “CD40L” groups that did not receive PE stimulation, whereas PE stimulation led to a >5-fold increase in signal, as indicated by the numbers below the ELISpot images. These findings demonstrate that the in vitro immunization methods described herein are applicable to multiple antigens and immunization strategies for the generation of antigenspecific B cells.Example 29: NOD2 agonist muramyl dipeptide (MDP) enhances antigen-specific antibody responses in vitro

[0399] The experimental design outlined in Fig. 17A was used to stimulate immunosuppressive cell / cytotoxic cell-depleted PBMCs with the antigen of interest, TL1 A,- 84 -508002954.1Attorney Docket No.: 144242-0301 in combination with N0D2 Agonist Muramyl dipeptide (MDP). Human PBMCs were treated with LLME to deplete immunosuppressive cells and cytotoxic cells and cultured for 7 days in RPMI 1640 medium supplemented with 10% human serum, IL-2, IL-4, and IL-21. Cells were stimulated on days 0 and 3 with MDP, TL1A, or a combination of TLlA and MDP. TL1A- binding IgD B cells were subsequently sorted and analyzed by flow cytometry. The gating strategy used for sorting in each condition was: Lymphocytes / Live / CD3“CD 14 C D 16-CD567CD 19+ / IgD7HS A7TL 1 A-PE+.

[0400] Figs. 20A-20B show flow cytometry plots of the selection of the TL1 A- specific B cells. In Fig. 20A, the first and second graphs from left to right show the full sorting profile for cells treated with MDP or TL1 A alone, respectively. The third through fifth graphs show the total number of sorted cells, demonstrating that the combination of TL1A and MDP yielded the highest number of TLlA-specific B cells compared to either treatment alone. These findings are further supported by Fig. 20B, which presents flow cytometry outcomes following PBMCs stimulation with the MDP alone (left graph), showing no TL1 A signal on the X axis; TL1 A alone (middle graph), showing a moderate signal; and the combination ofTLl A and MDP (right graph), which resulted in the strongest TL1 A- specific signal. Together, these results demonstrate that the in vitro immunization methods described herein can effectively incorporate MDP as an adjuvant to enhance the generation of antigen-specific B cells.Example 30: Oil-in-water nano-emulsion adjuvant AddaS03 enhances antigen-specific antibody responses in vitro

[0401] The experimental design outlined in Fig. 17A was used for the stimulation of immunosuppressive cell / cytotoxic-depleted PBMCs with the antigen of interest, TL1A, in combination with AddaS03. Human PBMCs were treated with LLME to deplete immunosuppressive cells and cytotoxic cells and cultured for 7 days in RPMI 1640 medium supplemented with 10% human serum, IL-2, IL-4, and IL-21. Cells were stimulated on day 0 and day 3 with AddaS03, TL1A, or a combination of TLlA and AddaS03. TLlA-binding CD I 9 IgD B cells were then sorted and analyzed by flow cytometry. The gating strategy used for the sorting in each condition was: Lymphocytes / Live / CD3“CD 14 CD 16 CD567CD 19+ / IgD7HS A7TL 1 A-PE+.

[0402] Fig. 21 A shows the full sort profiles of each population, while Fig. 21B shows FACS analysis clearly demonstrating that stimulation with the TL1 A antigen in combination- 85 -508002954.1Attorney Docket No.: 144242-0301 with the AddaS03 adjuvant results in a strong TL1 A-specific B cell signal. As shown previously, TL1 A stimulation alone induces the generation of antigen-specific B cells, however the addition of the AddaS03 adjuvant enhances this response by nearly 6-fold, as shown in Fig 21B. These results demonstrate that the methods described herein are effective for the selection and isolation of antigen-specific B cells with enhanced yield using the stimulation and sort strategy.Example 31: TLR9 ligand CpG ODN 2395 combined with checkpoint blockade via anti- CTLA-4 mAb enhances the generation of antigen-specific B cells upon in vitro immunization

[0403] The experimental design outlined in Fig. 17A was used for the stimulation of cytotoxic-depleted PBMCs with the antigen of interest, TL1A, combined with a checkpoint blockade agent, anti-CTLA-4 mAb. Human PBMCs were treated with LLME and used to generate lymphoid organoids (LNOs). LNOs were cultured for 7 days in complete RPMI- 1640 medium supplemented with 10% human serum, IL-2, IL-4, and IL-21. Cells were treated with anti-CTLA-4 mAb on day 0 and stimulated with TL1 A and CpG ODN 2395 on days 0 and 3. On day 7, cells were harvested, and TLlA-binding CD I 9 IgD live B cells were analyzed and sorted by flow cytometry. The gating strategy used for the sorting in each condition was Lymphocytes / Live / CD3-CD 14 CD 16-CD567CD 19+ / IgD7HS A7TL 1 A-PE+.

[0404] Fig. 22 depicts a series of flow cytometry plots for the selection of TL1A- specific B cells. As seen in Fig. 22, TL1 A combined with CpG ODN 2395 (first two graphs from left to right) and CpG ODN 2395 in combination with anti-CTLA4 mAb (third and fourth graphs from left to right) generated a strong antigenic response against TL1 A in B cells, as seen on the X axis. Accordingly, the methods of the present disclosure were shown to be effective for the selection and the isolation of TL1 A-specific B cells at an enhanced yield using this stimulation and sort strategy.Example 32: TL1A and CD40L induce the generation of TLlA-specific B cells in vitro

[0405] The experimental design outlined in Fig. 17A was used for the stimulation of cytotoxic-depleted PBMCs with the antigen of interest, TL1A, combined with CD40L. Human PBMCs were treated with LLME and used to generate lymphoid organoids (LNOs). LNOs were cultured for up to 14 days in complete RPML1640 medium supplemented with 10% human serum, IL-2, IL-4, and IL-21. Cells were stimulated with the antigen and adjuvant on day 0 and day 3 for 7-day cultures; and on day 0, day 3 and day 7 for 14-day- 86 -508002954.1Attorney Docket No.: 144242-0301 cultures. On both day 7 and day 14, cells were harvested, and TLlA-binding CD I 9 IgD live B cells were analyzed and sorted by flow cytometry. The gating strategy used for the sorting in each condition wasLymphocytes / Live / CD3“CD 14 CD 16-CD567CD 19+ / IgD7HS A7TL 1 A-PE+.

[0406] Fig. 23 shows the successful generation and isolation of TLlA-specific B cells via FACS from LNOs stimulated with CD40L adjuvant and TL1 A. Notably, TLlA-specific B cells were detected up to 14 days following the initial stimulation with antigen and adjuvant (bottom three graphs), whereas previous results only showed the presence of antigen-specific B cells up to 7 days post initial stimulation. These findings indicate that the methods described herein enable extended production of antigen-specific B cells, as confirmed by FACS and ELISpot.Example 33: NTA(Ni)-liposomes enhance the generation of antigen-specific B cells upon in vitro immunization

[0407] The experimental design outlined in Fig. 17A was used for the stimulation of immunosuppressive cell / cytotoxic cell-depleted PBMCs with the antigen of interest, TL1A, in combination with NTA (Ni)-liposome adjuvant. Human PBMCs were treated with LLME and used to generate lymphoid organoids (LNOs), which were cultured for 7 days in complete RPMI 1640 medium supplemented with IL-2, IL-4, and IL-21. Cells were stimulated on day 0 and day 3 with His-tagged TL1 A alone or in combination with NTA(Ni)- liposomes. On day 7, cells were harvested, and TLlA-binding CD I 9 IgD live B cells were analyzed and sorted by flow cytometry. The gating strategy used for the sorting in each condition was: Lymphocytes / Live / CD3-CD 14 CD 16-CD567CD 19+ / IgD7HS A7TL 1 A-PE+.

[0408] Fig. 24 demonstrates the effect of stimulation of the LNOs with a combination of the antigen TL1 A with the NTA (Ni)-liposome adjuvant. As shown in the third and fourth graphs (from left to right), a higher fraction of TLlA-specific B cells were sorted following stimulation with TL1 A-NTA (Ni)-liposome conjugate, compared to the TL1 A alone, as shown in the first and second graphs (from left to right). These results indicate that the methods described herein effectively enhance antigen-specific antibody response and the yield of antigen-specific B cells.Example 34: TLR9 ligand CpG ODN 2006 combined with checkpoint blockade via anti- PD-1 mAb enhances the generation of antigen-specific B cells upon in vitro immunization- 87 -508002954.1Attorney Docket No.: 144242-0301

[0409] The experimental design outlined in Fig. 17A was used for the stimulation of immunosuppressive cell / cytotoxic cell-depleted PBMCs with the antigen of interest, here TL1 A, in combination with CpG ODN 2006 and anti-PD-1 mAbs as a checkpoint blockade strategy. Human PBMCs were treated with LLME and used to generate lymphoid organoids (LNOs), which were cultured for 7 days in complete RPMI 1640 medium supplemented with IL-2, IL-4, and IL-21. Cells were treated with anti-PD-1 mAb on day 0 and stimulated with TL1 A and CpG ODN 2006 on days 0 and 3. On day 7, TL1 A-binding CD I 9 IgD live B cells were analyzed and sorted by flow cytometry. The gating strategy used for the sorting in each condition was: Lymphocytes / Live / CD3-CD 14 CD 16-CD567CD 19+ / IgD7HS A7TL 1 A-PE+.

[0410] Figs. 18A-18C show that stimulation with CPG ODN 2006 and TL1 A alone resulted in the generation of TLlA-specific B cells, as validated by FACS and ELISpot. Fig. 25 further shows that treatment of LNOs with an anti-PDl antibody (humanized IgG4 anti- PD-1 mAb Pembrolizumab, HY-P9902, MedChemExpress) enhanced the presence of TL1A- specific B cells by 2-fold (fourth graph from left to right) as compared to treatment without the antibody (second graph from left to right). These results confirm that the methods described herein enhanced both the antigen-specific antibody response to in vitro immunization and the yield of antigen-specific B cells using the sorting strategy described above.Example 35: Anionic liposomes (DOPG:DOPC and DOPS:DOPC) combined with CD40L enhance CD19-specific antibody responses in vitro

[0411] The experimental design outlined in Fig. 17A was used for the stimulation of cytotoxic-depleted PBMCs with the antigen of interest, here CD 19, in combination with the anionic liposomes DOPG:DOPC or DOPS:DOPC and CD40L. Human PBMCs were treated with LLME to generate lymphoid organoids (LNOs), which were cultured for 7 days in complete RPMI 1640 / 10% HS media supplemented with IL-2, IL-4, and IL-21. LNOs were stimulated with positively charged CD 19 ECD alone or CD 19 ECD complexed with DOPG:DOPC or DOPS:DOPC liposomes in the presence of CD40L. CD19-binding IgD“ live B cells were analyzed and sorted by flow cytometry. The gating strategy used for the sorting in each condition was:Lymphocytes / Live / CD3“CD 14 C D 16-CD567CD 19+ / IgD7HS A7CD 19-PE+ / CD 19-AF647+. A dual-color sorting strategy was employed using CD 19 protein fluorescently labeled with PE and AF 647 as detection reagents.- 88 -508002954.1Attorney Docket No.: 144242-0301

[0412] Fig. 26 shows a series of FACS plots for the quantification of CD19-specific B cells. As shown, stimulation with the CD 19 extracellular domain (ECD) alone generates a minimal response (first two plots from left to right). In contrast, CD 19 ECD in combination with the adjuvants DOPG:DOPC or DOPS:DOPC and CD40L generated a substantially higher frequency of antigen-specific B cells, as indicated by the dual-positive population (PE+ / AF647+) in the upper right quadrant of each FACS plot (third through sixth plots from left to right). These results further demonstrate that the methods described herein are effective for generating antigen-specific B cells across multiple target antigens, and that CD40L, when used in combination with other adjuvants, can enhance B cell response and improve the overall yield of the methods.Example 36: The universal T cell epitope PADRE and QP VLP enhance CD19-specific antibody responses in vitro

[0413] Previous examples have shown the generation antigen-specific B cells following stimulation with adjuvants and protein antigens such as CD3e, TL1 A, and CD19. The experimental design outlined in Fig. 17A was applied to stimulate immunosuppressive cell / cytotoxic cell-depleted PBMCs with the antigen of interest, here CD 19, conjugated to the Pan-DR Epitope peptide (PADRE), a synthetic peptide designed to bind broadly to human MHC class II molecules, particularly HLA-DR. Stimulation was optionally performed in combination with Qbeta VLP. Human PBMCs were treated with LLME to generate LNOs, which were cultured for 7 days in complete RPMI 1640 / 10% HS media supplemented with IL-2, IL-4, and IL-21. Cells were then stimulated with CD 19 ECD, mixed PADRE-tagged CD 19 ECD peptides (CAGASPKLYVWAKDRPEIWEGEPPGGPCAGAAKFVAAWTLKAAA (SEQ ID NO:19); CAGAPPRDSLNQSLSQDLTMAPGSGGPCAGAAKFVAAWTLKAAA (SEQ ID NO:20);CAGASPKLYVWAKDRPEIWEGEPPRRPPRDSLNQSLSQDLTMAPGSGGPAKFVAAW TLKAAA (SEQ ID NO: 21)), or PADRE-tagged CD 19 ECD peptides combined with Qbeta VLP. CD19-binding IgD live B cells were analyzed and sorted by flow cytometry. The same sorting strategy described in Example 35 was also applied for the selection and isolation of CD19-specific B cells.

[0414] The results shown in Fig. 27 demonstrate an increased signal of CD19-specific B cells in the response to stimulation with the CD19-PADRE peptides (third and fourth plots from left to right). Similarly, stimulation with the adjuvant bacteriophage Qbeta (Qbeta) also - 89 -508002954.1Attorney Docket No.: 144242-0301 resulted in a higher signal (fifth and sixth plots) compared to the CD 19 ECD alone (first two plots). These findings indicate that the methods described herein effectively generate antigenspecific B cells with enhanced yield through stimulation with adjuvants and antigen-specific synthetic peptides.Example 37: Antigen conjugation with 8MTT enhances antigen-specific antibody responses in vitro

[0415] The experimental design outlined in Fig. 17A was used for the stimulation of immunosuppressive cell / cytotoxic cell-depleted PBMCs with the antigen of interest, here Activin receptor type IIB (ACTRIIB), conjugated with 8MTT. Human PBMCs were treated with LLME to generate LNOs, which were then cultured for 7 days in complete RPMI 1640 / 10% HS media supplemented with IL-2, IL-4, and IL-21. LNOs were stimulated with either free ACTRIIB ECD or 8MTT-conjugated ACTRIIB ECD in the presence of CD40L.

[0416] Fig. 28A shows that stimulation with ACTRIIB plus CD40L (third and fourth plots from left to right) resulted in a modest antigen-specific response, whereas challenge with ACTRIIB-8MTT conjugates plus CD40L (first and second plots) led to a significantly stronger antigen-specific antibody response as evidenced by the higher number of sorted cells. These results demonstrate that conjugation with 8MTT enhances the antigen-specific antibody response and that the stimulation and sorting methods described herein effectively enable the isolation of ACTRIIB-specific B cells at an improved yield.

[0417] Fig. 28B shows that stimulation with ACTRIIB plus CD40L (third and fourth plots from left to right) resulted in a modest B cell differentiation whereas challenge with ACTRIIB-8MTT conjugates plus CD40L (first and second plots) led to a significantly stronger B cell differentiation as evidenced by higher proportion of CD19+CD27+differentiated B cells among both total CD19+B cells and sorted ACTRIIB-specific B cells.Example 38: A cell membrane immunization approach delivers antigen-specific class- switched B cells to a multi-pass transmembrane GPCR protein in vitro

[0418] Prior examples have demonstrated efficacy for the methods of the present disclosure using an antigen in the form of a protein ECD, or a peptide conjugate, or an antigen-adjuvant conjugate. This examples further demonstrates that the methods of the present disclosure can be applied using a cell membrane fraction containing the antigen of interest as a vehicle for immunization and generation of antigen-specific B cells. Figs. 29A- 29B show the experimental method applied. The cell membrane fraction was isolated from a - 90 -508002954.1Attorney Docket No.: 144242-0301 stable GIPR-expressing HEK293 cell line using the Minute™ Plasma Membrane Protein Isolation and Cell Fractionation Kit (Invent Biotechnologies), following the manufacturer’s protocol. Human PBMCs were treated with LLME to generate LNOs, which were cultured for 7 days in complete RPMI 1640 / 10% HS media supplemented with IL-2, IL-4, and IL-21. LNOs were stimulated with either the cell membrane fraction of GIPR-expressing HEK293 cells or GIPR ECD protein in the presence of CD40L. CD19+IgD GIPR-specific B cells and IgG+B cells within this population were identified and sorted by FACS. The gating strategy used for the sorting was:Lymphocytes / Live / CD3 CD14 CD16 CD56 / CD19 IgD / HSA / GIPR-PE+. HSA was used to exclude cells non-specifically stained by the GIPR tetramer.

[0419] Fig. 29C (first and second plots from left to right) shows that GIPR-specific B- cells were successfully isolated from LLME-treated PBMCs challenged with a membrane fraction derived from GIPR-expressing HEK293 cells. Fig. 29C (fifth and sixth plots) shows that GIPR-specific B-cells were also isolated from LLME-treated PBMCs challenged with GIPR-ECD protein. Surprisingly, a higher proportion of GIPR-specific B cells that are IgG+was observed in the group challenged with the membrane fraction compared to the group challenged with the GIPR-ECD protein. These findings indicate that the membrane fractionbased antigen delivery method enhances the antigen-specific response.Example 39: Sensorgrams depicting the binding affinity of in vitro immunization- derived Prellis antibodies against the target antigen TL1A

[0420] Additional experiments were conducted to demonstrate that antibodies generated using the methods described herein possess desirable properties, including strong binding affinity. Prellis3371 was derived from LNOs on day 7 that were treated with LLME + DOTAP 3 (5pg / ml), and Prellis3378 was derived from LNOs on day 7 that were treated with LLME+DOTAP 3(5pg / ml) + CpG A and B. The sensorgrams shown in Fig. 30 were obtained using the Carterra surface plasmon resonance (SPR) platform per the manufacturer’s instructions to determine the binding affinity between the TL1 A antigen and the indicated antibodies. Fig. 30 shows a strong affinity between Prellis antibodies Prellis3371 (KD 83nm ± 1.5nm) and Prellis3378 (KD 78nm ± l. lnm) and TL1A. This data, in combination with all prior figures shows that the antibodies derived using the methods outlined in Fig. 17A are antigen-specific and possess high binding affinities. Consequently, the methods of the present disclosure are effective for generating antigen-specific cells, and thus antigen-specific antibodies.- 91 -508002954.1Attorney Docket No.: 144242-0301

[0421] Sequences for Prellis3371 and Prellis3378 are provided below:- 92 -508002954.1Attorney Docket No.: 144242-0301Equivalents

[0422] The disclosure set forth above may encompass multiple distinct inventions with independent utility. Although each of these inventions has been disclosed in its preferred form(s), the specific embodiments thereof as disclosed and illustrated herein are not to be considered in a limiting sense, because numerous variations are possible. The subject matter of the inventions includes all novel and nonobvious combinations and subcombinations of the various elements, features, functions, and / or properties disclosed herein. The following claims particularly point out certain combinations and subcombinations regarded as novel and nonobvious. Inventions embodied in other combinations and subcombinations of features, functions, elements, and / or properties may be claimed in this application, in applications claiming priority from this application, or in related applications. Such claims, whether directed to a different invention or to the same invention, and whether broader, narrower, equal, or different in scope in comparison to the original claims, also are regarded- 93 -508002954.1Attorney Docket No.: 144242-0301 as included within the subject matter of the inventions of the present disclosure.- 94 -508002954.1

Claims

1. Attorney Docket No.: 144242-0301ClaimsWhat is claimed is1. An in vitro (ex vivo) method of generating (human) antibody(ies), comprising:(i) harvesting donor immune cells;(ii) challenging the donor immune cells with an antigen, wherein the antigen is encapsulated in or conjugated with liposome-based adjuvants, oil-in-water emulsion adjuvants, virus-like particles, TLR-ligands, carrier proteins, carrier polysaccharides and / or phycoerythrin (PE); and(iii) sorting and sequencing resulting antibodies.

2. The method according to claim 1, further comprising assembling the harvested donor immune cells into at least one lymph node organoid (LNO), wherein assembly occurs between steps (i) and (ii).

3. The method according to any of the above claims, wherein the carrier proteins comprise or consist of keyhole limpet hemocyanin (KLH), non-toxic diphtheria toxin mutant (CRM197), bovine serum albumin (BSA), Tetanus toxoid (TT), non-toxic tetanus toxoid mutant (8MTT), Diphtheria toxoid (DT), Haemophilus protein D (PD), the outer membrane protein complex of serogroup B meningococcus (OMPC), phycoerythrin (PE), or ovalbumin (OVA), or wherein the carrier polysaccharides comprise or consist of polydextran.

4. The method according to any of the above claims, wherein the virus-like particles comprise or consist of Qbeta virus-like particle (Qbeta VLP).

5. The method according to any of the above claims, further comprising treating the donor immune cells to remove immunosuppressive cells and cytotoxic cells.

6. The method according to claim 5, wherein treating donor immune cells comprises or consists of administering a lysosomotropic agent, optionally wherein the lysosomotropic agent is L-leucyl-L-leucine methyl ester (LLME), or negative selecting donor immune cells with anti-CD56 mAb-, anti-CD8 mAb-, and anti-CD14- 95 -508002954.1Attorney Docket No.: 144242-0301 mAb-conjugated magnetic beads, or combining administering LLME and negative selection with anti-CD8 mAb-conjugated magnetic beads.

7. The method according to any of the above claims, further comprising stimulation of the cells, optionally wherein the stimulation comprises stimulation of the cells with CD40 ligand (CD40L) or toll-like receptor (TLR) ligands, optionally wherein the TLR ligands are selected from the group consisting of GpG ODNs or muramyl dipeptide (MDP).

8. The method according to any of the above claims, wherein the liposome- or lipoparticle-based adjuvants comprise or consist of DOTAP lipoparticles (or other lipoparticles) / TLR ligands.

9. The method according to any of the above claims, further comprising conjugating antigen(s) with CD40L.

10. The method according to claim 9, wherein the antigen and CD40L are conjugated through streptavidin, streptavidin-nanoparticle, streptavidin-liposome, orNTA(Ni)- liposome.

11. The method according to any of the above claims, wherein the antigen is conjugated to anti-CD4 mAb, anti-CD3 mAb, or a bispecific anti-CD3 / anti-CD28 antibody through a biotin-streptavidin protocol with streptavidin-nanoparticle.

12. The method according to any of the above claims, further comprising conjugating the antigen with anti-CD3 or anti-CD4 mAb through streptavidin or streptavidin conjugated nanoparticles.

13. The method according to any of the above claims, further comprising additionally challenging donor immune cells with the antigen(s) conjugated with class B CpG ODN 2006 or class C CpG ODN 2395 through streptavidin-nanoparticle.- 96 -508002954.1Attorney Docket No.: 144242-030114. The method according to any of the above claims, further comprising adding an immune checkpoint inhibitor, optionally wherein the immune checkpoint inhibitor comprises anti-CTLA-4 and / or anti-PD-1 blocking antibodies, in the cell culture media.

15. The method according to any of the above claims, wherein donor immune cells are harvested from umbilical cord blood (UCB).

16. The method according to any of the above claims, wherein donor immune cells are tonsil cells.

17. The method according to any of the above claims, wherein the donor immune cells are challenged with an antigen foregrounded with DOTAP liposome-antigen complexes and / or Qbeta VLP-antigen conjugates.

18. The method according to any of the above claims, further comprising polarizing T cells in the donor immune cells.

19. The method according to claim 18, wherein the T cells are polarized to T helper 2 (Th2) cells.

20. The method according to claim 18, wherein the T cells are polarized to T helper 17 (Thl7) cells.

21. The method according to any of the above claims, further comprising priming T cells with antigen loaded dendritic cells (DCs).

22. The method according to any of the above claims, further comprising mixing antigen primed Th2 cells generated by DCs with B cells from the donor immune cells, the mixing occurring in the LNO.- 97 -508002954.1Attorney Docket No.: 144242-030123. The method according to any of the above claims, further comprising the generation of Langerhans cells (LCs) from adult monocytes or cord blood pluripotent CD34+cells to be loaded with antigen.

24. The method according to any of the above claims, further comprising the generation of follicular helper T cell (Tfh) in vitro from naive CD4 T cells isolated form adult or cord blood mononuclear cells.

25. The method according to claim 24, further comprising activating Tfh using antigen loaded LCs.

26. The method according to any of the above claims, further comprising mixing antigen- activated Tfh with B cells from the same donor, wherein the mixing occurs within the LNO.

27. The method according to any of the above claims, wherein the antigen is selected from the group consisting of CD3e, TL1 A, PE, CD 19 ECD, GIPR ECD and ACTRIIB, and / or wherein the antigen is a component of a cell membrane fraction, the antigen is contained within a cell membrane fragment, or the antigen is present in the cell membrane of an intact cell that expresses the antigen.

28. The method according to any of the above claims, wherein the method is effective for generating antigen-specific cells for about 7 to about 14 days following antigen challenge.

29. An in vitro (ex vivo) method of generating (human) antibody(ies), comprising challenging at least one LNO derived from harvested donor immune cells that were pre-treated to remove immunosuppressive cells and cytotoxic cells, with an antigen; optionally wherein the antigen is encapsulated in or conjugated with liposome-based adjuvants, oil-in-water emulsion adjuvants, virus-like particles, TLR-ligands, carrier proteins, carrier polysaccharides and / or PE.

30. The method according to claim 29, wherein the pre-treatment to remove immunosuppressive cells and cytotoxic cells from the harvested donor immune cells comprises or consists of administering a lysosomotropic agent, optionally wherein the- 98 -508002954.1Attorney Docket No.: 144242-0301 lysosomotropic agent is LLME, or negative selecting donor immune cells with anti- CD56 mAb-, anti-CD8 mAb-, and anti-CD14 mAb-conjugated magnetic beads or combining administering LLME and negative selection with anti-CD8 mAb- conjugated magnetic beads.

31. The method according to claim 30, wherein the lysosomotropic agent is LLME.

32. A kit comprising an adjuvant, at least one agent for the removal of immunosuppressive cells and cytotoxic cells, and instructions for using said agent to generate LNOs from a population of harvested donor immune cells.- 99 -508002954.1

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